<p> MARINE BIOTECHNOLOGY SUPPLEMENTARY MATERIAL</p><p>BIODEGRADATION OF THE PYRETHROID PESTICIDE ESFENVALERATE BY MARINE- DERIVED FUNGI WILLIAN G. BIROLLI1, NATALIA ALVARENGA1, MIRNA H. R. SELEGHIM2 and ANDRÉ L. M. PORTO1*</p><p>1Laboratório de Química Orgânica e Biocatálise, Instituto de Química de São Carlos, Universidade de São Paulo, Avenida João Dagnone, 1100, Ed. Química Ambiental, Jd. Santa Angelina, 13563-120, São Carlos, SP, Brazil.</p><p>2Departamento de Ecologia e Biologia Evolutiva, Universidade Federal de São Carlos, Via Washington Luís, Km 235, 13565-905, São Carlos, SP, Brazil</p><p>Address correspondence to André L. M. Porto, Instituto de Química de São Carlos, Universidade de São Paulo, Laboratório de Química Orgânica e Biocatálise, Av. João Dagnone, 1100, Ed. Química Ambiental, Jd. Santa Angelina, 13563-120, São Carlos, SP, Brasil. Phone: +55 16 3373 8103; Fax: +55 16 3373 9952; E-mail: [email protected]</p><p>Content</p><p>Electronic Supplementary Material (ESM) 1 – Analytical curves and linear estimations</p><p>It must be emphasized samples were tenfold concentrated in the extraciton, from 50 mL to 5 mL.</p><p>Linear standard curves were generated for Esf (Equation 1), using standard solution of 50, 300, 550, 800</p><p>1 and 1050.0 mg L-1 [c=(x+41097)/3503] of esfenvalerate in methanol (HPLC grade). For PBAc, two standard curves were constructed: 5, 12, 19, 26 and 33 mg L-1 [c=(x+1677)/4762] and 25, 80, 135, 190 and 250 mg L-1 [c=(x-3358)/4554]. For PBAlc [c=(B10+408)/6320] and PBAld [c=(x+1253)/4659] standard curve of 5, 12, 19, 26 and 33 mg L-1 were used. For CLAc, two standard curves were prepared from 5, 12, 19, 26 and 33 mg L-1 [c=(B10+61.8)/684] and 25, 80, 135, 190 and 250 mg L-1[c=(B10-</p><p>577)/691] (S.I.1).</p><p> c= (x-B)/A (Equation 1)</p><p>Where: c = analyte concentration, mg L-1</p><p> x = experimental area</p><p>B = intercept</p><p>A = slope</p><p>A linear estimate was calculated (Equation 2) for concentrations lower than 5 mg L-1 of PBAld </p><p>[c=(x.5)/2103], PBAc [=(x.5)/2049], PBAlc [c=(x.5)/29213] and ClAc [c=(x.5)/3598].</p><p> c= (x.5)/Y (Equation 2) Where: c= analyte concentration, mg L-1</p><p> x= experimental are</p><p>Y=5 mg L-1 standard solution area</p><p>An standard curve was obtained for Esf (Fig, S.I.1-1) using standard solutions of 50, 300, 550, 800 and</p><p>1050.0 mg.L-1 [c=(x+41097)/3503] of esfenvalerate in methanol.</p><p>ESM-1.Fig. S1. Standard curve to evaluate Esf concentration.</p><p>2 Two standard curves was obtained for PBAc. (Fig, S.I.1-2 and Fig, S.I.1-3) using 5, 12, 19, 26 and 33 mg.L-1 [c=(x+1732)/4762] and 25, 80, 135, 190 e 250 mg.L-1 [c=(x-3357,9)/4554,7] of PBAc in methanol.</p><p>ESM-1.Fig. S2. Standard curve to evaluate PBAc concentration (5-33 mg.L-1).</p><p>ESM-1.Fig. S3. Standard curve to evaluate PBAc concentration (25-250 mg.L-1).</p><p>Two standard curves was obtained for ClAc. (Fig, S.I.1-4 and Fig, S.I.1-5) using 5, 12, 19, 26 and 33 mg.L-1 [c=(B10+61.8)/683,7] and 25, 80, 135, 190 e 250 mg.L-1 [c=(B10-576,8)/691,3] of CLAc in methanol.</p><p>ESM-1.Fig. S4. Standard curve to evaluate CLAc concentration (5-33 mg.L-1).</p><p>ESM-1.Fig. S5. Standard curve to evaluate CLAc concentration (25-250 mg.L-1).</p><p>An standard curve was obtained for PBAld (Fig, S.I.1-6) using standard solutions of 5, 12, 19, 26 and 33 mg.L-1 [c=(x+1279)/4659] of PBAld in methanol.</p><p>ESM-1.Fig. S6. Standard curve to evaluate PBAld concentration (5-33 mg.L-1).