Quantitative Identification of Biogenic Nonextractable Pesticide

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Quantitative identification of biogenic nonextractable pesticide residues in soil by 14C-analysis Claudia Poßberg, Burkhard Schmidt, Karolina Nowak, Markus Telscher, Andreas Lagojda, and Andreas Schaeffer Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 18 May 2016 Downloaded from http://pubs.acs.org on May 18, 2016

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Environmental Science & Technology

Biomass ACS Paragon Plus Environment


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Quantitative identification of biogenic non-

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extractable pesticide residues in soil by 14C-analysis

3

Claudia Poßberg1 • Burkhard Schmidt1 • Karolina Nowak1,2 • Markus Telscher3 • Andreas

4

Lagojda3 • Andreas Schaeffer*,1,4,5

5

1

6

1, 52074 Aachen, Germany

7

2

8

Biotechnology, 04318 Leipzig, Germany

9

3

Bayer CropScience AG, Alfred-Nobel-Str. 50, 40789 Monheim am Rhein, Germany

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4

State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment,

11

Nanjing University, Nanjing 210093, P. R. China

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5

13

P. R. China

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KEYWORDS Non-extractables residues, NER, biogenic residues, soil, pesticides, 14C-analysis,

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radioactive labelling

16

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RWTH Aachen University, Institute for Environmental Research (Biology 5), Worringer Weg

Helmholtz-Centre for Environmental Research – UFZ, Department of Environmental

College of Resources and Environmental Science, Chongqing University, Chongqing 400030,

ABSTRACT

1 ACS Paragon Plus Environment

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Quantification of non-extractable residues (NER) of pesticides in soil is feasible by use of

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radioactively labelled compounds, but structural information of these long-term stabilized

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residues is usually lacking. Microorganisms incorporate parts of the radiolabeled (14C-) carbon

20

from contaminants into microbial biomass, which after cell death enters soil organic matter, thus

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forming biogenic non-extractable residues (bioNER). The formation of bioNER is not yet

22

determinable in environmental fate studies due to a lack of methodology. This paper focuses on

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the development of a feasible analytical method to quantify proteinaceous carbon, since proteins

24

make up the largest mass portion of bacterial cells. The test substance 14C-bromoxynil after 56

25

days forms more than 70% of NER in soil. For further characterisation of NER the amino acids

26

were extracted, purified, and separated by two-dimensional thin-layer chromatography (TLC).

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Visualization of the 14C-amino acids was performed by bioimaging, unambiguous identification

28

by GC-MS and LC-MS/MS. Our analysis revealed that after 56 days of incubation about 14.5%

29

of the 14C-label of bromoxynil was incorporated in amino acids. Extrapolating this content based

30

on the amount of proteins in the biomass (55%), in total about 26% of the NER is accounted for

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by bioNER and thus is not environmentally relevant.

32

INTRODUCTION

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Xenobiotics in soil dissipate by movement , binding and degradation processes that depend on

34

the physicochemical properties of the substance and the soil, and on environmental conditions.1

35

If radioactively labelled compounds are used to establish a mass balance of their fate in soil,

36

combustion of the thoroughly extracted soil usually reveals a third component besides

37

extractable and volatile mineralized residues, i.e., the so called non-extractable residues (NER)

38

which are formed via biological and physical-chemical processes.



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NER are hardly distinguishable from soil organic matter (SOM), which forms a huge natural

40

resource.2 Thus, the structural elucidation of the residues comprising the NER is an analytical

41

challenge which results in that in most studies the structural composition remains unidentified.3

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A variety of chromatographic, spectroscopic and spectrometric analyses have been applied to

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study the nature of NER. In a recent review, these attempts have been summarized.4 As a

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conclusion, NER can be differentiated into xenobiotic residues, either entrapped (type I NER) in

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the structural voids of the soil, covalently bound to humic matter (type II NER), or so-called

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biogenic residues (type III NER).4-7 Carbon or nitrogen from certain pesticides can be used by

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metabolic or co-metabolic degradation for synthesis of the cell constituents of microorganisms,

48

e.g., amino acids, fatty acids. After the death and cell lysis, these compounds are incorporated

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into SOM forming ultimately biogenic residues.4 This has been demonstrated in studies on the

