# Contribution of Atmospheric Fallout to the Soil-Root-Leaf Transfer of PAHs in Higher Plants
## 2 Materials and Methods
### Analysis of PAHs  
Gas chromatography (GC-MS) method was used to determine PAH content in aerosol extract according to MSZ 1484-6:2003 standard [36] (MSZ 1484-6:2003: Testing of waters. Determination of polycyclic aromatic hydrocarbons (PAH) content by gas chromatography-mass spectrometry, LOD: 0.005 ug/L per component) and plant samples analysis based on MSZ EN 15527:2009 [37] (Characterization of waste. Determination of polycyclic aromatic hydrocarbons (PAH) in waste using gas chromatography-mass spectrometry (GC/MS, LOD: 0.1 ug/kg per component). In our study, 19 PAHs were analyzed: Naphthalene (Nap), 2-Methyl-naphthalene (Methyl-Nap), 1-Methyl-naphthalene (Me-Nap), Acenaphthylene (Acy), Acenaphthrene (Ace), Fluorene (Flu), Phenanthrene (Phen), Anthracene (Ant), Fluoranthene (Flt), Pyrene (Pyr), Benzo[a]anthracene (B(a)a), Chrysene (Cry), Benzo[b]fluoranthene (B(b)f), Benzo[k]fluoranthene (B(k)f), Benzo[a]pyrene (B(a)p), Benzo[e]pyrene (B(e)p), Indeno(l,2,3-cd)pyrene (IP), Dibenzo[a,h]anthracene (D(a,h)a), and Benzo(g,h,i)perylene (B(g,h,i),p). Different plant parts were separately grinded in a ceramic mortar with 10 g anhydrous sodium sulphate. An amount of 10 mL acetone was added, and the samples were spiked with 100 uL of 0.01 ug/mL deuterated PAH surrogate mixture containing Naphtalene-d8, Acenaphthene-d10, Phenanthrene-d10, Chryzene-d12, Benzo[a]pyrene-d12, and Perylene-d12 (Restek Corporation, Bellefonte, PA, USA) and extracted with 20 mL n-hexane 3 times with ultrasonic extraction for 20 min. In a dry nitrogen stream, the extract was concentrated to 1 mL, and silica gel and alumina oxide clean-up was performed. For the GC-MS analysis, HP-6890 gas chromatograph was coupled with an HP-5973 (Agilent Technologies, Palo-Alto, CA, USA) quadrupole mass spectrometer (low-resolution single MS). From a standard PAH mixture, a five-point calibration for each of the target chemical compound was established, detected over a concentration range of 0.5-5.0 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/L. All data were corrected for the average value of the blanks. Each compound and surrogate standards relative response factors was calculated for every calibration level using the following equation  
\[\mathrm{Rfi}=(\mathrm{Ai}*\mathrm{Cs})/(\mathrm{As}*\mathrm{Ci})\]  
where \(\mathrm{Rfi}\) = response factor of analyte \(\mathrm{i}\);  
\(\mathrm{Ai}\) = peak response of compound \(\mathrm{i}\);  
\(\mathrm{Cs}\) = concentration of internal standard (\(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g, or \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/L);  
\(\mathrm{As}\) = peak response Internal Standard; and  
\(\mathrm{Ci}\) = concentration of analyte or surrogate (\(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g, or \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/L). The limit of detection (LOD) values for PAHs in plant sample were as follows: Nap 0.0100 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g; Methyl-Nap 0.0120 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g; Me-Nap 0.0110 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g; Acy 0.0105 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g; Ace 0.0120 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g; Flu 0.0110 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g, Phen 0.0100 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g, Ant 0.0110 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g, Flt 0.0100 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g, Pyr 0.0110 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g, B(a)a 0.0110 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g, Cry 0.0130 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g, B(b)f 0.0110 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g, B(k)f 0.0110, B(e)p 0.0130 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g, B(a)p 0.0125 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g, IP 0.0140 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g, D(a,h)a 0.0150 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g, B(g,h,i)p 0.0140 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/g.