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Glyphosate spraying and earthworm Lumbricus terrestris L. activity: Evaluating short-term impact in a glasshouse experiment simulating cereal post-harvest
European Journal of Soil Biology 96 (2020) 103148
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Glyphosate spraying and earthworm Lumbricus terrestris L. activity: Evaluating short-term impact in a glasshouse experiment simulating cereal post-harvest
T
Visa Nuutinena,∗, Marleena Hagnera,b, Heikki Jallia, Lauri Jauhiainena, Sari Rämöa, Ilkka Sarikkaa, Jaana Uusi-Kämppäa a b
Natural Resources Institute Finland (Luke), FI-31600, Jokioinen, Finland Ecosystems and Environment Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, FI-15340, Lahti, Finland
A R T I C LE I N FO
A B S T R A C T
Handling editor: Stefan Schrader
A glasshouse experiment was conducted to study if glyphosate spraying has immediate short-term effects on the growth, reproduction and residue incorporation of the earthworm species Lumbricus terrestris L. The experiment simulated post-harvest conditions in no-till cereal cultivation, where L. terrestris often abound, and glyphosate is widely used. Pairs of adult, field-collected L. terrestris (median total mass 11.2 g fresh weight) were kept in fine sand-filled cylinders, with chopped wheat straw on the soil surface as feed. The treatment cylinders were sprayed with glyphosate at normal field application rate [Rodeo® XL 3.0 l ha−1, resulting in 1080 g a.i. ha−1 and “Contact” surfactant (0.5 l ha−1)]; the controls were sprayed with water (N = 12 for both). The treatment and control cylinders were maintained for two months at +15 °C, approximate 60% air humidity and 12h:12h light:dark cycle. All individuals survived the experiment. There was no difference in L. terrestris mass change over the experiment, with an average weight change of +0.96% in glyphosate treatment and −0.93% change in the control (p = 0.66). Glyphosate treatment and control did not differ in cocoon production rate (31 vs 28 cocoons cylinder−1 respectively; p = 0.30). The straw incorporation was slightly but not significantly lower in glyphosate-treated soil (decline in surface straw mass 27.5% vs. 30.5%; p = 0.07). The glyphosate treatment applied represents a commonly used practice in spring cereal cultivation, and it did not affect negatively L. terrestris. The varying study results on the effects of glyphosate on earthworms – and on L. terrestris in particular – may partly arise from notable variation in application rates and the formulation-surfactant combinations used in different experiments which needs to be considered in the ecological risk assessment of glyphosate use.
Keywords: Earthworms Glyphosate No-till risk assessment Cocoons
1. Introduction The increased abundance of soil macrofauna, especially earthworms, is a typical change in conversion from mouldboard ploughing to no-tillage or conservation agriculture of arable fields [1]. According to a recent meta-analysis, earthworm densities are 137% and 127%, and biomasses 196% and 101% higher under no-till and conservation tillage, respectively, compared to ploughed soils [2]. The pronounced increase of earthworm biomass in no-till fields relates to the increased proportion of large, deep burrowing and surface feeding anecic earthworms such as Lumbricus terrestris L. They especially benefit from low physical disturbance of soil and from the availability of plant residues on the soil surface. Anecic earthworms improve arable soil conditions by, for instance, enhancing soil macroporosity, conductivity and
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aereation and by incorporating surface residues and producing nutrient rich casts [3,4] thus having a relatively strong positive effect on the growth of different crop plants [5]. Reduced tillage often requires the use of herbicides, particularly to control difficult rhizome-spreading weeds such as couch grass (Elymus repens) [6]. The extensive adoption of less intensive tillage is one of the main reasons for the explosive increase in the use of glyphosate, which is currently the most used herbicide in the world [7]. Studies on the effects of glyphosate on earthworms mainly involve ecotoxicological laboratory tests where compost worms (Eisenia sp.) have been used as the study model. Several investigations have reported negative effects of glyphosate on, for instance, the growth, reproduction and behaviour of Eisenia sp. (reviewed in Refs. [8–10]). Transfer from these results to the effects on earthworms in the field is difficult, because Eisenia sp. do
Corresponding author. E-mail address: visa.nuutinen@luke.fi (V. Nuutinen).
https://doi.org/10.1016/j.ejsobi.2019.103148 Received 15 May 2019; Received in revised form 11 December 2019; Accepted 19 December 2019 1164-5563/ © 2019 Elsevier Masson SAS. All rights reserved.
European Journal of Soil Biology 96 (2020) 103148
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not normally occur in field soils [11], and they are less sensitive to pesticides than species found in cultivated fields [12]. Common earthworm species in arable soils have therefore been recommended as complementary species for testing the effects of agrochemicals on soil biota [13]. According to the recent research synthesis of Briones and Schmidt [2] glyphosate has no negative effect on earthworms in field conditions: their meta-analysis suggested that use of glyphosate did not lower but rather enhanced the increase of earthworm density and biomass in notill fields. However, Gaupp-Berghausen et al.‘s [14] glasshouse study with A. caliginosa (Sav.) and L. terrestris reported that L. terrestris activity, measured as the rate of surface cast production, ceased after glyphosate (Roundup®) spraying of plants growing in mesocosms. Thus, despite the evidence of no effect on earthworm abundances in the field, glyphosate spraying may have immediate activity lowering impacts on L. terrestris. This is of particular concern, because spraying may often coincide with periods of high L. terrestris activity. For example, the autumn post-harvest glyphosate applications which are done to control E. repens, the most common grass weed in Finnish cereal fields [15], take place when cool and moist conditions and the lengthening of the daily dark period encourage the nocturnal surface activity of L. terrestris [16]. Consequently, their foraging on and incorporation of surface crop residues may be compromised [17,18] and glyphosate spraying may also disturb L. terrestris reproduction by interfering with mating, which occurs on the soil surface [19]. We investigated the short-term impacts of glyphosate application on L. terrestris in a greenhouse experiment simulating post-harvest conditions in no-till arable soil. We aimed at producing applicable knowledge for the conditions of Northern Europe and used a regionally common glyphosate surfactant combination with a realistic application rate. The specific aims were to find out if glyphosate (i) lowers the rate of residue incorporation L. terrestris, (ii) affects their mass change and (iii) influences the reproduction of by lowering cocoon production rate.
