Seasonal effects on the outcome of reproduction tests 1 with silver nanoparticles, silver nitrate and the 2 Collembola Folsomia candida

Background Toxicity of silver nanoparticles (AgNP) are increasingly studied due to a rise in application 14 in various products. Various studies on AgNP toxicity with terrestrial and aquatic 15 organisms confirmed their negative effects. In our previous experiments, strong variability 16 was observed in the reproduction of Collembola in different seasons. To investigate the 17 effects of silver nanoparticles (AgNP) on the reproduction of Collembola in different 18 seasons, Folsomia candida were exposed to AgNP and silver nitrate (AgNO 3 ) at a 19 concentration of 30 mg/kg dry soil for 28 days. The reproduction tests were repeated 20 during different seasons throughout one year in order to assess if animals’ sensitivity 21 varied with the season. Significantly lower reproduction was found in the control in winter with only 101 (± 7) 24 juveniles per adult, compared to 126-158 individuals in other seasons. Strong toxic effects 25 (inhibition of reproduction by up to 50%) were observed during summer, spring and 26 autumn in both treatments. However, AgNP showed no toxic effects on the reproduction 27 of F. candida in winter. The relative toxicity of both substances varied with the seasons: 28 AgNP were more toxic than AgNO 3 in spring and summer, and less toxic in autumn and 29 winter. These findings indicate that seasonal effects on the reproduction of Folsomia candida are 32 significant. Moreover, we demonstrated the reproductive toxicity of AgNP in soil at a much 33 lower concentration than reported thus far. These effects can mainly be attributed to soil 34 conditions, which raises concern whether these commonly used test substrates are really 35 protective. hypothesized that (a) the reproduction of is different during four seasons and (b) the toxic effects of AgNP and AgNO 3 vary between seasons.


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The application of silver nanoparticles (AgNP) is strongly increasing in several areas 40 electrical, medicine, food and textile products. AgNP can be released from these products 41 during washing [1], disposal, and industry wastewater [2,3]to the environment. Up to 90% 42 6 in a breeding container for 3 days to lay eggs and then were removed. After hatching, four 108 9-12 day-old juveniles were placed randomly in each test vessel. The vessels were 109 incubated in a climate chamber (Sanyo MLR-350H) at 20°C with a 12-hour light/12-hour 110 dark cycle with 80% humidity and 500 Lux illumination. During the test, 5 pieces of dried 111 baker's yeast (Dr. Oetker) were added to the animals twice a week, and the old food 112 bunches were removed. The test vessels were aerated twice a week, and moisture 113 content of the soil was kept constant at 50% of the maximum water holding capacity by 114 replenishing the water loss once a week. After 28 days of exposure, 100 mL deionized 115 water was added to each test container, and the soil was transferred to a plastic container. 116 F. candida floating on the surface of the dispersion were visible after adding 2 drops of ink 117 to the water. A picture was taken of each container, in order to count the juveniles using 118 Image J 1.46r software package. The same procedure was repeated four times (spring, 119 summer, autumn and winter) during one year to examine seasonal effects. 120

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Statistical analyses were performed with SPSS 17.0 and R 3.4.0. For the reproduction test, 122 data were log-transformed to obtain normal distribution (according to Shapiro-Wilk test), 123 and a general linear model (GLM) was used to analyse the main effect and interactions of 124 treatment and season as influencing factors. For comparing the toxicity within each 125 season, additionally one-factorial models were run with R. 126

