Hazard assessment of a silver nanoparticle in soil applied via sewage sludge

Ecotoxicological effects of NM-300K and silver nitrate on soil microorganisms

Ecotoxicological tests of pristine NM-300K and silver nitrate on sewage sludge microorganisms

Long-term effects of NM-300K and silver nitrate in soil using manually spiked sludge and sewage treatment plant simulation

Potential ammonium oxidation

The criterion of validity was passed with ≤15% coefficient of variation between the replicates of the control at all measurement points for both spiked sludge and STP simulation. In all treatments with spiked sludge, the potential ammonium oxidation was inhibited after 11 days. The test system seemed to be disturbed by the introduction of sewage sludge, but no effects due to the test materials were observed (data not shown).

Comparing the controls with and without sludge of the test performed with manually spiked sludge (long-term test 1), nitrite production in the sludge control was stimulated through the addition of sewage sludge (32, 60 and 100 days); however, after 180 days, no significant difference was observed (Additional file 6: Table S7). For both NM-300K and silver nitrate, nitrite production was comparable to the sludge control after 32 and 60 days. Statistical differences compared to the sludge control were measured in both treatments with NM-300K (3.4 mg/kg dry sludge, 33.2%) and silver nitrate (4.0 mg/kg dry sludge, 41.3%) for the first time after 100 days (Figure 1). After 180 days, 51.4% inhibition of nitrite production was calculated for the NM-300K concentration of 3.4 mg/kg dry soil. The results after 100 and 180 days for NM-300K were in a comparable range to the results with pristine NM-300K after 28 days. Pristine NM-300K was found to inhibit nitrite production by 45% at 1.67 mg/kg dry soil (Additional file 1: Table S2). Both concentrations of silver nitrate led to a significant decrease of the nitrite production by 37.9% to 85.5% (Figure 1). Effects observed with silver nitrate after 180 days are comparable with those for pristine silver nitrate over the whole test period (28 days).

For STP-spiked sludge (long-term test 2), there was higher nitrite production in the sludge control compared to the control, but the effect had evened out by days 60 and 100, and was inhibited by day 140 (Additional file 6: Table S7). After 32 days, at higher concentrations of NM-300K and at the tested silver nitrate concentration, the nitrifiers were inhibited (Figure 2). With increasing incubation time, effects also occurred at lower concentrations of NM-300K (except 0.3 mg/kg dry soil) and continued to increase until the end of the test. Nitrite production was inhibited at high soil concentrations of 9.0 mg/kg dry soil for NM-300K (25%) and 3.4 mg/kg dry soil for silver nitrate (39%) after 32 days. Concentration-response relationships for NM-300K were also found on days 60, 100 and 140 in the two highest test concen-trations (Figure 2). By test end, nitrite production was inhibited 52% at 2.5 mg/kg dry soil, but the effect was not significant due to the high coefficient of variation. The effect of silver nitrate on nitrite production increased to 88.0% at a concentration of 3.4 mg/kg dry soil by day 140 (Figure 2).

The results after 100 and 140 days for NM-300K were comparable to the results for pristine NM-300K after 28 days. Nitrite production was inhibited 45.4% by NM-300K at 1.67 mg/kg dry soil (Additional file 1: Table S2). The effect of silver nitrate was comparable to the effect of pristine silver nitrate over the whole test period (82.4% inhibition by 5.0 mg/kg dry soil after 28 days).

C-transformation

The validity criterion was met with a coefficient of variation between the replicates of the control of ≤15% at all measurement points for both manually spiked and STP-spiked sludge.