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biodegradation of several pesticides and pharmaceuticals labelled with stable isotopes in soil,

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where the contribution of microbial biomass residues to NER in soil was quantified.8-12 Amino

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acids (AA) account for 10-20% of total C in SOM and are mainly incorporated as polymers in

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proteins, protein-humic complexes or peptides.13 In microbial biomass, AA are the most

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abundant components: microbial biomass contains about 55% of proteins of the dry weight of

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bacterial cells.14

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Until now, biogenic residues have been characterized using stable isotope tracers (13C or 15N)8,

57

9, 15

and radioactive derivatives, e.g., by fumigation-extraction.16 However, stable isotope tracers

58

are usually not used in the standardized studies for the regulatory risk assessment of chemicals.

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Rather, such experiments, in which quantitative recovery of the fate of pesticides are needed,

60

rely on the use of radiotracers (14C) but the method for ready 14C-analyses of biogenic residues is

61

missing.


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We here report an analytical method, to unambiguously identify and quantify the formation of 14

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biogenic type III of

C-labelled pesticides based on chromatographic separation (two-

64

dimensional thin layer chromatography, 2D-TLC) and mass spectrometry (LC-MS/MS, GC-MS)

65

verification. We propose the use of

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negligible low natural background and is therefore detectable in very low concentrations in

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liquid and solid matrices. (II) The fate studies actually required for pesticides authorization

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processes are usually conducted using

69

usuallyavailable.

14

C for two reasons: (I) The radioactive label has a

14

C labelled compounds and such derivatives are

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Bromoxynil was selected as a model substance due to formation of high NER content and

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considerable mineralization in a relative short period of time.3, 17 The substance was expected to

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generate high amounts of biogenic residues. Preliminary experiments to develop a method to

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analyse and quantify the formation of

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glucose as a substrate. The objective of the present investigation was to elaborate and establish a

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method for ready

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assessment of non-extractable residues.

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MATERIAL AND METHODS

14

C labelled amino acids were performed by use of

14

C

14

C-analyses of biogenic pesticide residues as important tool for the risk

78

Chemicals

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[UL-Ring14C-]-3,5-dibromo-4-benzonitrile ([14C]-bromoxynil; radiochemical purity >98%,

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specific radioactivity 604 MBq mmol-1) and the non-labelled reference substances bromoxynil ,

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3,5-dibromo-4-hydroxybenzoic acid and 3,5-dibromo-4-hydroxybenzamide were obtained from

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Bayer Crop Science Division (Monheim, Germany). Uniformly labelled AA (valine,

83

phenylalanine, leucine and isoleucin, radiochemical purity > 98.7%, specific radioactivity 10 to

84

17 MBq µmol-1) were obtained from Hartmann Analytic, Germany. Non-labelled amino acids of



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85

technical purity were obtained by Sigma Aldrich, Germany. All other chemicals used were

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analytical grade. Glucose D-[14C(U)] (radiochemical purity 99%, specific radioactivity 370 MBq

87

mmol-1) was obtained from ARC, St. Louis, USA.

88 89

Soil

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A sandy loam soil from grass land (depth: 0–30 cm, Monheim am Rhein, Germany),

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containing a rather high content of soil organic matter and microbial activity, was used for

92

biodegradation experiments. The soil was sieved (< 2 mm) and stored at 4°C (max. 3 months).

93

Prior to the start of the experiments, the soil samples were equilibrated for 7 days at room

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temperature (20°C) and a water content of about 50% of maximum water holding capacity

95

(WHC), measured according to Alef, 1991.18 The soil characteristics were as follows: 76% sand,

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17% silt, 7% clay, 0.16% total nitrogen, 1.9% organic carbon, pH (CaCl2) 6.1.