powder mixed in 10 l of water) [20] applied to the soil surface in lateOctober 2016 at a broad-leaved forest margin in Jokioinen (60°48′28,740″ N, 23°28′46,308″ E). Glyphosate had never been used at the site. Emerging L. terrestris were immediately rinsed in water and inoculated in groups of 6–8 individuals in 10 l buckets filled with moist, unsieved experimental soil. Prior to the start of the experiment, the buckets were stored for three months at +6 °C and in constant darkness. To keep the earthworms in good condition, horse manure (dried for a few days at +60 °C and remoistened) was added as feed on the soil surface ad libitum. Fifty-six L. terrestris with well-developed clitellum and in good condition were weighed (median fresh mass 5.4 g, range 3.2–8.7 g), and placed individually in small numbered vials with water. Individuals were randomly assigned and introduced into number-coded soil cylinders, with two individuals per cylinder. To keep total L. terrestris mass relatively similar in the cylinders, a deviation from completely random pair formation was made by forming six pairs using the largest and smallest individuals, then the second largest and the second smallest individuals and so on. The median total L. terrestris fresh mass was 11.2 g per cylinder (min 9.5, max 13.6 g). To prevent the escape of earthworms, a plastic collar which reached 0.2 m above the upper rim of the cylinder was firmly attached to it with adhesive tape (Appendix Fig. A1). The cylinders were allowed to stabilise for five weeks in experimental conditions (below). During the stabilising period, L. terrestris were fed horse manure with 20 g (dwt; pre-treated as above) applications per cylinder on the soil surface at the start and during the third week of the period. All germinating plant seedlings were picked away from the cylinders weekly. This was also done during the subsequent experimental period. 2.3. Treatments The surface residue used in the experiment was wheat (Triticum aestivum) straw baled in the late summer harvest of 2016. The harvest had not received glyphosate treatment during the growing season. The straw was air-dried and cut in 3 cm pieces. The ash-free dry weight of the straw was determined from five grounded samples by thermogravimetric analyses (TGA701, Leco, UK). A day before the experimental treatment, the manure remaining on the soil surface was carefully displaced from the cylinder soil surfaces using a vacuum cleaner with low suction. The next day, a few hours before the spraying treatments, 10 g of the cut straw was spread evenly on the soil surface of each cylinder (Fig. 1a). The experimental cylinders were randomly allocated in control and glyphosate treatment groups (N = 14) so that both groups had three randomly chosen cylinders with the pair of exceptionally large and small L. terrestris. The groups were taken in turns outside the greenhouse where the treatments were conducted. To allow efficient exposure of the cylinder surfaces to the sprayings, the plastic collars were lowered to a height of 5 cm. Glyphosate was sprayed in accordance with the GEP (Good Experimental Practice) protocol used by Natural Resources Institute Finland, adopted from EEC Directive 93/71/EEC. First, the control cylinders (arranged in a rectangle) were sprayed with clean water, and then the similarly arranged treatment cylinders with glyphosate. The glyphosate treatment imitated normal autumn couch grass control spraying in Finland. The herbicide was Rodeo® XL (360 g l−1 glyphosate (as potassium salt)) and the dose used was 3.0 l ha−1 (1080 g a.i. ha−1). The adhesive agent used was “Contact” (active ingredient: isodecyl alcohol ethoxylate) in a dose of 0.5 l ha−1. A 2-m-wide portable spraying boom was used, and the amount of spray water equalled 200 l ha−1 (for details see Ref. [21]). To evaluate the efficiency of the glyphosate treatment, glyphosate concentration at the 0–0.25 m soil layer was later measured from five pairs of control and treatment pairs (3.5 months after the finish of the experiment from soil stored at +6 °C). The measurement was conducted using Natural Resources Institute Finland's in-house method based on the determination
2. Materials and methods 2.1. Experimental soil and vessels Soil was collected in mid-October 2016 from 0.1 to 0.2 m depth of a cereal field (60°51′10,641″ N, 23°28′0,646″), when the soil was under post-harvest stubble. Uppermost 0.1 m layer of soil was not used to avoid usage of previously glyphosate affected topsoil (below). Topsoil textural composition at the site is 14.8% clay, 13.8% silt, 70.1% fine sand and 1.3% sand. The main chemical characteristics at the 0.1–0.2 m layer were pH 5.9 (1:5H2O), C 25 g kg−1, P 18.9 mg l−1 and conductivity 1.3 × 10−4 S cm−1. A glyphosate application (Rodeo XL®, 1080 g a.i. ha−1) had been conducted on the field six weeks before the collection of the soil. The likely initial presence of glyphosate in experimental soil was not regarded as detrimental for the main aim of the study, which was the investigation of immediate behavioural response of L. terrestris to glyphosate spraying on the residue layer and soil surface. The field-collected soil was sieved through 7 mm mesh and moistened to an initial moisture content of 21.7% v/v. The mesh used in soil sieving was sufficiently large to allow the passage of L. terrestris cocoons, but no cocoons were found in the wet sieving (3.5 mm mesh) of three 10.8 kg samples taken from the batch of sieved soil. PVC cylinders of 0.15 m diameter and 0.4 m height, closed at their bottoms with plastic caps, were used as experimental vessels. Altogether, 28 cylinders were filled with 10.8 kg of soil to obtain 1.2 g cm-3 bulk density so that the soil surface was 0.02 m below the cylinder's upper rim (Appendix Fig. A1). 2.2. L. terrestris collection and inoculation L. terrestris were collected with mustard solution (60 g of mustard 2
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emergence. At the end of the experiment, the soil moisture content of the top 0–15 cm layer was measured from four randomly chosen control and treatment cylinders with TDR (Model no. 6050X1 Trase System, Soilmoisture Equipment Corporation, Santa Barbara, California, USA). The mean moisture contents (SD) were 26.25% (1.82) and 26.98% (0.93) for control and glyphosate treatment, the difference being statistically indiscernible (two-sided p = 0.67, Wilcoxon two-sample test; n = 4). The soil temperature in treatment and control follow-up cylinders remained close to +15 °C during the experiment (Appendix Fig. A2). The readings in the control cylinder were on average 0.14 °C higher. This difference was probably due to the control and treatment follow-up cylinders being positioned at opposite sides of the table. 2.5. Ending of the experiment At the end of the experiment, all the straw remaining on the soil surface (Fig. 1b) was carefully collected in paper bags using forceps. When a piece of straw was partly below the soil surface or covered by earthworm casts, the visible part was collected by cutting it with scissors (Fig. 1c). The samples were air-dried and weighed. The ash-free dry weight of each sample was measured from a milled sample of straw using TCA, and the dry weight of straw remaining on the soil surface was converted into ash-free weight. The cylinders were turned upside-down and gradually emptied into a large plastic vessel. L. terrestris individuals were hand-sorted from the soil, washed immediately in tap water, dried on a paper towel and weighed. The presence of spermatophores, which indicates individuals’ recent copulation, was recorded. From six randomly chosen pairs of treatment and control cylinders, all the soil was wet-sieved for L. terrestris cocoons in a 3.5 mm sieve.