The addition of manually spiked sludge increased microbial biomass after 11 days (non-significant due to high coefficient of variation). For the entire test period, no significant differences between the controls with and without sludge were observed (Additional file 6: Table S7). There were no differences between the effects of different concentrations of NM-300K and silver nitrate compared to the sludge control after 11 and 32 days. At the highest concentration of silver nitrate (4.0 mg/kg dry soil), 21.7% inhibition of microbial biomass was determined after 60 days (Figure 3). At all concentration levels of NM-300K and silver nitrate, microbial biomass was inhibited 23.3% to 30.8% by day 100 (Figure 3). After 180 days, microbial biomass was significantly inhibited by all concentrations of NM-300K and silver nitrate on one level (approximately 42%). This effect is similar to the effect of pristine NM-300K, where inhibition of microbial biomass of 21% to 44% at concentrations of 1.67 to 5.0 mg/kg dry soil for NM-300K was observed. Effects observed with silver nitrate after 180 days were only comparable with those found for pristine silver nitrate after 24 h (60% inhibition at 5.0 mg/kg dry soil).

For STP-spiked sludge at test start (day 11), the comparison of both controls showed that microbial biomass was stimulated by the addition of sewage sludge, but there was no influence by NM-300K or silver nitrate. After 32 days, that stimulation by sewage sludge had slightly decreased while NM-300K at 0.3 and 2.5 mg/kg dry soil (15.1% and 20.0%) showed statistically different inhibition of microbial biomass (Additional file 6: Table S7). No inhibition was observed in the higher soil concentrations of NM-300K (5.2 and 9.0 mg/kg dry soil) or the replicate with silver nitrate. At day 60, a concentration-effect relationship was determined for the 2.5 and 5.2 mg NM-300K/kg dry soil concentrations (25% inhibition) and 9.0 mg/kg dry soil (50% inhibition). These effects were also observed for pristine NM-300K at day 7 and day 28. Silver nitrate inhibited microbial biomass by 43.3% (Figure 4). The inhibitory effect of silver nitrate at day 60 was also comparable with the effect found in the test with pristine silver nitrate after 24 h (60% inhibition at 5.0 mg/kg dry soil). With progressive test duration, inhibition caused by low soil concentrations of NM-300K (0.3 and 2.5 mg/kg dry soil) and silver nitrate decreased (day 100) to a level similar to that of the sludge control. By day 100, only the two highest soil concentrations of NM-300K continued to inhibit microbial biomass, by 52% and 64.4% (Figure 4). At the end of the test, after 140 days, no inhibition was observed.

Hazard assessment

Due to the lack of a PEC for NM-300K, no classic risk characterisation could be performed. Instead, a hazard assessment was conducted, and the maximum concentration of AgNPs in sewage sludge in which no effects are expected on the organisms of the terrestrial ecosystem was determined.

The results of the conducted tests show good comparability between the effects of pristine NM-300K and long-term tests with NM-300K (application via sewage sludge) on soil microorganisms and in further tests with plants (data not shown). In both test types (with pristine NM-300K and in long-term tests), nitrifiers were the most sensitive to NM-300K. Due to the larger dataset from tests with pristine NM-300K (more concentrations tested; thus, a NOEC and EC₁₀ could be determined), we decided to prepare the hazard assessment based on the results from tests with pristine NM-300K.

From our results, a PNEC was calculated with consideration for the effects of AgNPs on different trophic levels (microorganisms in the soil, plants and earthworms). The calculation was based on the lowest soil concentration causing an organism response. These organisms were the nitrifiers, with an EC₁₀ after 28 days (end of test) of 0.5 mg/kg dry soil for NM-300K.

The hazard assessment was based on EC₁₀ values of at least three tests with three different organisms from three different trophic levels, which resulted in an assessment factor of 10. On this basis, a PNEC in soil of 0.05 mg/kg dry soil was calculated for NM-300K. The calculated PNEC was transferred to the sludge pathway. Based on the German sewage sludge ordinance (5 tons of dry sludge can be applied per hectare in 3 years, see the ‘Methods’ section), 1.67 g dry sludge/kg dry soil could be applied as sludge to the test soil. The concentration of NM-300K that can be applied via 1.67 g dry sludge without passing the limit of 0.05 mg/kg dry soil was calculated. The PNEC in soil (for the sewage sludge pathway) corresponds to a concentration of 30 mg/kg dry sludge of NM-300K in a single application.