</p><p>3 An standard curve was obtained for PBAlc (Fig, S.I.1-7) using standard solutions of 5, 12, 19, 26 and 33 mg.L-1 [c=(B10+407,9)/6320,2] of PBAlc in methanol.</p><p>ESM-1.Fig. S7. Standard curve to evaluate PBAlc concentration (5-33 mg.L-1).</p><p>Electronic Supplementary Material 2 – Fungal control and biodegradation chromatograms</p><p>ESM-2.Fig. S1. (A) HPLC-UV chromatogram of esfenvalerate (100 mg L-1) biodegradation for 14 days with orbital shaking (130 rpm and 32°C), for the strain Acremonium sp. CBMAI 1676 and the abiotic control. (B) Exapanded view between 9 and 18 min.</p><p>4 ESM-2.Fig. S2. (A) HPLC-UV chromatograms of esfenvalerate (100 mg L-1) biodegradation for 14 days with orbital shaking (130 rpm and 32 °C), for the strain Acremonium sp. CBMAI 1676 and the fungal control. (B) Expanded view between 9 and 18 min.</p><p>5 Electronic Supplementary Material 3 – Numeric values of the biodegradation over time</p><p>6 ESM-3.Fig. S1. Chromatograms of the biodegradation for (A) 0, (B) 7, (C) 14, (D) 21 and (E) 28 days by the fungus strain Acremonium sp. CBMAI 1676.</p><p>ESM-3.Table S1. Biodegradation of esfenvalerate (100 mg.L-1) by Acremonium sp. CBMAI 1676 after 7, 14, 21 and 28 days (32 °C, 130 rpm).</p><p>7 Time (days) Compound 0 7 14 21 28 Esfenvalerate (mg L-1) Esf (Residual) 98.3±2.2 74.3±2.9 64.8±6.7 56.6±6.0 48.0±6.7 PBAld 1.1±0.1 NDb NDb NDb NDb PBAc 0.1±0.0 5.0±0.2 7.4±0.3 11.0±0.6 16.6±0.9 PBAlc NDa NDa NDa NDa NDa CLAc 0.1±0.0 4.2±0.2 6.3±0.3 9.7±0.6 13.4±1.1 ND: Not detected. aThe detection limit for PBAlc was 0.02 mg L-1, thus c<0.02 mg L-1. bThe detection limit for PBAld was 0.03 mg L-1, thus c<0.03 mg L-1.</p><p>ESM-3.Table S2. Biodegradation of esfenvalerate (100 mg.L-1) by Westerdykella sp. CBMAI 1679 after 7, 14, 21 and 28 days (32 °C, 130 rpm). Time (days) Compound 0 7 14 21 28 Esfenvalerate (mg L-1) Esf (Residual) 98.3±2.2 84.3±7.4 69.5±3.7 63.9±2.1 56.3±4.7 PBAld 1.1±0.1 NDb NDb NDb NDb PBAc 0.1±0.0 0.8±0.1 3.1±1.5 5.0±1.1 6.6±2.4 PBAlc NDa NDa NDa NDa NDa CLAc 0.1±0.0 2.5±0.3 7.1±1.6 9.4±1.6 9.3±1.1 ND: Not detected. aThe detection limit for PBAlc was 0.02 mg L-1, thus c<0.02 mg L-1. bThe detection limit for PBAld was 0.03 mg L-1, thus c<0.03 mg L-1.</p><p>ESM-3.Table S3. Biodegradation of esfenvalerate (100 mg.L-1) by Microsphaeropis sp. CBMAI 1675 after 7, 14, 21 and 28 days (32 °C, 130 rpm). Time (days) Compound 0 7 14 21 28 Esfenvalerate (mg L-1) Esf (Residual) 98.3±2.2 91.5±2.6 77.9±4.1 66.9±5.4 52.2±2.5 PBAld 1.1±0,1 NDb NDb NDb NDb PBAc 0.1±0.0 0.6±0,1 0.6±0.2 1.2±0.1 2.7±0.4 PBAlc NDa 0.2±0,1 NDa NDa NDa CLAc 0.1±0.0 1.0±0.1 2.7±0.1 5.4±0.2 6.6±0.6 ND: Not detected. aThe detection limit for PBAlc was 0.02 mg L-1, thus c<0.02 mg L-1. bThe detection limit for PBAld was 0.03 mg L-1, thus c<0.03 mg L-1.</p><p>ESM-3.Table S4. Abiotic control of esfenvalerate (100 mg L-1) after 7, 14, 21 and 28 days (32 °C, 130 rpm).</p><p>8 Time (days) Compound 0 7 14 21 28 Esfenvalerate (mg L-1) Esf (Residual) 98.3±2.2 97.5±0.8 97.6±0.6 97,9±0,4 97,1±0,9 PBAld 1.1±0.1 1.0±0.1 0.8±0.1 0.7±0.1 0.5±0.1 PBAc c<0.1a c<0.1a c<0.1a c<0.1a c<0.1a PBAlc NDc NDc NDc NDc NDc CLAc c<0.1b c<0.1b c<0.1b c<0.1b c<0.1b ND: Not detected. c.: concentration aThe compound PBAc was detected. However, its concentration was below 0.1 mg.L-1 and was not determined. bThe compound CLAc was detected. However, its concentration was below 0.1 mg.L-1 and was not determined. cThe detection limit for PBAlc was 0.02 mg L-1, thus c<0.02 mg L-1.