2.1 Experimental setup
The general experimental setup regarding the lysimeters, the used soil and the amount of AgNM applied via sewage sludge in 2014 have been published in Schlich et al. (2017). In the following section, all methods are briefly described. The focus in the current study was on the second application of AgNM via sewage sludge to the existing lysimeters and the preparation of new lysimeters with AgNO3 or controls with pure sewage sludge without AgNM or AgNO3 application.
The experiments were conducted using a reference soil (RefeSol 01A, pHCaCl2 = 5.61, Corg = 9.3 g/kg, clay content = 61 g/kg), which was classified as Cambisol (Ad-hoc-AG Boden, 2006). For detailed physicochemical properties, refer to Table S1. The soil was selected as reference soils by the German Federal Environment Agency, and it matches the properties stated in various OECD terrestrial ecotoxicological guidelines (e.g., tests with plants and soil microflora).
2.1.2 Silver nanomaterials and silver nitrate
A colloidal silver dispersion (NM-300K), with a nominal silver content of 10% (w/w) and a transmission electron microscope diameter smaller than 20 nm, was used as AgNM. The particles are dispersed in a mixture of stabilizing agents (NM-300K DIS) comprising 4% (w/w) each of polyoxyethylene glycerol trioleate and polyoxyethylene sorbitan monolaurate according to Klein et al. (2011). Silver nitrate from Merck (Merck KGaA, Darmstadt, Germany) was used as a pure material in the crystalline structure.
2.1.3 Sewage sludge
Sewage sludge fed municipal sewage was freshly gathered at the WWTP of Schmallenberg (Germany). The sewage sludge met the requirements of the German Sewage Sludge Ordinance (AbfKlärV, 1992) regarding the metal content (lead, cadmium, chromium, copper, nickel, mercury, zinc) of sewage sludge used as fertilizer on agricultural land. For the application of AgNM and AgNO3, sewage sludge was sieved to particles smaller than 2 mm and then stored in a vessel under permanent moderate stirring and aeration (2.5 mg O2/L).
AgNM and AgNO3 were spiked into sewage sludge by a ratio dry matter of sewage sludge sufficient to receive the desired nominal concentrations after application via sewage sludge. After the addition of either AgNM or AgNO3 into sewage sludge, the sludge remained in the vessel for another 16 h under aeration and moderate stirring to allow transformation reactions of the AgNM or AgNO3 with the surrounding media and the interaction with the sewage sludge. To separate water and sewage sludge, the same flocculant as 2014 (0.2% cationic polyacrylamide solution, Sedifloc 154, Kemira Germany GmbH, Frankfurt) was applied.
2.1.4 Setup of lysimeters
The experiment is an extension of the lysimeter experiment described by Schlich et al. (2017). In the first part of the experiment, AgNM was incorporated into soil via sewage sludge. Over a period of three years, both the effect of AgNM on soil microorganisms and their fate in the uppermost 40 cm of the lysimeter and in leachate were investigated. In the second part of the experiment, sewage sludge (with and without AgNM) was again applied to the already existing lysimeters to investigate the influence of repeated AgNM input via sewage sludge on the fate and effect of AgNM. In addition, the fate and effect of AgNO3 in a lysimeter experiment was considered in comparison to AgNM.
Control lysimeters (unspiked sewage sludge, L1, L27) and lysimeters with AgNM concentrations of 2 (L2) and 8 (L6) mg Ag/kg dry matter soil (DMS) were tested in the first part of the experiment (Schlich et al. 2017). Again, 1.67 g dry matter sewage sludge/kg DMS was applied to the lysimeters (L1, L2, L6) in May 2018. In L2, AgNM was mixed to achieve a nominal concentration of 8 mg/kg DMS. Here, the aim was to investigate whether a comparable effect could be achieved as it was observed for lysimeter 6 (8 mg Ag/kg DMS) in the first part of the experiment. In addition, pure sewage sludge was applied to L6 with 8 mg Ag/kg DMS to observe whether the detected strong inhibition further increased over time or if it reached a steady state or even decreased. In addition, a new lysimeter experiment was initiated to obtain data on the comparability of effects observed due to AgNO3 and AgNM. For the control (L27), only sewage sludge was applied to the soil, and for L28, a concentration of 2 mg Ag/kg DMS was introduced into the soil by AgNO3-spiked sewage sludge.
The artificially filled lysimeters (0.9 x 0.9 x 0.9 m; ~ 1 t DMS) are cubic shaped and made of high-grade stainless steel. The volume of approximately 1 m3 of the soil presents a homogeneous and integrating system. For microbial determinations, samples can be collected at distinct locations to consider potential inhomogeneity. Since the aim of the study was to investigate the effects of AgNM and AgNO3 applied via sewage sludge to soil on soil microorganisms, no lysimeter containing only soil was included. There was no effect on the soil microflora (ammonia oxidizing bacteria and microbial respiration) due to NM-300K DIS (Schlich et al. 2013), the dispersant of NM-300K. Therefore, no separate lysimeter containing only the dispersing agent has been conducted.
According to the German sewage sludge ordinance (AbfKlärV, 1992), sludge can be applied on argicultural land at an amount of 5 t/ha in three years. Following current practice, it was assumed that the complete amount of sewage sludge would be applied at once. In addition, a soil depth of 20 cm (in accordance with agricultural practice) and a soil bulk density of 1.5 g/cm³ (OECD Guideline 216, 2000; OECD Guideline 217, 2000) were assumed for the calculation of the amount of sewage sludge that could be applied to the soil.