Fate and behaviour of NM-300K in a STP

The test materials were spiked into sewage sludge in two different ways, manually over 2 h and through the simulation of a STP over 10 days. Simulating a STP leads to homogeneous distribution of silver in soil more so than manual spiking. However, the main advantage of testing by manual spiking was the faster incorporation of silver into sludge (2 h). Independent of the application method (two tests with manual spiking and two tests with a simulation of a STP), the results were reproducible and comparable. At environmentally relevant concentrations, over 90% of the introduced NM-300K was absorbed into the sludge (either in the nanoparticulate or ion form - we were unable to differentiate). Micro-organism degradation activity in the sludge was not affected by the addition of NM-300K at any stage. These results are supported by studies described in the literature.

Batch experiments by Tiede et al. [20] also found that after 6 h of contact time between AgNPs (particle size 20 to 60 nm) and sludge, more than 90% of the added AgNPs were found in the sludge (at 0.5, 5 and 10 mg/L). Less than 10% of the AgNPs were found in the supernatant. The interactions were explained by absorption of particles to sludge. Absorption and precipitation reactions of silver ions from AgNPs with chloride, sulphide, and organic material are possible reasons for the interaction of sewage sludge and AgNPs.

Our fate results are also supported by two other studies [21, 22]. In a study by Kaegi [21] using model STPs, 85% of the AgNPs added to the denitrification process were detected in sludge over 43 days. Of the remaining AgNPs, 5% were in the effluent and another 5% were detected in the STP. The remaining 5% of the AgNPs could not be detected and were absent from the mass balance [21]. A study by Shafer et al. [22] about the fate of silver in STPs also confirmed our results. Here it has been shown that 92% to 99% of the inlet concentration of silver was removed by the STP. Other experiments showed that independent of the AgNP properties (size and coating), particles absorb to the sludge by around 90% [23, 24], which we also observed.

The mentioned studies confirm the results of the investigations on the fate of the AgNP NM-300K in sewage sludge. Due to the high absorption of AgNPs to sludge, regardless of size or coating, application of sewage sludge to agricultural land will result in deposition of AgNPs.

The respiration inhibition test [19] showed that NM-300K was less toxic than silver nitrate (NM-300K: EC₅₀ 43 to 67 mg/L; silver nitrate: EC₅₀ 2 to 3 mg/L). In a study by Choi et al. [25], the effect of AgNP (average particle size 14 nm, stabilized with PVA), silver chloride colloids and silver nitrate to the nitrifiers in sewage sludge was studied. At a concentration of 1 mg/L, nitrifying organisms were inhibited by 86%. Silver chloride colloids and silver nitrate inhibited the nitrifiers by 46% and 42%, respectively. The effect caused by silver nitrate was in a comparable range to our results, while the AgNP inhibition effect was stronger. Differences can be explained not only by different properties of the applied AgNP but also by the preparation of the sewage sludge. Choi et al. [25] conducted tests with enriched nitrifiers and washed sewage sludge, whereas in the tests with NM-300K, the sludge was only washed once with isotonic solution. It cannot be excluded that different amounts of substances reacting with AgNP were present due to the different preparation procedures. Another study of Choi et al. [26] showed that small amounts of sulphide can decrease the inhibitory effect of AgNPs, delivering an explanation for the decreased toxicity in the tests with NM-300K.

The respiration inhibition test was conducted prior to the simulated STPs to measure the potential influent concentration and its effect on sludge microorganisms. Our results indicate that the OECD 209 (1984) respiration inhibition test represents a good test for a first observation and allows extrapolation of effects which might occur in a simulated sewage treatment plant.

Effects of NM-300K and silver nitrate in soil added via spiked sludge (long-term tests)

Comparison of the tests shows that the effects caused by the pristine material over 28 days occurred earlier than in the long-term tests with sludge over 140 or 180 days. The sewage sludge masked the effect of NM-300K and silver nitrate until the sludge was degraded by microorganisms.