97 98

Incubation of 14C-Glucose in a Rhodococcus wratislaviensis culture (method development)

99

Regarding the presumed formation of biogenic residues from bromoxynil, the methods

100

required, i.e. hydrolysis of proteins, purification of resulting AA, and corresponding analysis,

101

were developed using a bacterial culture of Rhodococcus wratislaviensis (Rhw) cultivated in a

102

medium containing – besides sucrose – 14C-glucose as carbon and energy sources. Rhw is a soil

103

bacterium capable to utilize a number of xenobiotics in soil as carbon and energy source.19 A

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culture of the soil bacterium Rhw (DSM – 44107; DMSZ, Braunschweig, Germany) was

105

prepared with DifcoTM Czapek Dox (Nordwald, Hamburg, Germany) medium in a 2 ml

106

centrifugation tube and pre-incubated at 28°C. After one day of pre-incubation (during log-

107

phase) 58 KBq

14

C-glucose (28.6 µg) were added to 1 ml of the suspension. After 2 days, the


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108

culture was frozen to stop the incubation. The cell suspension was thawed in a sonication bath

109

for 15 min at 20°C. Then, 0.3 mg of the liquid sample were removed, divided into 3 subsamples,

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which were combusted in a biological oxidizer (OX501, Zinsser Analytic, Frankfurt, Germany)

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in order to determine the radioactivity contained in the sample. The remaining sample of the cell

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suspension was mixed with 1 ml of acetonitrile and centrifuged (11,000 x g). The supernatant

113

was examined for 14C by means of a liquid scintillation counting (Hidex 300 SL, Turku, Finland)

114

and contained 15 % of the radioactivity (not analysed further). The pellet of cells and

115

precipitated proteins was hydrolysed (see below)..

116 117

Spiking and incubation of soil with [14C]bromoxynil (fate study)

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In order to determine the fate of bromoxynil in soil, 450 µl of an ethanol/water solution (7/3,

119

v/v) of bromoxynil (labelled and non-labelled) was applied to a 3 g soil aliquot resulting in a

120

concentration of 20 µg g-1 soil (1 KBq). The concentration was chosen high enough to allow

121

determination of bioNER and low enough to exert no toxic impact on microorganisms.17, 20 After

122

evaporation of the ethanol at room temperature, the aliquot was stirred with a spatula, transferred

123

to the incubation bottle (250 ml) and mixed with 17 g of fresh non-treated soil. The water content

124

was adjusted to 50% of WHCmax. The incubation flasks were capped by means of a chimney

125

glass tube containing (15 g) soda lime for trapping 14CO2 and unlabelled CO2. The assays were

126

incubated at 20°C in the dark for 1, 7, 14, 28 and 56 days with 3 parallels per incubation period.

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Every 3 to 7 days the 14CO2-traps were replaced with fresh traps after flushing the system with

128

humid air. The soil water content was adjusted weekly according to the loss of weight of the soil.

129 130

Spiking and incubation of soil with [14C]-bromoxynil (bio-NER study)



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Either [14C]-bromoxynil or non-labelled bromoxynil was dissolved in 70% ethanol solution.

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Labelled and non-labelled bromoxynil were applied to 3 g of the soil sample (fresh weight).

133

After evaporation of ethanol, the soil aliquot was stirred with a spatula and transferred to the

134

incubation bottle and mixed with 9 g of fresh non-treated soil. The resulting spiked soil samples

135

were then adjusted to 60% WHCmax and incubated for 14, 28 and 56 days in the dark. Soda lime

136

traps were not installed. Initial radioactivity added to each soil sample was 74.6 KBq g-1,

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corresponding to a bromoxynil concentration of 16,5 µg g-1.

138 139

Extraction procedure

140

For both the fate study and the bioNERstudy, 1 g of the soil was subjected to combustion

141

analysis in order to determine the total radioactivity remaining in the soil. For the bioNERstudy,

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another 1 g of the soil was used for acidic hydrolysis for release of the AA (see below). The soil

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sample of each assay was extracted for 24 h with 100 ml of methanol using a Soxhlet apparatus.

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The extracted soil was then examined by combustion analysis for non-extractable residues.

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Another 1 g of the soil in the bio-NER study was used for a further acidic hydrolysis to release

146

the AA contained in the NER fraction. As determined in a preliminary experiment, the recovery

147

of the extraction method was 98 ± 2% of applied 14C.

