Study system and experimental setup

The experiment was performed between March and July 2013 in a greenhouse at the University of Natural Resources and Life Sciences Vienna (BOKU), Austria. We used 36 plastic pots (volume: 45 l, diameter: 42 cm, depth: 39 cm) filled with a 70 : 30 (vol/vol) mixture of soil from an arable field (soil type: Haplic Chernozem; BOKU Experimental Farm Groß-Enzersdorf) and quartz sand (grain size 1.4–2.2 mm) to create mesocosms. The substrate was homogenized using a concrete mixer, sieved (10 mm mesh size) and filled into the pots at a bulk density of 1.3 g cm−3. The chosen mesh size might not completely retain juvenile earthworms or cocoons already present in the field soil; however because of the thorough mixing and random distribution of the substrate among the experimental pots homogeneity across treatments can be assumed. The substrate had the following characteristics: total C = 4.41 ± 0.06 mg g−1, total N = 0.16 ± 0.01 mg g−1 (C:N ratio 27.6), K = 3.18 ± 0.12 mg g−1, P = 0.62 ± 0.04 mg g−1 and pH (CaCl 2 ) = 7.45 ± 0.02 (mean ± SE). To provide an initial food source for the endogeic earthworms (see below), 1.5 g dry mass of shredded grassland hay l−1 soil was added to all mesocosms.

The mesocosms were planted with three types of plant species: the grass Dactylis glomerata L., the leguminous herb Trifolium repens L. and the non-leguminous herb Taraxacum officinale F.H.Wigg. The three species are common weeds on agricultural fields (e.g. arable land, vineyards) across Central Europe. Plants were germinated from seeds obtained from a commercial supplier specialized in wild plant populations (Rieger-Hofmann GmbH, Blaufelden-Rabholdhausen, Germany). When seedlings were 1 cm high, 17 seedlings per species were transplanted to the pots in a triangular pattern (5.5 cm between plant individuals; plant density: 51 plants mesocosms−1). During the experimental period, each mesocosm was irrigated equally using an automatic sprinkler system; mesocosms were placed on slats, allowing for free drainage.

Three weeks after planting, three earthworm treatments (n = 12) were established. The thirty-six mesocosms either received five specimens of adult vertically burrowing (anecic) Lumbricus terrestris L. (Lt) mesocosm−1 (25.5 ± 0.7 g mesocosm−1; ~183 g m−2), ten adult/sub-adult specimens of the horizontally burrowing (endogeic) Aporrectodea caliginosa Savigny (Ac) mesocosm−1 (12.09 ± 0.30 g mesocosm−1; ~87 g m−2), or no earthworms (NoEw). All earthworms were carefully rinsed, dried with filter paper and weighed before insertion; earthworm stockings for L. terrestris are in the upper range of natural abundance in temperate arable fields42. A. caliginosa was hand-collected from garden soil by one coauthor (JGZ) near the city of Eisenstadt (Burgenland, Austria), L. terrestris was purchased in a fishermen bait shop in Vienna. Earthworms were stored in boxes filled with the soil mixture for 5 days before transferred into mesocosms. All earthworms appeared to be in good health and buried themselves into the soil within a few minutes. Two times during the experiment 7.0 g of shredded hay were applied on the soil surface of each mesocosm, providing an additional food source. In order to prevent earthworms from escaping the mesocosm, drainage holes at the bottom of all pots were covered with garden weed fleece and the upper rim of all pots was extended with a 20 cm high, slightly outward bending barrier of transparent plastic film brushed with soft soap39.

Eight weeks after planting, mature plants (D. glomerata was about 40 cm high, T. repens 19 cm, T. officinale 31 cm) of half of the mesocosms were treated with the herbicide ‘Roundup®’ (treatment +H), whereas the other half of the mesocosms remained untreated (treatment –H). Each +H mesocosm was sprayed with 7.2 ml of ‘Roundup® Alphée’ (glyphosate concentration 7.2 g l−1; Scotts Celaflor, Mainz, Germany) on two consecutive days (in sum 14.4 ml) and 10 ml of ‘Roundup® Speed’ (glyphosate concentration 7.2 g l−1; Scotts Celaflor, Mainz, Germany) two days afterwards. In total for all applications, 176.12 ml m−2 of herbicide was applied which is 53% lower than the recommended plant-based application rate of 1000 plants l−1 for ‘Roundup® Speed’ and 62% lower than the recommended dose of 800 plants l−1 for ‘Roundup® Alphée’ (Monsanto Co., St. Louis/Missouri, USA). The manufacturer recommends both herbicides to be used as an areal application prior to planting new lawns or garden beds, however it is unclear how often these products are actually applied together. Both products are sold in ready-to-use spray bottles - according to the manual we sprayed the products homogeneously to wet all weed leaves. The manufacturer suggests repeated treatments especially for perennial weeds (www.monsanto.com).