Observations of microbial biomass over time in manually spiked sludge match those of the pristine material. The pristine material leads to inhibition of microbial biomass at the beginning of the test, which declined until no effect was observed at test end (28 days). Through STP, no inhibition was observed at test start due to the masking of NM-300K and silver nitrate by sludge. After 60 days, the easily degradable portion of the sludge was degraded and the NM-300K and silver nitrate inhibited microbial biomass. The observed effects decreased (100 days) until no effect was observed at test end (140 days). Effects of STP-spiked sludge occurred later in the test, and lack of full recovery of microbial biomass was most likely because of the limited duration of the experiment (max. 180 days). Different seasonal test start dates (manual spiking: July 2011, STP simulation: March 2012) and subsequent field sampling times and vegetation differences may have affected the composition of the soil microflora that resulted in the different time course.

The obtained results show that there is no difference in effects between manually spiked sewage sludge and sludge spiked via a STP. The main advantage of spiking via a STP is the homogeneous distribution of AgNPs in sludge and, after addition of the sludge, in soil. Another advantage is the reaction of AgNPs with the synthetic sewage that allowed potential transformation reactions with compounds of the synthetic sewage to occur. A disadvantage of this method is the long period of spiking over 10 days, while manual spiking needs only 2 h.

The tests with pristine NM-300K and silver nitrate showed that the highest toxicity was to microorganisms of the soil, the primary destruents. The nitrifiers were the most sensitive organism in tests over 28 days (NM-300K: EC₁₀ of 0.5 mg/kg dry soil). Microbial biomass was affected by NM-300K in a comparable range (EC₁₀ of 1.0 mg/kg dry soil) after 28 days. In the long-term tests, NM-300K effects on the nitrifiers and the microbial biomass were observed after 60 to 140 days at concentrations of about 1.4 mg/kg dry soil. The effect concentrations of silver nitrate were in a comparable range for both manually spiked sludge and STP-spiked sludge. Currently there are few studies on the behaviour of AgNPs on microorganisms in soil. Hänsch and Emmerling [11] examined the effect of AgNPs on the microbial biomass and basal respiration activity and the enzyme activity in the soil over 120 days. A commercially available nanosilver spray used to increase plant growth was investigated. Exact data on the particle size, coatings or additives were not listed. AgNP concentrations of 3.2, 32 and 320 mg/kg dry soil were studied. Due to a lack of information about the AgNP properties, a comparison of the results is difficult. However, concentrations at which effects were observed by Hänsch and Emmerling [11] are comparable with effect concentrations of NM-300K. The microbial biomass was significantly inhibited by increasing concentrations of nanosilver spray, while basal respiration was simultaneously stimulated. The microbial biomass was inhibited (about 50%) by a soil concentration of 0.32 mg/kg, which is comparable to effects caused by pristine NM-300K and those found in the long-term tests. The soil microbial diversity and enzyme activity were not significantly affected over 120 days. Only leucine aminopeptidase activity showed a weak, non-significant difference between days 0 and 14, suggesting that the N-cycle was most sensitive [11]. This is supported by measurements of soil microbial diversity carried out during the long-term tests.

Shin et al. [27] observed inhibition of the soil enzyme activity by AgNPs. Four soil enzymes of the nutrient cycle (urease, acid phosphatase, arylsulphatase, β-glucosidase) and two soil enzymes involved in general microbial activity (dehydrogenase and fluorescein diacetate hydrolase) were examined. Citrate-stabilised AgNPs with an average particle size of 9.9 nm (measured by transmission electron microscopy (TEM)) were considered. The enzyme activity was determined on days 0, 1 and 7. Urease activity was reduced at 1 mg/kg dry soil of AgNPs. The activity of other enzymes studied was also inhibited partly at 10 mg/kg dry soil (dehydrogenase) and 100 mg/kg dry soil by the AgNPs. Effects that occurred at test start increased up to day 7 of the test. Through the strong inhibition of urease activity, it was concluded that the nitrogen cycle in soil was strongly influenced by AgNPs. These results fit with the findings that have been observed in our experiments with nitrifiers (potential ammonium oxidation). Even in the long-term tests, the nitrogen cycle was most affected by NM-300K. The nitrifying microorganisms seem to represent the group that reacts most sensitively to AgNPs. The effects of NM-300K in soil observed in this research match the data already published.

