Biodegradation of the Pyrethroid Pesticide Esfenvalerate by Marine-Derived Fungi

MARINE BIOTECHNOLOGY SUPPLEMENTARY MATERIAL

BIODEGRADATION OF THE PYRETHROID PESTICIDE ESFENVALERATE BY MARINE- DERIVED FUNGI WILLIAN G. BIROLLI1, NATALIA ALVARENGA1, MIRNA H. R. SELEGHIM2 and ANDRÉ L. M. PORTO1*

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.

2Departamento de Ecologia e Biologia Evolutiva, Universidade Federal de São Carlos, Via Washington Luís, Km 235, 13565-905, São Carlos, SP, Brazil

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]

Content

Electronic Supplementary Material (ESM) 1 – Analytical curves and linear estimations

It must be emphasized samples were tenfold concentrated in the extraciton, from 50 mL to 5 mL.

Linear standard curves were generated for Esf (Equation 1), using standard solution of 50, 300, 550, 800

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-

577)/691] (S.I.1).

c= (x-B)/A (Equation 1)

Where: c = analyte concentration, mg L-1

x = experimental area

B = intercept

A = slope

A linear estimate was calculated (Equation 2) for concentrations lower than 5 mg L-1 of PBAld

[c=(x.5)/2103], PBAc [=(x.5)/2049], PBAlc [c=(x.5)/29213] and ClAc [c=(x.5)/3598].

c= (x.5)/Y (Equation 2) Where: c= analyte concentration, mg L-1

x= experimental are

Y=5 mg L-1 standard solution area

An standard curve was obtained for Esf (Fig, S.I.1-1) using standard solutions of 50, 300, 550, 800 and

1050.0 mg.L-1 [c=(x+41097)/3503] of esfenvalerate in methanol.

ESM-1.Fig. S1. Standard curve to evaluate Esf concentration.

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.

ESM-1.Fig. S2. Standard curve to evaluate PBAc concentration (5-33 mg.L-1).

ESM-1.Fig. S3. Standard curve to evaluate PBAc concentration (25-250 mg.L-1).

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.

ESM-1.Fig. S4. Standard curve to evaluate CLAc concentration (5-33 mg.L-1).

ESM-1.Fig. S5. Standard curve to evaluate CLAc concentration (25-250 mg.L-1).

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.

ESM-1.Fig. S6. Standard curve to evaluate PBAld concentration (5-33 mg.L-1).

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.

ESM-1.Fig. S7. Standard curve to evaluate PBAlc concentration (5-33 mg.L-1).

Electronic Supplementary Material 2 – Fungal control and biodegradation chromatograms

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.

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.

5 Electronic Supplementary Material 3 – Numeric values of the biodegradation over time

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.

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).

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.

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.

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.

ESM-3.Table S4. Abiotic control of esfenvalerate (100 mg L-1) after 7, 14, 21 and 28 days (32 °C, 130 rpm).

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.

Electronic Supplementary Material 4 – High resolution mass spectra

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).

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

(15.8 min). Thus, the mass spectra of these compounds were interpreted and when possible compared to analytical standards.

ESM-4.Fig. S1. HPLC-TOF total ion chromatograms of esfenvalerate biodegradation by strain Microsphaeropsis sp CBMAI 1675 and the fungal control.

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.

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.

Peak C showed an ion with m/z 215.07±0.05 and isotopic pattern for the molecular formula

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.

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.

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).

ESM-4.Fig S2. Mass espectrum of (A) compound B and (B) analytical standard of CLAc. Elution time was 12.9 min.

ESM-4.Fig S3. Mass espectrum of (A) compound C and (B) analytical standard of PBAc. Elution time was 9.7 min.

11 ESM-4.Fig S4. Mass espectrum of (A) compound D and (B) analytical standard of PBAlc. Elution time was 9.4 min.

ESM-4.Fig S5. (A) Mass espectrum of compound E, hydroxyPBAc and (B) its isotopic pattern. Elution time was 4.6 min.

12 ESM-4.Fig S6. Mass espectrum of (A) PBAld from the abiotic degradation and (B) analytical standard. Elution time was 17.2 min.

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.

Electronic Supplementary Material 5 – Quadrupole mass spectra

14 ESM-5.Fig S1. Mass espectrum of PBAc (A) in the esfenvalerate analysis and (B) the standard.

ESM-5.Fig S2. Mass espectrum of methylPBAc in the esfenvalerate analysis.

ESM-5.Fig S3. Mass espectrum of PPAc in the esfenvalerate analysis.

ESM-5.Fig S4. Mass espectrum of methylCLAc in the esfenvalerate analysis.

ESM-5.Fig S5. Mass espectrum of CLAc (A) in the esfenvalerate analysis and (B) the standard.

ESM-5.Fig S6. Mass espectrum of PBAld (A) in the esfenvalerate analysis and (B) the standard.

ESM-5.Fig S7. Mass espectrum of PBAlc (A) in the esfenvalerate analysis and (B) the standard.