Fig. 1. Residue related observations during and at the termination of the experiment. (a) and (b): Wheat straw cover on the soil surface of a glyphosatetreated temperature follow-up cylinder (no. 25) on day #5 and day #57, respectively, of the 61-day experiment. Incorporation of the straw by L. terrestris exposed the soil surface towards the end of the experiment. (c) Sampling of the remaining straw from the soil surface at the end of the experiment. (d) Incorporated straw (arrows) in the upper part of L. terrestris burrow (soil surface at the top).
of glyphosate residues as 9-fluorenmethyl chloroformate (FMOC-Cl) with multiple reaction modelling (MRM) by ultra-performance liquid chromatography (UPLC-MS/MS). Glyphosate concentration was 0.52 and 0.09 mg kg−1 (p < 0.01) in glyphosate and control treatments respectively. Values were within the glyphosate concentration range of 0.05–0.6 mg kg−1 reported for agricultural topsoil (0–0.15/0.20 m) in cereal cultivation in a recent European Union-wide survey [22]. After the treatments, the cylinders were taken immediately inside the greenhouse. They were arranged on a table in a column of twos with the treatment and control cylinders randomly chosen and subsequently randomly positioned on either the left or right side of the table, the arrangement corresponding with a row-column design. There were 12 experimental cylinders for control and treatment. Two extra pairs of control and treatment cylinders were used for soil temperature (0–5 cm depth) and moisture follow-ups (below).
2.6. Statistical analyses
2.4. Experimental period
3. Results
The duration of the experiment was 61 days, from 26 January to 27 March 2017. The room's air temperature was set at +15 °C, and air moisture content at 60% relative humidity. The light cycle in the room was 12h:12h (dark period from 6 p.m. to 6 a.m., < 0.001 lux) to allow a daily soil surface activity cycle for L. terrestris. Because of light reflectance from nearby glasshouses and from snow cover outside the building, darkness was not complete. However, the illumination during the dark period was low enough to allow the emergence of L. terrestris based on several direct observations of surface activity made over the experiment using red torch light. Towards the end of the experimental period, twilight conditions prevailed in the first 1–2 h of the night due to the lengthening of the day. The moisture content of the soil was followed by weekly weighing of two follow-up cylinders, and the mean weight loss was compensated by watering all cylinders with the corresponding amount of water. During the last two weeks of the experiment, the soil surface was sprayed every evening with 10 ml of water to further encourage L. terrestris
All L. terrestris were alive at the end of the experiment. There was no difference in the weight change of the earthworms between the glyphosate-treated and control cylinders (+0.96% vs. 0.93%, p = 0.66; Fig. 2a). Likewise, there was no statistically discernible difference between glyphosate treatment and control in the number of cocoons produced per cylinder (31 vs 28 cocoons cylinder−1, p = 0.30; Fig. 2b). In two control and two glyphosate treatment cylinders, both L. terrestris individuals had two spermatophores indicating recent copulation. Active burial of straw by L. terrestris was shown by the gradual exposure of the soil surface during the experiment (Fig. 1 a-b) and the presence of straw in the upper parts of L. terrestris burrows at the end of the experiment (Fig. 1d). Overall, ca. 29% of initially applied straw was incorporated below surface level during the 61 days of the study. The incorporation rate of wheat straw was 3.0% points lower in the glyphosate treatment, but the difference between treatments was not statistically significant (27.5% vs 30.5%, p = 0.07; Fig. 2c). When one extreme observation was removed from the data and the initial
Statistical analysis of the data was based on the row-column design which takes into account variation within the glasshouse across two dimensions. In the analysis of surface straw mass change during the study, straw ash-free dry weight at the start of the experiment was used as a covariate. In addition, the mass change of L. terrestris over the study was analysed using the mass of the individuals in the cylinder as a covariate (two levels: mass of the individual is < 5 g or ≥ 5 g). Scatter plots of residuals and predicted values were drawn to evaluate visually the assumption of homoscedasticity between the predicted dependent variables and the errors of prediction. These plots revealed an extreme observation in the analysis of the straw incorporation rate data. The model was fitted both with and without it. Statistical analyses were performed using SAS/STAT software (version 9.4).
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4. Discussion We found no evidence of glyphosate spraying negatively affecting L. terrestris. All individuals survived the experiment, and the variation in earthworm final mass and cocoon production were not associated with the experimental treatments. There was an indication of a small negative effect of glyphosate spraying on the straw incorporation rate, but this effect was also statistically indiscernible. As the standard errors were relatively small for the mean estimates of all response variables, we consider that the statistically non-significant differences were not due to the low statistical power of the comparisons but rather implied absences of effects. One of the motivations of this study was to identify the extent to which the recently noticed [14] negative effect of glyphosate spraying on L. terrestris activity may impair one of their key ecosystem functions, the incorporation of surface residues. While the earlier study [14] measured egestion and the present study foraging, the two phenomena are intimately connected and both involve activity on or close to the soil surface, although by different ends of the animal. Both studies deal with immediate behavioural response, and it is conceivable that the effects of spraying on L. terrestris relate to sensory reaction to the glyphosate-affected soil surface and plant residues. The indication of a small, though non-significant decline in the straw incorporation rate in glyphosate-treated soil which we noticed may have resulted from reduced foraging immediately after spraying due to the avoidance of the herbicide-affected soil surface and straw. This type of avoidance, which can partly result from dermal exposure and irritation, may wane over time when the glyphosate concentration in residue is lowered by watering, resulting in a small net negative effect over the experiment. It is possible that both the relatively high glyphosate application rate and the more toxic surfactant contributed to the negative effects on L. terrestris activity observed by GauppBerghausen et al. [14]. In their study, the treatment mesocosms were first sprayed with 7.2 ml of ‘Roundup® Alphée’ on two consecutive days (in total 14.4 ml), and then two days later with 10 ml of ‘Roundup® Speed’. Therefore, in total 176.12 ml m−2 of herbicide (glyphosate concentration of the products 7.2 g l−1) was applied to the pot (diam. 0.42 m), resulting in a field application rate of 12.7 kg a.i. ha−1. This is a twelvefold dose compared, for instance, with the typical application rate in agricultural field use in Finland used in the present study (1.1 kg a.i ha−1). ‘Roundup® Alphée’ also contains POEA (polyethoxylated tallow amine) as a surfactant. POEA is regarded as more toxic than glyphosate, which has led to the banning of POEA containing glyphosate products in the EU [23,24]. We are not familiar with any studies on POEA's effects on earthworms, although it has been stated that a negative effect has been demonstrated [10]. The surfactant used in the present study was isodecyl alcohol ethoxylate-based, and we are unaware of any reports of its toxicity to earthworms. Stellin et al. [10] recently reported elevated mortality of L. terrestris with an increasing application rate of Roundup® 360 Power mixed in mesocosm soil. The gradient of glyphosate concentration used in their study was 0.59, 2.9, 5.79 and 11.59 g m−2. While these concentrations reportedly were in the range applied in North Italian vineyards, they are notably high and approximately 5–100 times higher than the application rate in cereal cultivation in Finland followed in the present study (0.11 g a.i. m−2). A notable difference with Gaupp-Berghausen et al.’ study [14] was the absence of growing plants from our experiment. The bearing of this on the response of L. terrestris to glyphosate spraying is difficult to judge. Despite the absence of growing weeds from our experiment, we consider that our experiment meaningfully simulated the cereal postharvest environment where L. terrestris vigorously forage on surface residues. The setting also avoided the difficult issue of what constitutes proper control when growing plants are included in experiments where glyphosate's effects on surface residue feeding earthworms are studied [25]. However, direct extrapolation from the present results to field
Fig. 2. L. terrestris response to glyphosate spraying in a nine-week glasshouse experiment. (a) Weight change in the experimental vessels over the settling and experimental periods (n = 12); (b) cocoons retrieved from the soil after the experiment (n = 6); (c) residue incorporation rate (AFDW = ash-free dryweight; n = 12). In all panels, model-based means and standard errors are shown.
