Materials
Test soil
The experiments were carried out using the reference soil RefeSol 01A (sieved ≤2 mm) as both the test and carrier soil [33]. RefeSol 01A is a loamy, medium-acidic and lightly humic sand (Additional file 9: Table S1). RefeSol soils were selected as reference soils by the German Federal Environment Agency (Umweltbundesamt) and are known to be suitable for testing the influence of substances on the habitat function of soils (bioavailability, effects on organisms). RefeSol 01A matches the properties stated in various OECD terrestrial ecotoxicological guidelines (e.g. tests with plants and soil microflora). The soils were sampled in the field and stored in high-grade stainless steel basins with drainage and ground contact on the grounds of Fraunhofer IME in Schmallenberg. During all experiments, red clover was shown on the stored soils and no pesticides were used. Appropriate amounts of soil were sampled 1 to 4 weeks before the test. If the soil was too wet for sieving, it was dried at room temperature to 20% to 30% of the maximum water holding capacity (WHCmax) with periodic turning to avoid surface drying. If the tests did not start immediately after sieving, the soil was stored in the dark at 4°C under aerobic conditions [34].
Sewage sludge
Sewage sludge, mainly fed with municipal sewage, was freshly gathered in the morning of the experiment at the sewage treatment plant of Schmallenberg (Germany). Previous measurements showed that the silver concentration in the sewage sludge was on average 1.8 mg/kg dry matter sludge. For dewatering the sludge, the cationic polyacrylamide SEDIFLOC 154 (3F Chimica Deutschland GmbH, Burgthann-Oberferrieden, Germany) was used. Addition of the flocculant was applied strictly according to the instructions of the local sewage treatment plant but varied slightly depending on dry matter concentration and composition of the sewage sludge.
Silver nanoparticle
The use of the silver nanoparticle NM-300K was stipulated by the OECD Sponsorship Programme [18]. NM-300K is a colloidal silver dispersion with a nominal silver concentration of 10% (w/w) and a particle size of approximately 15 nm with a narrow size distribution (99%). A second particle size of 5 nm, which is much less abundant (1%), was identified by TEM. NM-300K is a mixture of a stabilising agent (NM-300K DIS) comprising 4% (w/w) each of polyoxyethylene glycerol trioleate and polyoxyethylene sorbitan monolaurate (Tween-20) and AgNPs. The release of ions from NM-300K particles into the matrix under storage conditions was estimated to be less than 0.01% (w/w)[35].
Silver nitrate was purchased from Merck KGaA, Darmstadt, Germany. The application of the test materials to the test soil is described in Schlich et al. [10] and Hund-Rinke et al. [33], while the application to sewage sludge is described in the ‘Methods’ section.
Ecotoxicological effects of NM-300K and silver nitrate on soil microorganisms
In the following, all concentrations refer to the silver Ag content in the respective test substance. For the assessment of the effect of NM-300K and silver nitrate on the nitrifiers, a combination of two test guidelines was applied. The incubation was carried out in accordance with the OECD Guideline 216 [36]. Due to the high concentration of nitrate from the sewage sludge used in the long-term effect tests, measurement of the nitrate concentration was not performed. Instead, the nitrite concentration was determined with a short-term potential ammonium oxidation test performed in accordance with the ISO Guideline 15685 [37]. Tests were carried out using sieved and spiked soil. Four 250-mL Erlenmeyer flasks per treatment were filled with 25 g dry matter of spiked soil along with four matching controls. The vessels were incubated in the dark at 20°C ± 2°C for 24 h, after which the mineral test medium was added to make the volume up to 100 mL. The medium consisted of KH2PO4 (0.56 mM), K2HPO4 (1.44 mM), NaClO3 (5 mM) and (NH4)2SO4 (1.50 mM). The slurries were incubated on an orbital shaker at 25°C ± 2°C, after which 10-mL samples were removed at 2 and 6 h, supplemented with 10 mL of 4 mol/L KCl and filtered, and the nitrite levels in the filtrate determined photometrically (Cary 300 Scan UV–VIS Spectrophotometer, Varian Deutschland GmbH, Darmstadt, Germany) with a wavelength of 530 nm. The effect of NM-300K and silver nitrate on nitrifying microorganisms was examined by the transformation of ammonium after 24 h, 7 days and 28 days.