Hazard assessment

It is important for a risk characterisation to consider significant transformation processes and changes of the nanoparticle properties, e.g. in STPs [16]. The effects of these transformation processes in a STP, and also in soil and soil sludge mixtures, and the effects of NM-300K and silver nitrate were tested in long-term tests. A PNEC in soil of 0.05 mg NM-300K/kg dry soil was calculated. Via the sludge pathway, this value means sludge containing 30 mg NM-300K/kg dry sludge may be applied in a single application to soil before the microorganisms are affected.

The measurement of AgNP in sewage sludge is not provided under the German sewage sludge ordinance. Currently, due to a lack of measurement methods for routine monitoring, it is only possible to perform the determination of the total silver concentration in sewage sludge. A differentiation between silver, AgNPs and silver ions is linked to a complex test methodology (e.g. EDX, SP-ICP-MS or 4F) and unsuitable for routine monitoring of sewage sludge. The lack of adequate techniques for detection and quantification of nanoparticles at environ-mentally relevant concentrations in complex media hampers the assessment of the results [28].

For a preliminary hazard assessment, the effect data obtained with NM-300K were compared with available environmental concentrations and modelled values. The results of the existing data were compared to the total concentration of silver in the soil, even if the proportion of AgNPs of the total silver concentration was expected to be small. Additionally, the results were compared with modelled values for AgNPs in soil introduced within sewage sludge, in which case all AgNPs were included even those that are more or less toxic.

According to a literature review of the German Federal Environmental Agency, the silver concentration in nine municipal sewage treatment plants was determined. A silver concentration between 4 and 26.3 mg/kg dry sludge was found in 2002. In 2007 the silver concentration in the municipal wastewater treatment plant sludge in Karlsruhe, Pforzheim and Nuremberg was between 12 and 45 mg/kg dry sludge [29]. In our investigations the sludge from the municipal STP in Schmallenberg had a silver concentration of 1.8 mg/kg dry sludge. A study by the US EPA showed silver concentrations in the sewage sludge of 1.94 to 856 mg/kg dry sludge [30]. In this case various sites were studied. High concentrations can be explained by industrial silver entry. From these data, it can be concluded that in sludge of municipal STPs used for agricultural purposes, silver concentrations range from 2 to 45 mg/kg dry sludge.

For NM-300K, a derived PNEC in soil (via the sludge pathway) of 30 mg/kg dry sludge, with incorporation of the sewage sludge from municipal wastewater treatment plants, can be classed as a risk to soil organisms. However, this assessment is limited by the missing differentiation between total silver and AgNPs and the fact that the PNEC is for NM-300K only. Nevertheless, it should be kept in mind that the use and release of AgNPs into sludge will increase, and thus, the discrepancy will become smaller.

Model calculations for AgNPs resulted in a PEC for soil undergoing sludge application of 0.0015 mg/kg dry soil in Europe. For the USA, it was shown that the AgNP concentration increased in the period of 2008 to 2012 from 0.0023 to 0.0074 mg/kg dry soil [31]. In the present work, for NM-300K, a PNEC for soil of 0.05 mg/kg dry soil was determined. The concentration of AgNPs increased annually by about 0.001 mg/kg dry soil [31]. Assuming a similar continuous rise, the PNEC in soil would be exceeded in approximately 50 years. In this context the behaviour of AgNPs in soil must be considered. Low mobility of AgNPs in soil and repeated application of sewage sludge containing AgNPs to soil can cause an accumulation of AgNPs in soil. One study indicated that 80% of incorporated AgNPs in soil (particle size 19 nm) were not extractable in tests over 70 days, but were a constant source of bioavailable silver [32].

The lack of appropriate measurement techniques and the resulting lack of differentiation between the forms of silver hamper the preparation of a hazard assessment. Even if the amount of AgNPs in the total silver concentration in sewage sludge is marginal, accumulation of AgNPs in the soil to unacceptable levels over time due to repeated farm application of sludge will affect soil microorganisms. Without proper analytical measurement techniques, this issue cannot be fully addressed.

Materials

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 WHC_max 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 WHC_max 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).