differences in cylinder total L. terrestris biomasses were controlled for, the difference in the straw incorporation rate was smaller and the pvalue larger (2.0% points; p = 0.15). 4
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population growth of earthworms under reduced cultivation and no-till where glyphosate is widely used [2]. Our results indicate that immediate activity-lowering impacts on soil surface active L. terrestris can also be avoided. The varying responses of L. terrestris – and earthworms in general – to glyphosate herbicides in different studies may be partly due to the notable chemical variation in formulation-surfactant combinations and in their application rates. As Niemeyer et al. [34] also indicate, this needs to be carefully considered in the risk assessment of glyphosate use.
situations where weeds are present is not possible because glyphosate affected weeds and straw are likely to have different biochemical compositions and microbiological communities which may affect litter burial and consumption of L. terrestris. The evident welfare of L. terrestris in our experiment was possibly due to “carry-over effects” [26] from the settling period, where the earthworms were fed horse manure. Horse manure encourages L. terrestris growth [27], and the individuals may have gained weight during the conditioning period to the extent that subsequent weight loss during the experiment – likely to occur under the type of straw-only diet used [28] – went unnoticed. The average cocoon production rate we observed (approximately 7 cocoons ind.−1 month−1) is somewhat higher than the monthly rates reported earlier from comparable laboratory conditions [27,29]. While L. terrestris did mate during the experiment, this may also have been due to the positive carry-over effect from the favourable conditioning period – and even from the field – as L. terrestris are able to store sperm for several months for viable cocoon production [27,30].
Funding This study is part of the Glyfos II-project (Luke project #4100700052900), funded by the Ministry of Agriculture and Forestry of Finland. Declaration of competing interest The authors declare no conflict of interest.
5. Conclusions
Acknowledgements
The debate on the risks of glyphosate-based herbicides involves human health, environmental and socioeconomic issues. The question of impacts on soil life is only one – if important – aspect of the discussion. Current plant protection policies aim to minimise the harmful effects of pesticide use [31,32]. For the particular case we studied, the results suggest that this is possible in the case of glyphosate when recommended application rates are followed and particular glyphosate product and surfactant combinations are used. Our findings are also parallel with the recent field study of Hagner et al. [33] which indicated minor side effects of glyphosate spraying on soil micro- and mesofauna. The prevailing view is that glyphosate spraying does not impair the
We thank Samuli Klemelä for his help in the setting up of the experiment, Matti Eskola and Leena Ruokonen for their assistance in spraying, Mirva Ceder, Leena Holkeri and Outi Haapala for collaboration in laboratory analyses and Heikki Hiisilä for providing background information. Kartanonkylän Talli (Ypäjä) kindly provided the horse manure for L. terrestris maintenance. Comments by two anonymous referees and the Editor helped us to improve the manuscript. Interest and helpful comments by the project steering group are gratefully acknowledged.
Appendix
Fig. A.1. The experiment consisted of 28 PVC cylinders (⌀ 0.15, m height 0.4 m) filled with 10.8 kg arable field soil and closed at their bottoms with plastic caps. To prevent the escape of worms, a plastic collar, which reached 0.2 m above the upper rim of the cylinder, was attached to the cylinder with adhesive tape.
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Fig. A.2. Topsoil (0–5 cm) temperature over the experiment in treatment and control follow-up cylinders.
