A fluorometric method for the selective determination
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1 SUPPLEMENTARY INFORMATION A fluorometric method for the selective determination of lipid hydroperoxides in river water Michael Oluwatoyin Sunday and Hiroshi Sakugawa* Graduate School of Biosphere Science, Department of Environmental Dynamics and Management, Hiroshima University, Kagamiyama, Higashi-Hiroshima, Hiroshima , Japan. *Corresponding author: H. Sakugawa. address: hsakuga@hiroshima-u.ac.jp. Tel.: ; fax: CONTENT Fig. S1. Reaction of Liperfluo with Lipid hydroperoxide. Fig. S2. Map of sampling sites. Fig. S3. Flow diagram of the Flow injector analysis (FIA). Fig. S4. Three-Dimensional fluorescence spectra. Fig. S5. Fluorescence spectra of Liperfluo with various Lipid hydroperoxide concentrations. Fig. S6. Reduction of lipid hydroperoxide by TCEP in water. Fig. S7. Interference studies due to some river water ions. Fig. S8. Reaction of various hydroperoxides to the peroxidase-catalyzed POPHA method of hydroperoxides determination. Table S1. Chemical composition of water from the Kurose River sampled on March Table S2. Chemical composition of water from the Kurose River sampled on April
2 Fig S1. Reaction of Liperfluo with lipid hydroperoxide. Liperfluo, which is non-fluorescent selectively reacts with lipid hydroperoxides to form Liperfluo-Ox as a fluorescent product and a lipid alcohol. The selectivity of Liperfluo for lipid hydroperoxides over other reactive oxygen species such as H 2 O 2, OH., ROO., NO, O2.-, RO. has been well demonstrated by Yamanak et al (2012). In our study, we present further evidence of the selectivity of Liperfluo for lipid hydroperoxide over hydroperoxides such as methyl hydroperoxide, ethyl hydroperoxide present in river water.
3 Japan Hiroshima Prefecture N Higashi Hiroshima Kurose River K1 = Namitakiji; K2 = Tokumasa K3 = Izumi; K4 = Ochiai K5 = Hinotsume; K6 = Kurose Bunka Centre Fig. S2. Map of sampling stations along Kurose River. The Kurose River flows from the Kokuzo mountain with an elevation of 666 m to an urban area and passes through agricultural fields (mainly rice fields) before emptying into the Seto Inland
4 Sea. Sampling site K1 was located upstream in a forested area, and is considered to be unaffected by anthropogenic contamination. Sampling sites K2 to K6 were located within a densely populated urban area, and could be affected by agricultural and human activities (Anifowose et al., 2015). Sampling site K4 receives effluent from a wastewater treatment plant. Carrier: MQ water Pump (0.5 ml min - 1 ) Sample (100 µl) Injector Fluorescence detector (Ex/Em: 495/546 nm) Waste Recorder Fig. S3A. Flow diagram of the FIA system. Fig. S3B: A typical chart obtained from the FIA.
5 A Emission (nm) B Emission (nm) C Excitation (nm) Excitation (nm) Excitation (nm) Emission (nm) Fig. S4. Three-Dimensional fluorescence spectra of 2 µm Liperfluo: (A) in the absence of MLOOH (B) in the presence of 500 nm MLOOH in water. (C) Fluorescence of 1 µm Liperfluo- Ox standard. The fluorescence of (C) was observed in (B) as the product of reaction between Liperfluo and MLOOH in water.
6 Fluorescence intensity (A.U) Emission (nm) Fig. S5. Relationship between the fluorescence intensity (excitation/emission: 495 nm/546 nm, respectively) and MLOOH concentration for the reaction of 0, 100, 200, 300 and 500 nm MLOOH with 2 µm Liperfluo in water. Fluorescence intensity (A.U) Riverwater MilliQ TCEP concentraqon (mm) Fig. S6. TCEP reduction of LHPs. Reduction of fluorescence signal when 500 nm MLOOH in MilliQ and river water was treated with various concentrations of TCEP 0, 0.25, 0.5, 1, 10, 20 and 30 mm for 5 min prior to reaction with 2 µm Liperfluo.
7 Fluorescent intensity (A.U) MQ Cl- NO2- NO3- SO42- Mixture MQ Cl - NO 2 - NO 3 - SO 4 2- *Mixture Fig. S7. Interference studies due to some river water ions. *Mixture represents the mixture of these ions at the same concentrations studied individually Fluorescence intensity (A.U) Blank H2O2 MHP EHP MLOOH Hydroperoxides Fig S8: Response of 500 nm of various hydroperoxides to the peroxidase-catalyzed POPHA method of hydroperoxide determination.
8 Table S1. Chemical composition of water from the Kurose River sampled on March Site ph DOC (µmol C L -1 ) Anions (µm) Cl - NO 2 - NO 3 - SO 4 2- Cations (µm) Na + NH 4 + K + Mg 2+ Ca 2+ K1 * ND K K K K K Table S2. Chemical composition of water from the Kurose River sampled on April Site ph DOC (µmol C L -1 ) Anions (µm) Cl - NO 2 - NO 3 - SO 4 2- Cations (µm) Na + NH 4 + K + Mg 2+ Ca 2+ K ND K K K K K The ph was measured after filtration using a ph meter (D-51, Horiba, Japan). The ph range across the sampling sites was
9 Dissolved organic carbon (DOC) was measured using a TOC-VCSH analyzer (Shimadzu, Kyoto, Japan). Potassium hydrogen phthalate (Wako Pure Chemical Industries, Osaka, Japan) was used as the standard solution. Sampling site K4 had the highest DOC concentration. Ions in the samples were measured on by ion chromatography (ICS-1600, Dionex, Japan). The anion and cation standards used were purchased from Chameleon reagent. The NO 2 concentration is important as an indicator of environmental pollution. At sampling sites K1 and K2, the NO 2 concentrations were the lowest and the concentrations of LHPs were below the detection limit. By contrast, at sampling sites K3 K6, the NO 2 concentrations were high and LHPs were present at detectable concentrations.
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