Effects of Titanium Dioxide Nanoparticles on Red Clover and Its Rhizobial Symbiont

<div><p>Titanium dioxide nanoparticles (TiO₂ NPs) are in consideration to be used in plant protection products. Before these products can be placed on the market, ecotoxicological tests have to be performed. In this study, the nitrogen fixing bacterium <i>Rhizobium trifolii</i> and red clover were exposed to two TiO₂ NPs, i.e., P25, E171 and a non-nanomaterial TiO₂. Growth of both organisms individually and their symbiotic root nodulation were investigated in liquid and hydroponic systems. While 23 and 18 mg l^-1 of E171 and non-nanomaterial TiO₂ decreased the growth rate of <i>R</i>. <i>trifolii</i> by 43 and 23% respectively, P25 did not cause effects. Shoot length of red clover decreased between 41 and 62% for all tested TiO₂ NPs. In 21% of the TiO₂ NP treated plants, no nodules were found. At high concentrations certain TiO₂ NPs impaired <i>R</i>. <i>trifolii</i> as well as red clover growth and their symbiosis in the hydroponic systems.</p></div

Total TiO₂ suspended or sedimented in the hydroponic system.

<p>Red clover was exposed (n = 3) over 162 h to the two nanoparticles P25 and E171. TiO₂ amounts of the pooled stock suspension is shown at t = 0 in black. TiO₂ amounts of the top (white, 17 ml, in contact with roots) and bottom part (grey, 3 ml, including precipitate) are shown. Differences of the total TiO₂ NP amount (bottom and top part together) to the total Ti amount at t = 0 are indicated with asterisks (p<0.05). Error bars indicate standard deviations (n = 3).</p

Number of root tips and number of secondary roots of red clover in hydroponic system.

<p>Roots were assessed at the harvest (n = 6, mean ± standard deviation). Exposure concentrations (1<2) are described in detail in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155111#pone.0155111.t002" target="_blank">Table 2</a>.</p

Relative growth rates of <i>R</i>. <i>trifolii</i> in YMB medium over a 32 h exposure assessed by optical density.

<p><i>R</i>. <i>trifolii</i> growth rates were assessed in medium containing different actual concentrations and qualities of TiO₂ NPs (P25 filled diamond, E171 filled triangle, NNM TiO₂ inverted triangle, ZnSO₄*7H₂O circle) during the 34 h (n = 4). Stars indicate significant (p<0.05) differences from the control. Exponential growth curves are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155111#pone.0155111.s001" target="_blank">S1 Appendix</a>.</p

Length of the main root (white) and shoot (grey) at harvest (t = 4 weeks).

<p>Lengths are shown for the control, the different TiO₂ NP treatments in two concentrations 1 (low) and 2 (high) that are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155111#pone.0155111.t002" target="_blank">Table 2</a>, and the 16.1 mg l^-1 ZnSO₄ treatment (a) in presence of <i>R</i>. <i>trifolii</i> or (b) without <i>R</i>. <i>trifolii</i>. Significant (p<0.05) differences to the respective control (n = 6) are indicated with asterisks above the standard deviation error bars for shoots, and below the error bars for roots. The results of the same treatments with and without <i>R</i>. <i>trifolii</i> are not significantly different and neither were the controls, but the root length of ZnSO₄ with and without <i>R</i>. <i>trifolii</i> was different (p = 0.005).</p

Scanning electron microscopy image of dried red clover root.

<p>Root surface from a 24 mg l^-1 E171 treated plant is shown. The insert shows a magnification of 1, and from the spots 2 and 3 (+) X-ray fluorescence spectra were prepared revealing that spot 2 did not contain titanium while spot 3 contained titanium.</p

¹⁵N content (% of total N) of red clover shoots.

<p>Results are shown for the control, the different TiO₂ NPs in two concentrations (1 = low, 2 = high) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155111#pone.0155111.t001" target="_blank">Table 1</a> and the ZnSO₄ treatment (a) with addition of <i>R</i>. <i>trifolii</i> and (b) without <i>R</i>. <i>trifolii</i> inoculation. Error bars indicate standard deviations, asterisks show significant differences compared to the control (p<0.05) and number of replications are indicated on the graph (n). Number of replications varied because not for all samples the required amount of shoot biomass for ¹⁵N measurement was available.</p

Analytical data of the TiO₂ NPs suspended in YMB medium.

<p>Suspensions for the <i>R</i>. <i>trifolii</i> exposure experiment were assessed at the start of the experiment (t = 0) and at the end, i.e., after 34 h (n = 3).</p

Analytical data of the TiO₂ NP suspensions in Fåhraeus medium (FM).

<p>Suspensions of the hydroponic experiment were explored over four weeks (n = 3).</p