The treatments were replicated six times in a full-factorial design: three earthworm treatments (no earthworms, Lt, Ac) and two herbicide treatments (−H, +H). To encounter influences from microclimatic gradients, the mesocosms were placed in a randomized complete block design. Soil moisture in the upper 30 cm of each mesocosm was monitored using a TDR system (6050 × 1 Trase System I; Soil moisture Equipment Corp., Santa Barbara, CA, USA); soil temperature in each mesocosm was measured at 10 cm depth (Digital thermometer az-8851; Guangzhou Orimay Electronic Co, Guangzhou, China). Air temperature and relative humidity were monitored using tinytags (Gemini Data Loggers, West Sussex, UK) at 1.5 m above the floor. Environmental conditions during the experiment: daily air temperature 22.6 ± 2.3 °C, relative humidity 58.6 ± 5.8%, soil temperature 20.2 ± 1.2 °C and soil moisture 18.1 ± 2.6 vol% (means ± SE).

Measurements and analyses

The measurement of earthworm activity started ten days after the introduction of earthworms by collecting freshly produced casts on the soil surface in the morning. Surface casts were collected 20 times before, three times during and 20 times after the herbicide application; each time the casts were counted, collected, dried (50 °C, 48 h) and weighed. Cast production was expressed as number of casts produced m−2 day−1.

The water infiltration rate (l m−2 s−1) was measured two weeks after the final herbicide application by simulating a heavy rain shower of about 40 l m−2 (5.5 l mesocosm−1)14. The time from pouring the water onto the soil surface until the last visible water disappeared into the soil was recorded.

Plant-available nutrients in the soil were measured using ion exchange resin bags (Amberlite IRN-150; Alfa Aesar, Karlsruhe, Germany;43). The resin bags (7 × 7 cm nylon mesh bags, 50 μm mesh width, containing 4.5 g resin) were stored in 2 M KCl and were rinsed in deionized water. Five days prior to herbicide application one resin bag was installed per mesocosm at 10 cm depth. Excavation took place 30 days after the last herbicide application to prevent resin saturation in the rather nutrient rich soil. Collected bags were quickly rinsed in deionized water to remove adhering soil and kept refrigerated until further analysis. The solution was analyzed for NH 4 + and NO3¯ using a xMark Microplate Absorbance Spectrophotometer (BIO-RAD, Philadelphia, PA, USA) and PO 4 3¯ using an EnSpire Multimode Plate Reader (Perkin Elmer, Walthalm, MA, USA;44). Plant available NH4+ was below detection limit and was therefore not reported.

Decomposition rate in the soil was determined using the Tea Bag Index (TBI,45). Therefore, two plastic tea bags containing either green tea (Lipton Unilever, USA: EAN 87 22700 05552 5) or Rooibos tea (Lipton: EAN 87 22700 18843 8) were buried at 10 cm depth in each mesocosm. Tea bags were removed 70 days after insertion (30 days after the last herbicide application). For calculating the TBI, consisting of the two parameters k (decomposition rate) and S (stabilization factor), the recommended calculated hydrolysable fractions (H; 0.842 g g−1 for green tea; 0.552 g g−1 for rooibos tea) were used45. During decomposition, parts of the labile compounds stabilize and become recalcitrant46. This stabilization depends on environmental factors47 and results in a deviation of the actual decomposed fraction (i.e. limit value) from the hydrolysable (i.e. chemically labile) fraction. Stabilisation factor S is this deviation is interpreted as the inhibiting effect of environmental conditions on the decomposition of the labile fraction45.

Destructive harvest of the mesocosms took place 32 days after the last herbicide application. Mesocosms were flipped over on a 2 × 2 mm mesh screen. In addition, a total number of 292 cocoons of A. caliginosa and totally 25 cocoons of L. terrestris were collected during the harvest. They were stored separated by treatment in plastic boxes (26 × 16.5 × 12 cm, length × width × depth, respectively) and mixed into the soil substrate used for the main experiment and kept in a dark basement (mean temperature 15 °C, >70% relative humidity). After 15 weeks, the number of hatched earthworms was counted and the hatchling ratio per treatment was calculated.

Statistical analysis

Residuals of all variables were tested for homogeneity of variances and normality using the tests after Levene and Shapiro-Wilk, respectively. Assumption for normality were not fulfilled by earthworm surface casting activity and associated soil temperature and moisture. Treatment effects for parameters not fulfilling the assumption for parametric tests were analyzed using the two-sample Wilcoxon test. Effects on decomposition, soil nutrients and water infiltration rate were measured either by one-way or two-way analysis of variance (ANOVA). Each significant ANOVA result (P < 0.05) was followed by Pairwise t tests as post-hoc comparisons with sequential Bonferroni corrections to account for differences in herbicide effects within earthworm treatments. All statistical analyses were performed using R (version 3.0.1; The R Foundation for Statistical Computing; http://www.R-project.org). Values given throughout the text are means ± SE.