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promote the reduction of Fusarium biomass and deoxynivalenol content in wheat straw under field conditions, Soil Biol. Biochem. 43 (2011) 1858–1865. V. Nuutinen, K.R. Butt, Earthworm dispersal of plant litter across the surface of agricultural soils, Ecology 100 (2019) e02669. V. Nuutinen, K.R. Butt, Mating behaviour of the earthworm Lumbricus terrestris L. (Oligochaeta: Lumbricidae), J. Zool. (Lond.) 242 (1997) 783–798. A. Gunn, The use of mustard to estimate earthworm populations, Pedobiologia (1992) 65–67. M. Hagner, S. Hallman, L. Jauhiainen, R. Kemppainen, S. Rämö, K. Tiilikkala, H. Setälä, Birch (Betula spp.) wood biochar is a potential soil amendment to reduce glyphosate leaching in agricultural soils, J. Environ. Manag. 164 (2015) 46–52. V. Silva, L. Montanarella, A. Jones, O. Fernández-Uglade, H.G.J. Mol, C.J. Ritsema, V. Geissen, Distribution of glyphosate and aminomethylphosphonic acid (AMPA) in agricultural topsoils of the European Union, Sci. Total Environ. 621 (2018) 1352–1359. EFSA (European Food Safety Authority), Request for the evaluation of the toxicological assessment of the formulant POE-tallowamine, EFSA J. 13 (2015) 4303. European Union, Commission implementing regulation (EU) 2016/131 of I August 2016 amending Implementation Regulation (EU) No 540/2011 as regards the conditions of approval of the active substance glyphosate, Off. J.Eur. Union 2 (8) (2016) L 208/1. G. von Mérey, P.S. Manson, A. Mehrsheikh, P. Sutton, S.L. Levine, Glyphosate and aminomethylphosphonic acid chronic risk assessment for soil biota, Environ. Toxicol. Chem. 35 (2016) 2742–2752. C.M. O'Connor, D.R. Norris, G.T. Crossin, T.J. Cooke, Biological carry over effects: linking common concepts and mechanisms in ecology and evolution, Ecosphere 5 (2014) 28. K.R. Butt, Food quality affects production of Lumbricus terrestris (L.) under controlled environmental conditions, Soil Biol. Biochem. 43 (2011) 2169–2175. M. Nieminen, T. Hurme, J. Mikola, K. Regina, V. Nuutinen, Impact of earthworm Lumbricus terrestris living sites on the greenhouse gas balance of no-till arable soil, Biogeosciences 12 (2015) 5481–5493. K.R. Butt, J. Frederickson, R.M. Morris, The intensive production of Lumbricus terrestris L. for soil amelioration, Soil Biol. Biochem. 24 (1992) 1321–1325. K.R. Butt, V. Nuutinen, Reproduction of the earthworm Lumbricus terrestris Linné after the first mating, Can. J. Zool. 76 (1998) 104–109. European Union, Directive 2009/128/EC of the European Parliament and of the Council of 21 October 2009 establishing a framework for Community action to achieve the sustainable use of pesticides, Off. J.Eur. Union 24 (11) (2009) L 309/71. Finnish Safety and Chemical Agency (Tukes), The Finnish national action plan on the sustainable use of plant protection products for 2018–2022, https://tukes.fi/ documents/5470659/6372801/National+action+plan+-+Finland/75122d054d29-4371-ab61-29fbc60157fc/National+action+plan+-+Finland.pdf, (2018) 26. M. Hagner, J. Mikola, I. Saloniemi, K. Saikkonen, M. Helander, Effects of a glyphosate-based herbicide on soil animal trophic groups and associated ecosystem functioning in a northern agricultural field, Sci. Rep. 9 (2019) 8540. J.C. Niemeyer, J.B. de Santo, N. Guerra, A.M.R. Filho, T.M. Pech, Do recommended doses of glyphosate-based herbicides affect soil invertebrates? Field and laboratory screening tests to risk assessment, Chemosphere 198 (2018) 154–160.
Contents lists available at ScienceDirect
European Journal of Soil Biology journal homepage: www.elsevier.com/locate/ejsobi
Glyphosate spraying and earthworm Lumbricus terrestris L. activity: Evaluating short-term impact in a glasshouse experiment simulating cereal post-harvest
T
Visa Nuutinena,∗, Marleena Hagnera,b, Heikki Jallia, Lauri Jauhiainena, Sari Rämöa, Ilkka Sarikkaa, Jaana Uusi-Kämppäa a b
Natural Resources Institute Finland (Luke), FI-31600, Jokioinen, Finland Ecosystems and Environment Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, FI-15340, Lahti, Finland
A R T I C LE I N FO
A B S T R A C T
Handling editor: Stefan Schrader
A glasshouse experiment was conducted to study if glyphosate spraying has immediate short-term effects on the growth, reproduction and residue incorporation of the earthworm species Lumbricus terrestris L. The experiment simulated post-harvest conditions in no-till cereal cultivation, where L. terrestris often abound, and glyphosate is widely used. Pairs of adult, field-collected L. terrestris (median total mass 11.2 g fresh weight) were kept in fine sand-filled cylinders, with chopped wheat straw on the soil surface as feed. The treatment cylinders were sprayed with glyphosate at normal field application rate [Rodeo® XL 3.0 l ha−1, resulting in 1080 g a.i. ha−1 and “Contact” surfactant (0.5 l ha−1)]; the controls were sprayed with water (N = 12 for both). The treatment and control cylinders were maintained for two months at +15 °C, approximate 60% air humidity and 12h:12h light:dark cycle. All individuals survived the experiment. There was no difference in L. terrestris mass change over the experiment, with an average weight change of +0.96% in glyphosate treatment and −0.93% change in the control (p = 0.66). Glyphosate treatment and control did not differ in cocoon production rate (31 vs 28 cocoons cylinder−1 respectively; p = 0.30). The straw incorporation was slightly but not significantly lower in glyphosate-treated soil (decline in surface straw mass 27.5% vs. 30.5%; p = 0.07). The glyphosate treatment applied represents a commonly used practice in spring cereal cultivation, and it did not affect negatively L. terrestris. The varying study results on the effects of glyphosate on earthworms – and on L. terrestris in particular – may partly arise from notable variation in application rates and the formulation-surfactant combinations used in different experiments which needs to be considered in the ecological risk assessment of glyphosate use.
Keywords: Earthworms Glyphosate No-till risk assessment Cocoons
1. Introduction The increased abundance of soil macrofauna, especially earthworms, is a typical change in conversion from mouldboard ploughing to no-tillage or conservation agriculture of arable fields [1]. According to a recent meta-analysis, earthworm densities are 137% and 127%, and biomasses 196% and 101% higher under no-till and conservation tillage, respectively, compared to ploughed soils [2]. The pronounced increase of earthworm biomass in no-till fields relates to the increased proportion of large, deep burrowing and surface feeding anecic earthworms such as Lumbricus terrestris L. They especially benefit from low physical disturbance of soil and from the availability of plant residues on the soil surface. Anecic earthworms improve arable soil conditions by, for instance, enhancing soil macroporosity, conductivity and
∗
aereation and by incorporating surface residues and producing nutrient rich casts [3,4] thus having a relatively strong positive effect on the growth of different crop plants [5]. Reduced tillage often requires the use of herbicides, particularly to control difficult rhizome-spreading weeds such as couch grass (Elymus repens) [6]. The extensive adoption of less intensive tillage is one of the main reasons for the explosive increase in the use of glyphosate, which is currently the most used herbicide in the world [7]. Studies on the effects of glyphosate on earthworms mainly involve ecotoxicological laboratory tests where compost worms (Eisenia sp.) have been used as the study model. Several investigations have reported negative effects of glyphosate on, for instance, the growth, reproduction and behaviour of Eisenia sp. (reviewed in Refs. [8–10]). Transfer from these results to the effects on earthworms in the field is difficult, because Eisenia sp. do
Corresponding author. E-mail address: visa.nuutinen@luke.fi (V. Nuutinen).
https://doi.org/10.1016/j.ejsobi.2019.103148 Received 15 May 2019; Received in revised form 11 December 2019; Accepted 19 December 2019 1164-5563/ © 2019 Elsevier Masson SAS. All rights reserved.