The carbon transformation (substrate-induced respiration) was determined in accordance with the OECD Guideline 217 [38] using sieved and spiked soil. Four 500-mL Erlenmeyer flasks per treatment were filled with 100 g dry matter of spiked soil and 2.64 g of a glucose-talc mixture, along with four matching controls. The vessels were incubated in darkness at 20°C ± 1°C for 24 h. Respiration was measured during the incubation with an OxiTop Sensomat system (AQUALYTIC GmbH & Co., Langen, Germany). Through respiration, oxygen was consumed and the low pressure that occurred was measured. The resulting carbon dioxide was bound to potassium hydroxide and did not interfere with the measurement. From the microorganism respiration rate, the microbial biomass was calculated over the linear time scale in accordance with the ISO Guideline 14240–1 [39].
Three replicates for each control (with or without dispersant) and the different contents of NM-300K and silver nitrate in soil were tested. Both NM-300K and silver nitrate were tested at concentrations of 0.56, 1.67, 5.0 and 15.0 mg/kg dry soil and additionally at 0.19 mg/kg dry soil for silver nitrate.
Functional microbial diversity was measured by enzyme activity patterns in soil samples in accordance with the ISO/TS Guideline 22939 [40] using fluorogenic substrates as indicators. In this test 2 g dry matter soil for each concentration was mixed with 200 mL of ultra-high-quality water and homogenised for 3 min at 9,600 rpm with a homogeniser. Buffer solutions of 2-(N-morpholino)ethanesulfonic acid buffer with a pH of 6.1 and Tris buffer with a pH of 7.8 were prepared freshly before each use. Two substrates per nutrient cycle (C, N, P) with a concentration of 0.1 mmol/L were tested. As standards, 4-methylumbelliferone (MUF) and aminomethylcoumarin (AMC) were used. Tests were performed in 96-well microtiter plates filled with 50 μL of soil suspension, 50 μL of buffer and 100 μL of substrate. Fluorescence of the standards was measured in plates filled with 50 μL of soil suspension, 110 μL of buffer and 40 μL of MUF or AMC. After the preparation of the microtiter plates, the fluorescence was measured with a Synergy MX spectrophotometer (BioTek Germany, Bad Friedrichshall, Germany) at an excitation wavelength of 355 nm and an emission wavelength of 460 nm.
Ecotoxicological effects of NM-300K and silver nitrate on sewage sludge microorganisms
The respiration inhibition test was performed in accordance with the OECD Guideline 209 [19]. It was used as a screening test before the simulation of a sewage treatment plant to gain information about the effects caused by AgNP. The pH of the sewage sludge was between 7.5 ± 0.5. Control measurements were performed in replicates at the beginning and end of the test. As a reference substance, 3,5-dichlorophenol was used. Prior to the test, the sludge was sieved to ≤2 mm and continuously stirred and aerated. When the stirring and aeration were stopped for 15 min, the sludge was able to settle. The supernatant was removed and replaced with Ringer’s solution. After that, the sludge was stirred and aerated again. The dry matter content was adjusted to 4.0 ± 0.4 g/L to reach a final dry matter content of 1.6 g/L for the test. Synthetic sewage was prepared in accordance with the guideline. The tests were conducted in 1-L Erlenmeyer flasks into which distilled water and synthetic sewage were introduced, followed by the AgNP stock solution and 200 mL of sludge (dry matter content of 4.0 g/L) in 15-min intervals to a final volume of 500 mL. The mix was stirred at 300 rpm and aerated with 0.5 to 1 L O2/min for 3 h. The respiration rate was measured in an aliquot of 100 mL taken every 20 s, within 10 min, with an oxygen electrode.