148 149

Acid hydrolysis and purification of amino acids

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Amino acids were hydrolysed from soil or bacterial pellets using 6 M HCl as described

151

previously (Nowak et al, 2011).8 The hydrolysate was filtered, evaporated to dryness using a

152

rotary evaporator and purified over a cation exchange resin using oxalic acid and the AA were

153

ultimately eluted with ammonia solution (DOWEX 50 W X8; Roth, Karlsruhe, Germany).8 The


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w evaporatted to dryneess and re-ddissolved in a mixture oof methanol and ammonnia acetate eluate was

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buffer (MeOH:AA ( Ac, 1/1, v/vv). The exttraction andd purificatiion proceduures are skketched in

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Schemee 1.

157

Schemee 1: Soil exxtraction prrocedures annd analysis of amino acids to annalyse xenobbiotic and

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biogenicc residues after a incubattion with 14C-bromoxyn C nil.

159 160

TLC of o AA eluatte

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Two ddimensionall TLC was eexecuted onn cellulose pplates (200 x 200 mm, 0.25 mm, MachereyM

162

Nagel, D Düren, Germ many) by a method according to P Pillay.21 Plaates were deeveloped tw wice in the

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first dim mension withh butanol/accetone/amm monia/water (10/10/5/2,, v/v/v/v) annd once in tthe second

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dimensiion with isoopropanol/w water/formicc acid (20/5/1, v/v/v). B Between ruuns, plates w were dried

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over nigght at room temperaturee. Amino accid standardds were visualized usingg ninhydrinee, whereas

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the raddioactive sppots on pllates were analysed uusing a BioImager B BAS-1000 (Fujifilm,

167

Düsselddorf, Germaany). Radiooactive spotts were scrraped off, eextracted w with MeOH:AAc and

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analysedd by LC--MS/MS inn combinaation with radioanalyytical quanntification or, after

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derivatisation, by GC-MS. G

170

a of presumed p am mino acids iisolated by T TLC separaation Derivvatization annd GC-MS analysis



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The MeOH:AAc solutions containing amino acids were derivatised with ethylchloroformate

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according to Husek.22-24 Subsequent analysis by GC-Electron-Impact-MS was carried out on an

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Agilent 6890N gas chromatograph coupled to an Agilent 6873 mass spectrometer. Separation

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was done with Optima 35 MS column (30 m length x 0.25 mm I.D. 0.25 µm film thickness)

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purchased from Macherey-Nagel (Düren, Germany). Further information is given as Supporting

176

Information.

177

LC-MS and LC-MS/MS of amino acids

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The radioactive spots of valine, phenylalanine, leucine, isoleucine, alanine (identified by GC-

179

MS) and the observed bromoxynil degradate produced under the hydrolysis conditions were also

180

analysed by LC-MS/MS.

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The chromatographic separation was performed with a Phenomenex HPLC column Synergi RP

182

Hydro, 4 µm 150x2 mm (Phenomenex, Aschaffenburg, Germany).

183

The LC-MS/MS System consisted of an Agilent 1290 HPLC, (Agilent Technologies,

184

Waldbronn, Germany), linked to a Q-Exactive Plus Orbitrap mass spectrometer, (Thermo, San

185

Jose, CA, U.S.A). Timebased fractions were taken additionally into solid scintillation plates

186

(Luma Plate 384, Perkin Elmer, Waltham, MA, USA) using a micro fraction collector (Sun

187

Collect, Sun Chrom, Friedrichsdorf, Germany). Fractions of 10 µl, corresponding to 3 seconds of

188

the effluent, were collected. Luma Plates were measured using a MicroBeta2 plate counter

189

(Perkin Elmer, Waltham, MA, USA). The obtained histograms were used for qualitative

190

evaluation. For more details see Supporting Information.

191

RESULTS

192

General turnover mass balance of 14C- bromoxynil in soil


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Bromoxynil was chosen as a test substance because of its known fast mineralization and fast

194

formation of high amounts of NER.3, 17 This behaviour was investigated in a preceding fate study

195

as shown in Figure 1: mineralization was fast but reached only about 20% of applied

196

radioactivity after 50 days. After one week of incubation, 10.9 ± 0.5% of applied radioactivity

197

(AR) was mineralized, 12.5 ± 1.6% corresponded to extractable fraction and 70.2 ± 1.3%

198

remained as NER. Until the end of 56 days of incubation the amounts of NER remained constant

199

(70.8 ± 2.2% of AR), whereas the extractable counterparts decreased rapidly after 14 days

200

reaching ultimately 4.1 ± 0.6% on day 56. At the end, 19.0 ± 0.7% of the applied radioactivity

201

was mineralised. The kinetics of dissipation can be described best by DFOP (double first order in

202

parallel).25 This finding was confirmed in a second fate study comprising of more samples and

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with short incubation periods (data not shown). The DT50 of bromoxynil calculated from all data

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determined by means of DFOP kinetics was 2.0 days ( 1.8 days). % of AR 100 80 60 40 20 0 0

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20

40

60

Incubation period (days)

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Figure 1. Fate of bromoxynil in soil in percentage of the applied radioactivity (AR).

