We investigated the toxicity of AgNP and AgNO3 to F. candida in four repeats during one year. For the first time, strong toxic effects of AgNP at a concentration of 30 mg Ag/kg on the reproduction of F. candida were observed. This is in contrast to Waalewijn-Kool et al.  who found no effect on survival and reproduction for F. candida exposed to AgNP at a measured concentration of 673 mg Ag/kg dry soil, which was more than 20 times higher than the concentration in our study. Mendes et al. (2015)  found that NM-300K reduced F. candida reproduction by about 50%, yet at a concentration of 640 mg Ag/kg soil. Mainly three factors may explain the differences between these studies: (1) Waalewijn-Kool et al.  used paraffin-coated AgNP in a water-only dispersion, while NM-300K are uncoated and dispersed in a suspension that contains three organic agents; (2) The size of AgNP used by Waalewijn-Kool et al. was 3-8 nm AgNP, whereas NM-300K have a diameter of 15 nm.; (3) Loamy sand soil (LUFA- Speyer 2.2, Sp 2121, Germany, 2009) with a pHcacl2 of 5.5 was used by  and  whereas we used RefeSol 01A, a loamy, medium-acidic, and lightly humic sand with pH cacl2 of 5.67.
For NM-300K, an effect of the organic dispersion can be excluded, because tests have been made in advance to ensure that the dispersion showed no toxic effect of on the reproduction of F. candida (X. Zhang, unpublished data). McKee et al. (2019) studied the dispersion of NM-300K in OECD soil pore water and found that the dispersion caused significant immobilization of F. candida at 10 mg L-1 whereas no toxic effect occurred at 40 mg L-1. This is in line with our findings. Secondly (except for differences in release kinetics, see Engelke et al. 2014), particle size can also be excluded for nanoparticle reactivity increases with decreasing size . Therefore, coating and soil type might be the main reasons for the fate and toxicity of the particles found in our study. The presence of a coating is important because it can modify the particle structure, the electrostatic surface charge and therefore its potential toxicity over time . Nguyen et al. , for instance, found considerable differences in toxicity between AgNP coated with citrate and polyvinylpyrrolidone and uncoated AgNP to macrophages and epithelial cells. They reported that uncoated AgNP, at a concentration of 1 µg/ml, decreased cell viability by 20-40% and that 20 and 40 nm particles were 10% more cytotoxic than the 60 and 80 nm particles. In exposures to coated AgNPs, cell viability dropped at 25 µg Ag/ml or higher concentrations. Similar coating effects were observed with ZnO-NPs and F. candida  and with iron oxide nanoparticles and mouse fibroblast cells . There is strong support for the assumption that the different soil types were the main reason for the large difference in toxicity between our study and the one by Mendes et al (2015) , as various studies in our lab with Collembola (McKee et al. 2019) and enchytraeids (Voua Otomo et al, under revision) have rendered much stronger toxic effects of AgNP in RefeSol 01A than in Lufa 2.2 and artificial OECD soil. In fact, it is challenging to evaluate the results of different studies on F. candida if different clonal lineages were used in the different studies. A large number of studies have shown that different lineages of F. candida can exhibit very different life history traits, and that they also differ genetically in their ability to cope with environmental stress [39,46-48].
In the present study, the toxicity of AgNP varied significantly between repeats. Significant toxic effects of AgNP on reproduction were observed in April, July and October, but not in January, while AgNO3 caused toxic effects during all repeats. The reproduction of F. candida in the control in January was 19.5% - 35.9% lower than in July and October. On the other hand, in the soil spiked with AgNP, the reproduction of F. candida in January was even higher than in July. In the following, we discuss four possible explanations: fungi compromising Collembola reproduction by defensive strategies or being entomopathogenic, differences in dissolution kinetics of AgNP and AgNO3, avoidance behaviour and circannual biological rhythms.
Fungi observed in our test vessels during winter might account for the reduction of the reproduction of F. candida in January. It is possible that spores from fungi emitted in autumn or winter are brought into the laboratory by the ventilation system. We speculate that these fungi might have defence properties (toxins or crystals at the hyphal surface) that inhibited reproduction of F.candida [49,50]. An alternative explanation could be Entomopathogenic Fungi (EPF), although not all of them are toxic to Collembola [51,52]. Outbreaks of infection with entomopathogens such as Entomophthora muscae tend to occur in spring and autumn, and sporulation usually takes place in cool, humid conditions , it might explain why we observed Fungi in our test vessels in January and the reproduction of F. candida is significantly decreased in control. Interestingly, there was no significant reduction in reproduction in January in the soil treated with AgNP (Figure 1), most likely due to their continuous antimicrobial activity . On the one hand AgNP are capable of inhibiting fungi that compromise the reproduction of F. candida, on the other hand, the direct negative effects of silver on the Collembola would partly be masked by the indirect positive effect through its suppressing effect on such fungi.