Long-term effects of NM-300K and silver nitrate in sewage sludge using manually spiked sludge and sewage treatment plant simulation
In accordance with the OECD Guideline 303A [41], a simulation test for aerobic sewage treatment was performed. A lab-scale sewage treatment plant (behrotest® Laborkläranlage KLD 4N, behr Labor Technik GmbH, Düsseldorf, Germany) with a denitrification, nitrification and secondary clarifier was used. The validity of the test was reached when 80% of the introduced DOC concentration (100 mg/L) had been eliminated by the microorganisms. Six systems were running as a control, four systems with continuous addition (influent) of AgNP at 0.04, 0.4, 0.8 and 1.6 mg/L, and another with silver nitrate at 0.4 mg/L. The room temperature was kept at 20°C to 25°C. The oxygen level was controlled to range from 2 to 3.5 mg/L in the aeration vessel over the whole test period. The STP was set to run with a retention time of 6 h and a continuous flow of 750 mL/h containing a mixture of synthetic sewage, tap water and Ag stock dispersion mixed within a tube system (synthetic sewage was mixed with water and then with the AgNP resulting in a dilution of 1:10). The AgNPs were applied into the denitrification section. Synthetic sewage and AgNP stock dispersion were prepared as a tenfold concentration, as mentioned in the guideline, and stored at 4°C in a refrigerator. The pH of the sewage sludge in the non-aerated and aerated vessels was consistently measured. Continuous reflux from the nitrification section into the denitrification section of 1.75 L/h was performed to avoid a decrease of pH. At test start, the sewage sludge was introduced into the STP and tap water was added to end up with a dry matter content of 2.5 g/L. For 3 days, the sewage sludge was adapted to laboratory conditions by continuously feeding it with just synthetic sewage before the addition of the AgNP for 10 days started. The dry matter content of sludge was determined periodically. The concentration of nitrate, nitrite and ammonia was checked photometrically in the effluent with test kits (Nanocolor, Macherey-Nagel, Düren, Germany), and the flow rate was checked and adjusted if necessary. The DOC was measured daily in the influent flow and effluent.
Manually spiked sludge
Sewage sludge was freshly gathered from the municipal sewage treatment plant of Schmallenberg, Germany. AgNP and silver nitrate were added to the sludge under aeration and stirring. The procedure was divided into three steps to avoid the inhibition of sludge microorganisms. After a contact time of 2 h, the sludge was dewatered with the flocculant. The supernatant was removed and the sludge centrifuged for 15 min at 10,500 rpm. Again, the supernatant was removed and the dry matter content of the sludge measured.
The sludge was added to soil in accordance with the German sewage sludge ordinance, which states that 5 tons per hectare over 3 years can be spread on agricultural areas. Respectively, 1.67 g of dry matter sludge could be introduced into the soil, under the assumption of a soil depth of 20 cm and soil density of 1.5 g/m3. The intended silver concentration was derived from the results of the tests with pristine AgNP but is a concentration with environmental relevance. A silver concentration of 2.5 and 6.0 mg/kg dry matter soil was intended which corresponds roughly to 1,500 and 3,500 mg/kg dry matter sludge.
An amount of water to achieve a WHCmax in soil of 55% was used to suspend the sludge and to introduce the slurry into soil. The soil was mixed gently to distribute the sludge homogeneously. The test included a control with and without uncontaminated sludge. For each of the AgNP and silver nitrate concentrations and for both controls, two replicates containing 4 kg of dry matter soil were prepared and incubated. Each replicate was prepared separately. The soil was incubated at 20°C under dark conditions in 5-L polyethylene containers. Care was taken that the depth of the soil-sludge mixture was less than 15 cm to avoid anaerobic sections. The containers were closed with a lid with holes for oxygen exchange. The WHCmax was adjusted to 55% once per week, and during this procedure, the soil-sludge mixture was again mixed. After 11, 32, 60, 100 and 180 days, the microbial activity was measured with the short-term potential ammonium oxidation test (nitrite formation) and the C-transformation test (microbial biomass). Additionally, after 32 and 100 days, the enzyme activity patterns in soil were measured.
For analytical monitoring, the pH values were determined regularly with a 0.01 M CaCl2 solution. The pH values of the soil were measured in the first test after 11, 100 and 180 days in both controls and the highest concentration of each test object. After 32 and 60 days, six samples were taken from different randomised positions in the test container and analysed for total silver concentration.
Sewage treatment plant simulation
The aim of this experiment was to simulate realistic exposure pathways of NM-300K via sewerage in a municipal STP and sewage sludge on agricultural areas. Six simulated sewage treatment plants were conducted as described in the OECD Guideline 303A [41]. One control, four influent concentrations of NM-300K (0.04, 0.4, 0.8, 1.6 mg/L) and one of silver nitrate (0.4 mg/L) were dosed into the denitrification section of the STP continuously over 10 days. The influent concentrations lead to final silver concentrations in the sludge of around 240, 2,400, 4,800 and 9,600 mg Ag/kg dry sludge. Based on an entry of 1.67 g dry sludge/kg dry soil, the targeted concentrations were approximately 0.4, 4.0, 8.0 and 16.0 mg/kg dry soil for NM-300K and 4.0 mg/kg dry soil for silver nitrate. The sludge was prepared and introduced into soil as described for manually spiked sludge. In this test only one replicate per control and soil concentration of NM-300K and silver nitrate was applied. First, the sludge was divided into two portions and each was applied to 6 kg of dry soil. The soil was then pooled, mixed well and stored (in 20-L polyethylene containers) as a replicate. Measurements were conducted with the same tests as described for manually spiked sludge. The test duration was reduced to 140 days after reduced microorganism activity was observed in manually spiked sludge. For STP simulation, at each point of measurement (11, 32, 60, 100 and 140 days), pH values were determined in both controls and each concentration of NM-300K and silver nitrate.