</p><p>Electronic Supplementary Material 4 – High resolution mass spectra</p><p>9 For example, in the strain Microsphaeropsis sp. CBMAI 1675 analysis, five peaks were observed in the esfenvalerate biodegradation sample that were absent in the fungal control (Figure 3).</p><p>The peaks are identified as: A (elution time = 4.6 min), B (9.2 min), C (9.7 min), D (12.9 min) and E</p><p>(15.8 min). Thus, the mass spectra of these compounds were interpreted and when possible compared to analytical standards.</p><p>ESM-4.Fig. S1. HPLC-TOF total ion chromatograms of esfenvalerate biodegradation by strain Microsphaeropsis sp CBMAI 1675 and the fungal control.</p><p>The spectrum of compound A showed no consistent mass with degradation products of esfenvalerate, since peaks with m/z greater than 1200 and three charges were observed. Therefore, it is suggested that compound A is an unknown macromolecule that was induced by the presence of the pesticide. </p><p>Compound B showed the ion m/z 213.07±0.05, with mass and isotope pattern for C11H14ClO2, referring protonated CLAc. The compound identity was confirmed by using the analytical standard, which yielded a similar chromatographic retention time and mass spectrum.</p><p>Peak C showed an ion with m/z 215.07±0.05 and isotopic pattern for the molecular formula</p><p>C13H11O3 , referring to protonated PBAc. The identity of the compound was confirmed by testing the analytical standard, which gave a similar chromatographic retention time and mass spectrum.</p><p>Peak D showed an ion with m/z 183.07±0.05, mass and isotopic pattern for the molecular formula C13H13O2, referring to PBAlc. The identity of the compound was confirmed by comparing the analytical standard, which gave a similar chromatographic retention time and mass spectrum.</p><p>10 In the case of peak E, a greater signal was observed in the biodegradation sample. The presence of the ion m/z 231.06 ± 0.05, with mass and isotope pattern for the molecular formula C 14H15O4, suggest the formation of the protonated 3-( hydroxyphenoxy)benzoic acid (hydroxyl`PBAc).</p><p>ESM-4.Fig S2. Mass espectrum of (A) compound B and (B) analytical standard of CLAc. Elution time was 12.9 min.</p><p>. </p><p>ESM-4.Fig S3. Mass espectrum of (A) compound C and (B) analytical standard of PBAc. Elution time was 9.7 min.</p><p>11 ESM-4.Fig S4. Mass espectrum of (A) compound D and (B) analytical standard of PBAlc. Elution time was 9.4 min.</p><p>ESM-4.Fig S5. (A) Mass espectrum of compound E, hydroxyPBAc and (B) its isotopic pattern. Elution time was 4.6 min.</p><p>12 ESM-4.Fig S6. Mass espectrum of (A) PBAld from the abiotic degradation and (B) analytical standard. Elution time was 17.2 min.</p><p>13 ESM-4.Fig S7. Mass espectrum of (A) methylPBAc from biodegradation by Cladosporium CBMAI 1237 and (B) analytical standard. Elution time was 21.0 min.</p><p>Electronic Supplementary Material 5 – Quadrupole mass spectra</p><p>14 ESM-5.Fig S1. Mass espectrum of PBAc (A) in the esfenvalerate analysis and (B) the standard.</p><p>ESM-5.Fig S2. Mass espectrum of methylPBAc in the esfenvalerate analysis.</p><p>ESM-5.Fig S3. Mass espectrum of PPAc in the esfenvalerate analysis.</p><p>ESM-5.Fig S4. Mass espectrum of methylCLAc in the esfenvalerate analysis.</p><p>ESM-5.Fig S5. Mass espectrum of CLAc (A) in the esfenvalerate analysis and (B) the standard.</p><p>ESM-5.Fig S6. Mass espectrum of PBAld (A) in the esfenvalerate analysis and (B) the standard.</p><p>ESM-5.Fig S7. Mass espectrum of PBAlc (A) in the esfenvalerate analysis and (B) the standard.</p><p>15</p>