In January, the reproduction was significantly lower in the treatment of AgNO3, but there was no difference in the treatment of AgNP compared to control (Figure 2). We postulate that the different performance of both Ag forms is due to their reaction kinetics. AgNO3 dissociates readily in water, but only part of the Ag+ ions are bioavailable: they will react with anions in the soil solution, forming insoluble precipitates, or complexes with organic acids. In turn, AgNP dissolve slowly, constantly releasing new Ag+ ions. Therefore, over a longer period it is likely that more Ag+ is bioavailable from AgNP than from AgNO3.
Figure 3: Hypothetical model on the development of deleterious fungi in winter and spring in the different treatments as affected by the released Ag+ ions. There is a slow and continuous ion release from AgNP, whereas AgNO3 ions dissolve at test start. Numbers indicate different phases on fungi populations in the single treatments and tests: (1) Efficient control of the originally small population by continuous Ag+ ion release; (2) High mortality and exponential recovery due to high growth rate during winter.
But what are the reasons for those differences between repeats? The hypothetical model in Figure 3 illustrates why the treatment with AgNO3 had a negative effect on F. candida in January, not the one with AgNP: The presumed contamination with fungal spores should have been present in low numbers at the beginning of January, then might increase due to favourable conditions and decrease again in spring due to increasing temperature. The release of dissolved Ag+ upon adding AgNP to moist soil provides a low, but constant supply of Ag+ ions. The low Ag+ concentration should be sufficient to control the small initial fungi population in winter and to prevent their further increase, thus reducing the negative effect of fungi on the reproduction of F. candida. With AgNO3, the sudden release of dissolved Ag+ upon adding AgNO3 to moist soil would kill most of the present fungi, but the population might quickly recover thereafter (Figure 3).
Some studies explained the difference in toxicity between AgNP and AgNO3 by a release of Ag+ from the particles and by a slower assimilation of AgNP, which leads to lower toxic effects on soil fauna compared with AgNO3 [56-58]. Such differences in toxicity were also reported in studies with earthworms  . Similar results were observed in our study during autumn and winter. Stronger toxic effects were found in the treatment with AgNO3 than that with AgNP, which supports the ion release theory. However, the pattern was reversed in the repeats in April and July. We believe this is a combination of Ag+ release kinetics (see above) and avoidance behaviour. Avoidance studies in our laboratory gave hints that F. candida and enchytraeids avoid high, but not low concentration of Ag. Assuming that they sense rather the ions than the undissolved metal it is possible that they actively avoided (e.g. by staying mostly at the uncontaminated food patch on the surface) only the AgNO3 treatment but not the one with AgNP in our study. Thus, in the AgNP treatment the animals were exposed to low concentrations of Ag+ permanently released by the AgNP, reducing their reproduction.
Circannual biological rhythms might be another explanation for the different toxicity results. Rozen (2006) collected earthworms (Dendrobaena octaedra) from the field and cultured them in the laboratory under constant conditions. The author found that reproduction was highest in spring and summer, and dropped significantly in the winter months, which indicated that internal regulation of reproduction may exist in the earthworm D. octaedra . However, the mechanisms have not yet been understood. Nevertheless, we cannot fully elucidate what exactly caused the toxicity of AgNP in the present study. The hypothesis on the interaction with fungi should be tested in further investigations, to identify the fungi species present during winter.
Krogh (2008) summarized data of reproduction tests using F. candida and Folsomia fimetaria from 1994-1999 and found that the variability of reproduction in control is obvious and many factors contribute to the variability . To what extent should we trust these data in the face of different experimental results? By comparing the data from our four repeated experiments, we suggest that F. candida in each laboratory should have a database of average reproduction rate in the control, and this database should also contain information on metadata such as different test soils or strains. In order to increase the reliability of the experimental results beyond established validity criteria, we suggest that test results be disregarded when the reproduction rate in control is significantly different from the average reproduction rate established in the laboratory.