Hazard assessment
The risk characterisation was based on the directive ‘ECHA guidance on information requirements and chemical safety assessment’ [42]. Implementation of a classic risk characterisation requires the PEC and PNEC and the ratio of PEC to PNEC.
However, as a PEC for NM-300K was not available, the implementation of the risk characterisation had to be changed. A PNEC was created based on the results of the toxicity of NM-300K on organisms of the terrestrial ecosystem. The effect caused by NM-300K to different trophic levels was considered by observing effects on microorganisms in the soil, plants (data not published) and earthworms [10]. The NOEC or EC10 determined in the test with the most sensitive organism was selected for the determination of the PNEC. Depending on the number of organisms to which the effect of the chemical had been studied, an evaluation factor was applied to the effective concentration. In this study three organisms of three trophic levels were studied (microorganisms, plants, earthworms) that resulted in an evaluation factor of 10. Based on this, the PNEC of NM-300K in soil was determined. Additionally, the PNEC of NM-300K applied to soil via sewage sludge was calculated. The concentration of NM-300K in sludge up to which the PNEC of NM-300K in soil is not exceeded was calculated. Application of the sludge strictly followed the German sewage sludge ordinance according to which 5 tons of dry sludge per hectare over 3 years can be applied to agricultural areas. Considering a soil depth of 20 cm, a soil density of 1.5 g/m3 and incorporation of the maximum amount of sludge in a single application, a maximum of 1.67 g of dry sludge could be applied per kilogram of dry soil.
Due to the missing PEC, a limit for NM-300K application to agricultural land via sewage sludge was calculated instead of the classic risk characterisation with PEC and PNEC values.
Determination of Ag levels
Digestion was carried out in accordance with standardised guidelines [43, 44], using soil dried at 105°C for at least 12 h until it reached a constant weight. Approximately 3 g of the homogenised material was mixed with 28 g of aqua regia and incubated at room temperature for 16 h without agitation. The mixture was then heated under reflux for 2 h with glass chips and 1-octanol added to avoid over-boiling and foaming. The mixture was cooled to room temperature, made up to 100 mL and filtered (0.45-μm syringe filter, polyethersulfone membrane, Pall Corporation, Port Washington, NY, USA). The concentration of silver was determined by inductively coupled plasma optical emission spectrometry (ICP-OES) using an IRIS Intrepid II (Thermo Electron, Dreieich, Germany) with a matrix-adjusted calibration carried out for each measurement. Silver was detected at 328.068 nm and compared to the certified reference material TMDA-70 (certified with 10.9 μg/L Ag) as a quality assurance sample. According to the quality assurance requirement, the silver recovery was in the range of ±15% of the certified value. However, regarding Ag concentrations measured by ICP-OES, the mean recovery (accuracy) and precision of the non-digested CRM TMDA-70 measurements was 101% ± 2.9% (n = 4). The recovery for Merck IV standard solution samples containing 50 μg/L was 101% ± 2.7% (n = 4) and 94.7% ± 0.7% for 500 μg/L. The instrument was calibrated prior to each measurement series. Silver concentrations in reagent blanks were always below the limit of detection (range 1.34 to 3.51 μg/L) in the corresponding measurement series. The limit of quantification ranged from 4.48 to 11.7 μg/L.
Statistical analysis
In tests with pristine silver nanoparticles (potential ammonium oxidation, microbial biomass and sewage sludge respiration inhibition), the NOEC were calculated by ANOVA followed by parametric pairwise comparisons of treatments to the control using the Dunnett, Welch, Williams or Fisher exact tests. Probit analysis was performed for the estimate of the EC
x
and dose–response curve. Due to fewer treatments and concentrations, the significance of variance between the different treatments was conducted by Student’s t tests in the long-term tests. Statistical analysis was carried out using ToxRat® Pro v2.10 software for ecotoxicity response analysis (ToxRat® Solutions GmbH, Alsdorf, Germany).