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not normally occur in field soils [11], and they are less sensitive to pesticides than species found in cultivated fields [12]. Common earthworm species in arable soils have therefore been recommended as complementary species for testing the effects of agrochemicals on soil biota [13]. According to the recent research synthesis of Briones and Schmidt [2] glyphosate has no negative effect on earthworms in field conditions: their meta-analysis suggested that use of glyphosate did not lower but rather enhanced the increase of earthworm density and biomass in notill fields. However, Gaupp-Berghausen et al.‘s [14] glasshouse study with A. caliginosa (Sav.) and L. terrestris reported that L. terrestris activity, measured as the rate of surface cast production, ceased after glyphosate (Roundup®) spraying of plants growing in mesocosms. Thus, despite the evidence of no effect on earthworm abundances in the field, glyphosate spraying may have immediate activity lowering impacts on L. terrestris. This is of particular concern, because spraying may often coincide with periods of high L. terrestris activity. For example, the autumn post-harvest glyphosate applications which are done to control E. repens, the most common grass weed in Finnish cereal fields [15], take place when cool and moist conditions and the lengthening of the daily dark period encourage the nocturnal surface activity of L. terrestris [16]. Consequently, their foraging on and incorporation of surface crop residues may be compromised [17,18] and glyphosate spraying may also disturb L. terrestris reproduction by interfering with mating, which occurs on the soil surface [19]. We investigated the short-term impacts of glyphosate application on L. terrestris in a greenhouse experiment simulating post-harvest conditions in no-till arable soil. We aimed at producing applicable knowledge for the conditions of Northern Europe and used a regionally common glyphosate surfactant combination with a realistic application rate. The specific aims were to find out if glyphosate (i) lowers the rate of residue incorporation L. terrestris, (ii) affects their mass change and (iii) influences the reproduction of by lowering cocoon production rate.
powder mixed in 10 l of water) [20] applied to the soil surface in lateOctober 2016 at a broad-leaved forest margin in Jokioinen (60°48′28,740″ N, 23°28′46,308″ E). Glyphosate had never been used at the site. Emerging L. terrestris were immediately rinsed in water and inoculated in groups of 6–8 individuals in 10 l buckets filled with moist, unsieved experimental soil. Prior to the start of the experiment, the buckets were stored for three months at +6 °C and in constant darkness. To keep the earthworms in good condition, horse manure (dried for a few days at +60 °C and remoistened) was added as feed on the soil surface ad libitum. Fifty-six L. terrestris with well-developed clitellum and in good condition were weighed (median fresh mass 5.4 g, range 3.2–8.7 g), and placed individually in small numbered vials with water. Individuals were randomly assigned and introduced into number-coded soil cylinders, with two individuals per cylinder. To keep total L. terrestris mass relatively similar in the cylinders, a deviation from completely random pair formation was made by forming six pairs using the largest and smallest individuals, then the second largest and the second smallest individuals and so on. The median total L. terrestris fresh mass was 11.2 g per cylinder (min 9.5, max 13.6 g). To prevent the escape of earthworms, a plastic collar which reached 0.2 m above the upper rim of the cylinder was firmly attached to it with adhesive tape (Appendix Fig. A1). The cylinders were allowed to stabilise for five weeks in experimental conditions (below). During the stabilising period, L. terrestris were fed horse manure with 20 g (dwt; pre-treated as above) applications per cylinder on the soil surface at the start and during the third week of the period. All germinating plant seedlings were picked away from the cylinders weekly. This was also done during the subsequent experimental period. 2.3. Treatments The surface residue used in the experiment was wheat (Triticum aestivum) straw baled in the late summer harvest of 2016. The harvest had not received glyphosate treatment during the growing season. The straw was air-dried and cut in 3 cm pieces. The ash-free dry weight of the straw was determined from five grounded samples by thermogravimetric analyses (TGA701, Leco, UK). A day before the experimental treatment, the manure remaining on the soil surface was carefully displaced from the cylinder soil surfaces using a vacuum cleaner with low suction. The next day, a few hours before the spraying treatments, 10 g of the cut straw was spread evenly on the soil surface of each cylinder (Fig. 1a). The experimental cylinders were randomly allocated in control and glyphosate treatment groups (N = 14) so that both groups had three randomly chosen cylinders with the pair of exceptionally large and small L. terrestris. The groups were taken in turns outside the greenhouse where the treatments were conducted. To allow efficient exposure of the cylinder surfaces to the sprayings, the plastic collars were lowered to a height of 5 cm. Glyphosate was sprayed in accordance with the GEP (Good Experimental Practice) protocol used by Natural Resources Institute Finland, adopted from EEC Directive 93/71/EEC. First, the control cylinders (arranged in a rectangle) were sprayed with clean water, and then the similarly arranged treatment cylinders with glyphosate. The glyphosate treatment imitated normal autumn couch grass control spraying in Finland. The herbicide was Rodeo® XL (360 g l−1 glyphosate (as potassium salt)) and the dose used was 3.0 l ha−1 (1080 g a.i. ha−1). The adhesive agent used was “Contact” (active ingredient: isodecyl alcohol ethoxylate) in a dose of 0.5 l ha−1. A 2-m-wide portable spraying boom was used, and the amount of spray water equalled 200 l ha−1 (for details see Ref. [21]). To evaluate the efficiency of the glyphosate treatment, glyphosate concentration at the 0–0.25 m soil layer was later measured from five pairs of control and treatment pairs (3.5 months after the finish of the experiment from soil stored at +6 °C). The measurement was conducted using Natural Resources Institute Finland's in-house method based on the determination
2. Materials and methods 2.1. Experimental soil and vessels Soil was collected in mid-October 2016 from 0.1 to 0.2 m depth of a cereal field (60°51′10,641″ N, 23°28′0,646″), when the soil was under post-harvest stubble. Uppermost 0.1 m layer of soil was not used to avoid usage of previously glyphosate affected topsoil (below). Topsoil textural composition at the site is 14.8% clay, 13.8% silt, 70.1% fine sand and 1.3% sand. The main chemical characteristics at the 0.1–0.2 m layer were pH 5.9 (1:5H2O), C 25 g kg−1, P 18.9 mg l−1 and conductivity 1.3 × 10−4 S cm−1. A glyphosate application (Rodeo XL®, 1080 g a.i. ha−1) had been conducted on the field six weeks before the collection of the soil. The likely initial presence of glyphosate in experimental soil was not regarded as detrimental for the main aim of the study, which was the investigation of immediate behavioural response of L. terrestris to glyphosate spraying on the residue layer and soil surface. The field-collected soil was sieved through 7 mm mesh and moistened to an initial moisture content of 21.7% v/v. The mesh used in soil sieving was sufficiently large to allow the passage of L. terrestris cocoons, but no cocoons were found in the wet sieving (3.5 mm mesh) of three 10.8 kg samples taken from the batch of sieved soil. PVC cylinders of 0.15 m diameter and 0.4 m height, closed at their bottoms with plastic caps, were used as experimental vessels. Altogether, 28 cylinders were filled with 10.8 kg of soil to obtain 1.2 g cm-3 bulk density so that the soil surface was 0.02 m below the cylinder's upper rim (Appendix Fig. A1). 2.2. L. terrestris collection and inoculation L. terrestris were collected with mustard solution (60 g of mustard 2
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emergence. At the end of the experiment, the soil moisture content of the top 0–15 cm layer was measured from four randomly chosen control and treatment cylinders with TDR (Model no. 6050X1 Trase System, Soilmoisture Equipment Corporation, Santa Barbara, California, USA). The mean moisture contents (SD) were 26.25% (1.82) and 26.98% (0.93) for control and glyphosate treatment, the difference being statistically indiscernible (two-sided p = 0.67, Wilcoxon two-sample test; n = 4). The soil temperature in treatment and control follow-up cylinders remained close to +15 °C during the experiment (Appendix Fig. A2). The readings in the control cylinder were on average 0.14 °C higher. This difference was probably due to the control and treatment follow-up cylinders being positioned at opposite sides of the table. 2.5. Ending of the experiment At the end of the experiment, all the straw remaining on the soil surface (Fig. 1b) was carefully collected in paper bags using forceps. When a piece of straw was partly below the soil surface or covered by earthworm casts, the visible part was collected by cutting it with scissors (Fig. 1c). The samples were air-dried and weighed. The ash-free dry weight of each sample was measured from a milled sample of straw using TCA, and the dry weight of straw remaining on the soil surface was converted into ash-free weight. The cylinders were turned upside-down and gradually emptied into a large plastic vessel. L. terrestris individuals were hand-sorted from the soil, washed immediately in tap water, dried on a paper towel and weighed. The presence of spermatophores, which indicates individuals’ recent copulation, was recorded. From six randomly chosen pairs of treatment and control cylinders, all the soil was wet-sieved for L. terrestris cocoons in a 3.5 mm sieve.