Reproduction in different treatments during four seasons
The controls in all reproduction tests met the validity criteria according to OECD guideline 129 232. Mortality did not exceed 20%, and did not differ between the treatments (p=0.628). 130 The mean number of juveniles per vessel was not lower than 100 for each replicate 131 control (n=6), and the coefficient of variation of reproduction was less than 30% (Table 1). 132 reproduction in the control was lowest during winter, followed by spring, summer and 137 autumn ( Figure 1). In the AgNP treatment, the highest reproduction was detected during 138 autumn whereas in soil spiked with AgNO3, F. candida reproduction did not differ between 139 the four seasonal tests (Table S1-2 in additional file).  Table S4 in 151 Appendix). AgNP were significantly (1) less toxic for the reproduction of F. candida than 152 AgNO3 in autumn and winter (p<0.05), (2) but more toxic in spring and summer (p<0.05, 153 who found no effect on survival and reproduction for F. candida exposed to AgNP at a 165 measured concentration of 673 mg Ag/kg dry soil, which was more than 20 times higher 166 than the concentration in our study. Mendes et al. (2015) [21] found that NM-300K 167 reactivity increases with decreasing size [28]. Therefore, coating and soil type might the 183 main reasons for the fate and toxicity of the particles found in our studies. The presence of 184 a coating is important, because it can modify the particle structure, the electrostatic 185 surface charge and therefore its potential toxicity over time [29]. Nguyen et al. [30], for 186 instance, found considerable differences in toxicity between AgNP coated with citrate and 187 polyvinylpyrrolidone and uncoated AgNP to macrophages and epithelial cells. They 188 reported that uncoated AgNP, at a concentration of 1 µg/ml, decreased cell viability by 189 20-40% and that 20 and 40 nm particles were 10% more cytotoxic than the 60 and 80 nm 190 particles. In exposures to coated AgNPs, cell viability dropped at 25 µg Ag/ml or higher 191 concentrations. Similar coating effects were observed in a study with ZnO-NPs and F. 192 candida [31] and in studies with iron oxide nanoparticles and mouse fibroblast cells [32]. 193 There is strong support for the assumption that the different soil types were the main 194 reason for the large difference in toxicity between our study and the one by

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In winter, the reproduction was significantly lower in the treatment of AgNO3, but there 223 was no difference in the treatment of AgNP compared to control (Figure 1). We postulate 224 that the seasonally different performance of both Ag forms is due to their reaction kinetics. 225 AgNO3 dissociates readily in water, newly dissolved but only part of the Ag + ions are 226 bioavailable; they will react with anions in the soil solution, forming insoluble precipitates, 227 or complexes with organic acids. In turn, AgNP dissolve slowly, constantly releasing new 228 Ag + ions. Therefore, over a longer period it is likely that more Ag + is bioavailable from 229 AgNP than from AgNO3. should have been present in low numbers at the beginning of winter, then increased due 241 to favourable conditions and decreased again in spring due to increasing temperature. 242 The release of dissolved Ag + upon adding AgNP to moist soil provides a low, but constant 243 supply of Ag + ions. The low Ag + concentration should be sufficient to control the small 244 initial EPF population in winter and to prevent their further increase. The negative effect of 245 EPF on the reproduction of F. candida was inhibited by AgNP, and a part of the AgNP was 246 attached to the EPF, so that the toxicity of AgNP on the reproduction during winter was 247 decreased. With AgNO3, the sudden release of dissolved Ag + upon adding AgNO3 to 248 moist soil will kill most of the present fungi, but the population will quickly recover during 249 winter (Figure 3). 250 On one hand, the likely appearance of EPF made the situation more complicated, and a 251 reduction in toxicity of AgNP may only be due to the interaction between EPF and silver as 252 discussed previously. On the other hand, some studies used the difference in toxicity 253 between AgNP and AgNO3 to demonstrate that the toxic effects of nanoparticles could 254 possibly be explained by a release of Ag + from the particles and by a slower assimilation 255 of AgNP, which leads to lower toxic effects on soil fauna compared with AgNO3 [39][40][41]. 256 Such differences in toxicity were also reported in studies with earthworms [16], [17]. 257 Similar results were observed in our study during autumn and winter. Stronger toxic 258 effects were found in the treatment with AgNO3 than that with AgNP, which supports the 259 ion release theory. However, the result was totally reversed in spring and summer. We 260 believe this is a combination of Ag + release kinetics (see above) and avoidance behaviour. 261

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Avoidance studies in our laboratory gave hints that F. candida and enchytraeids avoid 262 high, but not low concentration As of Ag. Assuming that they sense rather the ions than 263 the undissolved metal it is possible that they actively avoided (e.g. by staying mostly at the 264 uncontaminated food patch on the surface) only the AgNO3 treatment but not the one with 265 AgNP in our study. Thus, in the latter treatment the animals were exposed to low 266 concentrations of Ag + permanently released by the AgNP, reducing their reproduction. It is 267 possible that the EPF started developing already in autumn (not yet visible in the quickly 268 developing population, but perhaps supported by high population density) so that the conditions. The author found that reproduction was highest in spring and summer, and 275 dropped significantly in the winter months, which indicated that internal regulation of 276 reproduction may exist in the earthworm D. octaedra [42] conditions (compared to Lufa 2.2 and artificial OECD soil), this raises concern whether 288 these commonly used test substrates are really protective. This is also the first paper