Lambda 12

The chlorophyll and carotenoid contents were determined according to Lichtenthaler and Buschmann [26 (link),27 (link)]. The methodology of measuring anthocyanin contents followed the procedure reported by Drumm and Mohr [28 (link)]. Fresh leaves were homogenized in a mortar for anthocyanin extraction (extraction medium, 37% HCl: methanol; 1:99 [v/v]). The absorbance of the extracts was measured at 530 nm using a UV/VIS spectrometer (Lambda 12; Perkin–Elmer, Norwalk, CT, USA), with the anthocyanin contents calculated as absorbance per leaf area.
The methanol-soluble UV-B and UV-A–absorbing compounds were determined as described by Caldwell [29 (link)]. The UV-absorbing compounds were extracted from the homogenized fresh leaves using methanol: distilled water: 37% HCl (79:20:1 [v/v/v]). After that, the samples were centrifuged (2-16 PK; Sigma, Darmstadt, Germany), and extinction of the supernatants was measured from 280 nm to 400 nm at intervals of 1 nm, using a UV/VIS spectrometer (Lambda 12; Perkin–Elmer, Norwalk, CT, USA). The absorbances were determined from 280 nm to 320 nm for the UV-B–absorbing compounds and from 320 nm to 400 nm for the UV-A–absorbing compounds, and are calculated as absorbance per leaf area.

Antioxidant capacities of CD-NA and CD-SA complexes were measured using the TEAC assay protocol presented by Re et al. [37 (link)] with slight modifications. Here, the ABTS radical cation ABTS^•+ is obtained by the oxidation of ABTS (7 mM) with potassium persulfate (K₂S₂O₈, 2.45 mM) in distilled water (vol/vol reaction). The resulting solution was placed in the dark at room temperature for 12–16 h before use. CD-NA, CD-SA (0–5 μM, 50 μL), or Trolox standards (0–200 μM, 50 μL) were mixed with 1 mL of ABTS^•+ (absorbance maximum of 0.70 ± 0.02 at 734 nm). The absorbance of the mixture solution was recorded after 1 h of incubation at 30°C (734 nm, UV/VIS Lambda 12, PerkinElmer Inc., Norwalk, CT, USA). Inhibition percentage was calculated using 2. The Trolox equivalent antioxidant capacity (TEAC) was defined as the millimolar concentration of Trolox with the same antioxidant activity as 1 mM concentration of the samples [38 (link)].
$\begin{array}{c}\mathrm{S}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\left(\%\right)=\frac{{\left[{\mathrm{A}\mathrm{B}\mathrm{T}\mathrm{S}}^{+}\right]}_{\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}}-{\left[{\mathrm{A}\mathrm{B}\mathrm{T}\mathrm{S}}^{+}\right]}_{\mathrm{f}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{l}}}{{\left[{\mathrm{A}\mathrm{B}\mathrm{T}\mathrm{S}}^{+}\right]}_{\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}}}\ast 100.\end{array}$

Dissolved oxygen was measured with optodes (PreSens Precision Sensing, Germany), which were installed as flow through cells in the influent and effluent tubes. Except for total organic carbon (TOC) analyses, all samples were vacuum-filtered with 0.45 µm cellulose nitrate membranes (Sartorius, Germany). The TOC and DOC were quantified with a TOC Cube analyzer (Elementar, Germany) and further characterized with liquid chromatography and continuous organic carbon detection (LC-OCD, DOC-Labor Huber, Germany) (Huber et al. 2011 (link)). For quantifying the amount of double bonds and aromatic groups of the DOM, ultraviolet light (UV) absorption at 254 nm wavelength was measured with the spectral photometer Lambda 12 (Perkin Elmer, USA) using a cuvette of 1 cm optical length. The selected TOrCs were quantified by high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Besides calibrating TOrC concentration with nine standards, deuterated internal standards of 1 µg/L were used to comply with matrix effects. The method is described in detail by Hellauer et al. (2017b (link)).