Fig. 1. Residue related observations during and at the termination of the experiment. (a) and (b): Wheat straw cover on the soil surface of a glyphosatetreated temperature follow-up cylinder (no. 25) on day #5 and day #57, respectively, of the 61-day experiment. Incorporation of the straw by L. terrestris exposed the soil surface towards the end of the experiment. (c) Sampling of the remaining straw from the soil surface at the end of the experiment. (d) Incorporated straw (arrows) in the upper part of L. terrestris burrow (soil surface at the top).
of glyphosate residues as 9-fluorenmethyl chloroformate (FMOC-Cl) with multiple reaction modelling (MRM) by ultra-performance liquid chromatography (UPLC-MS/MS). Glyphosate concentration was 0.52 and 0.09 mg kg−1 (p < 0.01) in glyphosate and control treatments respectively. Values were within the glyphosate concentration range of 0.05–0.6 mg kg−1 reported for agricultural topsoil (0–0.15/0.20 m) in cereal cultivation in a recent European Union-wide survey [22]. After the treatments, the cylinders were taken immediately inside the greenhouse. They were arranged on a table in a column of twos with the treatment and control cylinders randomly chosen and subsequently randomly positioned on either the left or right side of the table, the arrangement corresponding with a row-column design. There were 12 experimental cylinders for control and treatment. Two extra pairs of control and treatment cylinders were used for soil temperature (0–5 cm depth) and moisture follow-ups (below).
2.6. Statistical analyses
2.4. Experimental period
3. Results
The duration of the experiment was 61 days, from 26 January to 27 March 2017. The room's air temperature was set at +15 °C, and air moisture content at 60% relative humidity. The light cycle in the room was 12h:12h (dark period from 6 p.m. to 6 a.m., < 0.001 lux) to allow a daily soil surface activity cycle for L. terrestris. Because of light reflectance from nearby glasshouses and from snow cover outside the building, darkness was not complete. However, the illumination during the dark period was low enough to allow the emergence of L. terrestris based on several direct observations of surface activity made over the experiment using red torch light. Towards the end of the experimental period, twilight conditions prevailed in the first 1–2 h of the night due to the lengthening of the day. The moisture content of the soil was followed by weekly weighing of two follow-up cylinders, and the mean weight loss was compensated by watering all cylinders with the corresponding amount of water. During the last two weeks of the experiment, the soil surface was sprayed every evening with 10 ml of water to further encourage L. terrestris
All L. terrestris were alive at the end of the experiment. There was no difference in the weight change of the earthworms between the glyphosate-treated and control cylinders (+0.96% vs. 0.93%, p = 0.66; Fig. 2a). Likewise, there was no statistically discernible difference between glyphosate treatment and control in the number of cocoons produced per cylinder (31 vs 28 cocoons cylinder−1, p = 0.30; Fig. 2b). In two control and two glyphosate treatment cylinders, both L. terrestris individuals had two spermatophores indicating recent copulation. Active burial of straw by L. terrestris was shown by the gradual exposure of the soil surface during the experiment (Fig. 1 a-b) and the presence of straw in the upper parts of L. terrestris burrows at the end of the experiment (Fig. 1d). Overall, ca. 29% of initially applied straw was incorporated below surface level during the 61 days of the study. The incorporation rate of wheat straw was 3.0% points lower in the glyphosate treatment, but the difference between treatments was not statistically significant (27.5% vs 30.5%, p = 0.07; Fig. 2c). When one extreme observation was removed from the data and the initial
Statistical analysis of the data was based on the row-column design which takes into account variation within the glasshouse across two dimensions. In the analysis of surface straw mass change during the study, straw ash-free dry weight at the start of the experiment was used as a covariate. In addition, the mass change of L. terrestris over the study was analysed using the mass of the individuals in the cylinder as a covariate (two levels: mass of the individual is < 5 g or ≥ 5 g). Scatter plots of residuals and predicted values were drawn to evaluate visually the assumption of homoscedasticity between the predicted dependent variables and the errors of prediction. These plots revealed an extreme observation in the analysis of the straw incorporation rate data. The model was fitted both with and without it. Statistical analyses were performed using SAS/STAT software (version 9.4).