Sewage sludge was applied as described in Schlich et al. (2017). Briefly, first sewage sludge was mixed over 30 minutes with 25 kg DMS, taken from the top layer (20 cm) of the lysimeters, to receive a homogeneous mixture of soil and sewage sludge containing either AgNM or AgNO3. Afterward, the mixture of soil-sewage sludge was spread on the top of the soil in the lysimeters and mixed into the top 20 cm with a spade. After the second application of sewage sludge in the lysimeters with AgNM (L1, L2, L6), the pH of the soil decreased (pH<5). Therefore, in November 2018, 75 g of CaO (Quicklime for soil improvement; purity 82% (CaO + MgO), Zement- und Kalkwerke Otterbein GmbH + Co. KG, Großenlüder-Müs, Germany) was added to each lysimeter containing AgNM and the control lysimeter to increase the pH values. No CaO was added to the new lysimeters (L27, L28) since the pH was comparable to the pH observed in the AgNM lysimeters in 2014. After sewage sludge (with or without AgNM or AgNO3) was incorporated into the soil (May 2018), leachate was sampled, but there was no sampling of the soil to allow a new equilibrium to be established.
In June 2018, summer wheat (Triticum aestivum ´Tybalt A`, Saaten Union GmbH Isernhagen, Germany) was sown followed by black fallow land from September 2018 to May 2019. The seeds were untreated for experimental purposes. Afterward, sugar beet (Hordeum vulgare ` SY Typee`, Syngenta, Maintal, Germany) was sown in May 2019 and harvested in September 2019. Plants were observed on a weekly basis and irrigated with the same amount (10-30 L) of tap water in dry periods (L1, 2, 6, 27 and 28: July 18 until August 3rd, 2018).
At harvest of the wheat, approximately 50% of the plants were taken, including their roots. The remaining plants were cut 5 cm above the ground. The roots remained in the soil to prevent the removal of AgNM or AgNO3 accumulated in the roots. The harvested wheat plants were divided into roots and shoots before they were stored at -21°C until analysis. The sugar beets were collected, and the soil was stripped away. The sugar beets were stored at 4°C in a refrigerator until analysis. After harvesting, five soil samples per lysimeter were taken using a soil sampler (Pürkhauer drilling stick) for the top 40 cm and divided in steps of 10 cm.
The leachate of each lysimeter was permanently collected, and the volume was determined. Water samples were taken regularly, filtered (0.45 µm polyethersulfon syringe filter, VWR International GmbH, Langenfeld, Germany), and preserved with 100 µl of 69% HNO3 suprapur (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) before analysis.
2.1.5 Ecotoxicological test system
The results of the first part of the experiment (Schlich et al. 2017) showed that AOB (potential ammonium oxidation, ISO Guideline 15685, 2012) were more strongly inhibited by AgNM than the substrate induced respiration (SIR, OECD 217, 2000). For this reason, in the second part of the experiment, we focused on the effect of AgNM and AgNO3 on AOB. In accordance with ISO 15685 (ISO Guideline 15685, 2012), the nitrite concentration was determined by a short-term potential ammonium oxidation test to observe the effects on soil nitrifying bacteria. The objective of ISO 15685 (2012) is to measure the ammonia oxidation potential, which provides an indication of the size of the ammonia oxidizing bacterial population.
2.1.6 Climate and soil conditions
The average monthly rainfall was between 40 and 199 mm, and the average monthly temperature was between -2.9°C and 17.5°C from June 2018 until October 2019. In L1, 2 and 6, the measured pH from June 2018 (addition of sewage sludge) until November 2018 (liming with CaO) was between 4.4 and 4.8 and increased to a maximum pH of between 6.1-6.6 in May 2019 (Table S2). In August 2019, the measured pH values were between 5.0 and 5.3. In the new lysimeters (L27 and 28) with a control and AgNO3, the pH was between 5.1 and 6.0 from June 2018 until May 2019 and decreased to a final pH from 4.9 to 5.0, which was comparable with L1, 2 and 6, until August 2018.
2.2.1 Soil, plant material, leachate
The soil pH was measured in 0.01 mol/L CaCl2 (Th. Geyer, Renningen, Germay) after 24 h of extraction using a inoLab pH 720 (WTW GmbH, Weilheim, Germany). According to DIN EN 16174:2012-11 (2012), aqua regia digestion (ARD) was applied to the ground soil samples. The Agtotal concentration after ARD was labeled as AgARD. According to Lowry et al. (2012), the dried and ground plant materials were digested with HNO3 (65 %, Suprapur, Merck, Darmstadt, Germany). The volume of concentrated HNO3 was increased from 1.5 ml to 4 ml to enable a complete dissolution of the plant tissues. The Ag concentration after HNO3 digestion was labeled AgHNO3. According to DIN 38402-11:2009-02 (2009), the leachates were filtered (0.45 µm, Graphic Controls, Buffallo, NY, USA) and acidified (69%, HNO3, Suprapur, Carl Roth GmbH & Co. KG, Karlsruhe, Germany) immediately after sampling. The measured Ag concentrations of the leachates were labeled AgDIN38402. Inductively coupled mass spectrometry (ICP-MS, 7700 Series, Agilent, Santa Clara, California, USA) and inductively coupled optical emission spectroscopy (ICP-OES, Ciros Vision, Spectro, Kleve, Germany) were used to determine the Ag concentrations in the leachates and in the digested soil and plant materials.