Uptake and Transformation of Hexachlorocyclohexane Isomers (HCHs) in Tree Growth Rings at a Contaminated Field Site

The potential transformation of hexachlorocyclohexane isomers (HCHs) within tree trunks could have a significant impact on the use of phytoscreening. However, the transformation mechanisms of HCH in trunks particularly in growth rings are not yet well understood. Therefore, a field study on an HCH-contaminated field site was conducted to investigate the fate of HCH, particularly α-HCH in tree trunks using multielement compound-specific isotope analysis (ME-CSIA) and enantiomer fractionation. The results indicate that α-HCH was transformed, as evidenced by higher δ13C and δ37Cl values detected across different growth ring sections and in the bark compared to those in muck and soil. Remarkably, in the middle growth ring section, δ13C values of HCH were only marginally higher or comparable to those in muck, whereas δ37Cl values were higher than those of the muck, indicating a different transformation mechanism. Moreover, the δ37Cl values of β-HCH also increased in the tree trunks compared to those in soil and muck, implying a transformation of β-HCH. Additionally, dual-element isotope analysis revealed that there are different transformation mechanisms between the middle growth rings and other sections. Our findings suggest that the transformation of HCHs in trunks could bias quantitative phytoscreening approaches; however, ME-CISA offers an option to estimate the degradation extent.

S2 20 S1 Field site 21 The site is located in Bitterfeld-Wolfen and belongs to the core center of the chlorine 22 chemistry of the former German Democratic Republic. This area belongs to the most 23 heavily HCH-contaminated sites in the world 1 . Beside the industrial manufacture of about 24 4500 chlorine-based chemical substances or associated consumer goods, HCH and DDT 25 were extensively synthesized in Bitterfeld-Wolfen between 1951− 1982 . Open-pit mines 26 were used in this period to dump chemical waste from industrial production without any 27 appropriate safety or environmental protection measures. Today most of the contaminated 28 soil in the production areas has been remediated or stabilized. However, the former loading 29 areas next to one previous factory still have some hot spots of soils heavily contaminated 30 by muck and now covered by a vegetation of bushes and trees with an estimated age of 31 more than 20 years. The trees selected for the present study were growing at the slope of a 32 hill which downhill a loading area with a rail way connection. The waste materials were 33 formally transported by train to the nearby waste deposit in exploited brown coal open pit 34 mines. We have conduct a previous study to analyze the transformation of HCH within 35 annual cycles in trees using leaves, branches and fruits as well as soil and muck as 36 substrates 4 .

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The tree trunks were firstly separated into different parts based on the tree growth rings 40 and frozen at −20 °C overnight and then freeze-dried in a freeze-dryer at −35°C and 0.310 41 mbar for 48 h. The bark samples are taken separately and up to 4-5 tree rings we combined 42 as one section which are shown in Figure S1. The growth ring samples were taken with a 43 wood driller as shown in Figure S2.

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The wood cutting from drilling were powdered using a grinder (Retsch of HCH in tree trunks, 5 repeats of one sample from a tree ring section of identical age 53 were conducted using the extraction process above and the extracts were combined for 54 further treatment. A small amount of DCM was added to re-dissolve the extracted materials.

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Then a small amount of Florisil was added to absorb the extracted materials and the DCM 56 was evaporated to dryness for further clean-up.

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The oven temperature was held at 45 °C for 5 min, increased at 8 °C min -1 to 180 °C and 74 then at 2 °C min -1 to 195 °C, finally increased at 8 °C min -1 to 220 °C and with a hold of 2 75 min. Samples were measured using splitless mode with an injector temperature of 250 °C.

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The injection volume was 1 µL and each sample was measured in triplicates.

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Isotope Analysis. Carbon isotope composition. A Zebron ZB1 column (60 m × 0.32 mm × 78 1 µm; Phenomenex, Germany) operated with a constant carrier gas flow of 2 mL min -1 was 79 applied for chromatographic separation. The oven temperature was initially held at 40 °C 80 for 5 min, then increased at 10 °C min -1 to 175 °C followed by an increase at 2 °C min -1 to 81 200 °C with a hold of 10 min, and finally increased at 15 °C min -1 to 300 °C and held for 82 2 min. All samples were injected in splitless mode into a split/splitless injector hold at 250 83°C. Each sample was analyzed in triplicate. The quality of the isotope data was controlled 84 by analyzing a HCH standard with a known isotope composition, as described elsewhere indicates the preferential transformation of (+) enantiomer, and an EF (-) <0.5 indicates the 102 preferential transformation of (-) enantiomer. 7 The oven temperature was initially held at 103 70 °C for 4 min, then increased at 10 °C min -1 to 120°C, at 1 °C min -1 to 169 °C, and finally 104 increased at 10 °C min -1 to 220 °C and held for 8 min isothermally.

S4 Calculation of Biotransformation of HCH based on the Rayleigh Equation.
107 For the quantification of α-HCH transformation by isotope analysis, the simplified 108 Rayleigh equation was applied for calculation biodegradation (B%), as shown in eq 1. δ 0 109 is the initial or referential carbon isotope values of the whole study and in current study δ 0 110 represents the carbon and chlorine isotope value of HCH-muck with a value of -27.2 ‰ 111 and -1.9 ‰, respectively. Δδ is the difference of carbon isotope value of environmental 112 samples between the HCH-muck. ε c /ε cl are the carbon/chlorine isotope fractionation factors.

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The reconstructed concentration (RC) was calculated using the percentages of 117 biodegradation and the residual concentration in different sections, which is shown in eq 118 2.

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(2) RC( / ) = residual concentration ( / ) (100 -)% Figure S1. The sampling procedure of tree trunks based on the tree growth rings. Around 4 growth rings were defined as one sample. The number 1, 2 ,3 and 4 in the picture represents the bark sample, outer, inner and middle tree growth ring section, respectively.  Figure S2. Example for the drilling procedure of tree trunks based on the tree growth rings.
MS-3 S10 Table S1. The different section of tree trunks with corresponding number for the age of the growth rings for each section.
The reconstructed values of initial concentration calculated employing low fractionation factor which was found in aerobic pathway give unreasonably high initial concentration when the degradation exceed 99.99%. On one hand the