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4. Discussion We found no evidence of glyphosate spraying negatively affecting L. terrestris. All individuals survived the experiment, and the variation in earthworm final mass and cocoon production were not associated with the experimental treatments. There was an indication of a small negative effect of glyphosate spraying on the straw incorporation rate, but this effect was also statistically indiscernible. As the standard errors were relatively small for the mean estimates of all response variables, we consider that the statistically non-significant differences were not due to the low statistical power of the comparisons but rather implied absences of effects. One of the motivations of this study was to identify the extent to which the recently noticed [14] negative effect of glyphosate spraying on L. terrestris activity may impair one of their key ecosystem functions, the incorporation of surface residues. While the earlier study [14] measured egestion and the present study foraging, the two phenomena are intimately connected and both involve activity on or close to the soil surface, although by different ends of the animal. Both studies deal with immediate behavioural response, and it is conceivable that the effects of spraying on L. terrestris relate to sensory reaction to the glyphosate-affected soil surface and plant residues. The indication of a small, though non-significant decline in the straw incorporation rate in glyphosate-treated soil which we noticed may have resulted from reduced foraging immediately after spraying due to the avoidance of the herbicide-affected soil surface and straw. This type of avoidance, which can partly result from dermal exposure and irritation, may wane over time when the glyphosate concentration in residue is lowered by watering, resulting in a small net negative effect over the experiment. It is possible that both the relatively high glyphosate application rate and the more toxic surfactant contributed to the negative effects on L. terrestris activity observed by GauppBerghausen et al. [14]. In their study, the treatment mesocosms were first sprayed with 7.2 ml of ‘Roundup® Alphée’ on two consecutive days (in total 14.4 ml), and then two days later with 10 ml of ‘Roundup® Speed’. Therefore, in total 176.12 ml m−2 of herbicide (glyphosate concentration of the products 7.2 g l−1) was applied to the pot (diam. 0.42 m), resulting in a field application rate of 12.7 kg a.i. ha−1. This is a twelvefold dose compared, for instance, with the typical application rate in agricultural field use in Finland used in the present study (1.1 kg a.i ha−1). ‘Roundup® Alphée’ also contains POEA (polyethoxylated tallow amine) as a surfactant. POEA is regarded as more toxic than glyphosate, which has led to the banning of POEA containing glyphosate products in the EU [23,24]. We are not familiar with any studies on POEA's effects on earthworms, although it has been stated that a negative effect has been demonstrated [10]. The surfactant used in the present study was isodecyl alcohol ethoxylate-based, and we are unaware of any reports of its toxicity to earthworms. Stellin et al. [10] recently reported elevated mortality of L. terrestris with an increasing application rate of Roundup® 360 Power mixed in mesocosm soil. The gradient of glyphosate concentration used in their study was 0.59, 2.9, 5.79 and 11.59 g m−2. While these concentrations reportedly were in the range applied in North Italian vineyards, they are notably high and approximately 5–100 times higher than the application rate in cereal cultivation in Finland followed in the present study (0.11 g a.i. m−2). A notable difference with Gaupp-Berghausen et al.’ study [14] was the absence of growing plants from our experiment. The bearing of this on the response of L. terrestris to glyphosate spraying is difficult to judge. Despite the absence of growing weeds from our experiment, we consider that our experiment meaningfully simulated the cereal postharvest environment where L. terrestris vigorously forage on surface residues. The setting also avoided the difficult issue of what constitutes proper control when growing plants are included in experiments where glyphosate's effects on surface residue feeding earthworms are studied [25]. However, direct extrapolation from the present results to field
Fig. 2. L. terrestris response to glyphosate spraying in a nine-week glasshouse experiment. (a) Weight change in the experimental vessels over the settling and experimental periods (n = 12); (b) cocoons retrieved from the soil after the experiment (n = 6); (c) residue incorporation rate (AFDW = ash-free dryweight; n = 12). In all panels, model-based means and standard errors are shown.
differences in cylinder total L. terrestris biomasses were controlled for, the difference in the straw incorporation rate was smaller and the pvalue larger (2.0% points; p = 0.15). 4
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population growth of earthworms under reduced cultivation and no-till where glyphosate is widely used [2]. Our results indicate that immediate activity-lowering impacts on soil surface active L. terrestris can also be avoided. The varying responses of L. terrestris – and earthworms in general – to glyphosate herbicides in different studies may be partly due to the notable chemical variation in formulation-surfactant combinations and in their application rates. As Niemeyer et al. [34] also indicate, this needs to be carefully considered in the risk assessment of glyphosate use.
situations where weeds are present is not possible because glyphosate affected weeds and straw are likely to have different biochemical compositions and microbiological communities which may affect litter burial and consumption of L. terrestris. The evident welfare of L. terrestris in our experiment was possibly due to “carry-over effects” [26] from the settling period, where the earthworms were fed horse manure. Horse manure encourages L. terrestris growth [27], and the individuals may have gained weight during the conditioning period to the extent that subsequent weight loss during the experiment – likely to occur under the type of straw-only diet used [28] – went unnoticed. The average cocoon production rate we observed (approximately 7 cocoons ind.−1 month−1) is somewhat higher than the monthly rates reported earlier from comparable laboratory conditions [27,29]. While L. terrestris did mate during the experiment, this may also have been due to the positive carry-over effect from the favourable conditioning period – and even from the field – as L. terrestris are able to store sperm for several months for viable cocoon production [27,30].
Funding This study is part of the Glyfos II-project (Luke project #4100700052900), funded by the Ministry of Agriculture and Forestry of Finland. Declaration of competing interest The authors declare no conflict of interest.
5. Conclusions
Acknowledgements
The debate on the risks of glyphosate-based herbicides involves human health, environmental and socioeconomic issues. The question of impacts on soil life is only one – if important – aspect of the discussion. Current plant protection policies aim to minimise the harmful effects of pesticide use [31,32]. For the particular case we studied, the results suggest that this is possible in the case of glyphosate when recommended application rates are followed and particular glyphosate product and surfactant combinations are used. Our findings are also parallel with the recent field study of Hagner et al. [33] which indicated minor side effects of glyphosate spraying on soil micro- and mesofauna. The prevailing view is that glyphosate spraying does not impair the
We thank Samuli Klemelä for his help in the setting up of the experiment, Matti Eskola and Leena Ruokonen for their assistance in spraying, Mirva Ceder, Leena Holkeri and Outi Haapala for collaboration in laboratory analyses and Heikki Hiisilä for providing background information. Kartanonkylän Talli (Ypäjä) kindly provided the horse manure for L. terrestris maintenance. Comments by two anonymous referees and the Editor helped us to improve the manuscript. Interest and helpful comments by the project steering group are gratefully acknowledged.
Appendix
Fig. A.1. The experiment consisted of 28 PVC cylinders (⌀ 0.15, m height 0.4 m) filled with 10.8 kg arable field soil and closed at their bottoms with plastic caps. To prevent the escape of worms, a plastic collar, which reached 0.2 m above the upper rim of the cylinder, was attached to the cylinder with adhesive tape.
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Fig. A.2. Topsoil (0–5 cm) temperature over the experiment in treatment and control follow-up cylinders.
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