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Extractable radioactivity,

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radioactivity: sum of 14CO2, NER and extractable radioactivity.

radioactivity mineralised to 14CO2,

NER,

recovery of



209

14

210

After 1 day of ccultivation on sucrose and

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C-am mino acids method m devvelopment based on the single-cultuured Rhw 14

C-gllucose, the Rhw culturre was exam mined for

211

radioacttivity and suubjected to hydrolysis. The purifieed eluate (caation exchannge chromaatography)

212

of the am mino acids (AA eluate)) contained 50% (15 KBq) K of the radioactivity r y that was iintroduced

213

into the hydrolysis procedure and was annalysed by ttwo-dimensional radio--TLC analyysis. As an

214

examplee, the TLC aanalysis dissplaying thee distributionn of 23 radiioactive spoots is shownn in Figure

215

3. Comppared to a ccorrespondinng TLC anaalysis with non-labelled AA as reference stanndards, 12

216

(out of 223) radioacttivity spots were identiffied by TLC C co-chromaatography, bby their masss spectra,

217

and GC C-retention times. t As ann example, a GC-MS cchromatograam and the correspondding mass-

218

spectra of proline are given aas Supportinng Informaation (Figurres S1 and S2). The other spots

219

(Figure 2) could nnot be identiified. Trypttophane, cysteine and methionine are degradded during

220

hydrolyysis. Asparaagine and glutamine, reespectively,, were hydrrolyzed durring this treeatment to

221

aspartic acid and gllutamic acidd, and thus, appeared inn these fractiions.

222


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Figure 2. Two-dimensional radio-TLC analysis of the AA eluate resulting from a culture of

224

Rhodococcus wratislavensis grown on

225

(unlabelled) sucrose. TLC conditions and identification see Material and Methods. Asp aspartic

226

acid, Glu glutamic acid, Gly glycine, Ser serine, Ala alanine, Pro proline, Thr threonine, Tyr

227

tyrosine, Val valine, Phe phenylalanine, Leu leucine and Ile isoleucine.

14

C-glucose as carbon and energy source besides

228

14

C-amino acids analyses in soil incubated with 14C-bromoxynil

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Based on the results of the fate studies, incubation periods of 14, 28 and 56 days were selected

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for the subsequent experiment targeting the quantitative analysis of radiolabelled AA presumably

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formed during the incubation with 14C-bromoxynil. Both, the extracted and the non-extracted soil

232

were hydrolysed and purified.

233

After 14, 28 and 56 days of bromoxynil incubation, the soils contained 94, 77 and 47%,

234

respectively, of the applied radioactivity (SBE, soil before extraction). With regard to hydrolytic

235

treatment of non-extracted and Soxhlet extracted soil, Table 1 summarizes percentages of

236

radioactivity found in fractions a) from the amino acid eluate (AA eluate) of the cation exchange

237

column derived from non-extracted soil (ESBE, containing both extractable and non-extractable

238

residues), b) from the Soxhlet extracted soil (NER), and c) from the corresponding eluate of

239

cation exchanger chromatography of extracted soil (EExS), b) and c) containing non-extractable

240

residues only. In the course of incubation, percentages of radioactivity detected in the AA eluate

241

of non-extracted soil decreased from 49.1% to 21.1% of the radioactivity present in soil before

242

extraction, this correspond to 46.2% to 9.9% of the applied radioactivity. In contrast, the

243

radioactive fraction of the eluate of the extracted soil (EExS) remained quite constant (6.0% after

244

14 days, 11.3% after 28 days and 7.1% of applied radioactivity after 56 days). These amounts

245

correspond to 23.7%, 23.5%, and 17.1% of the NER fraction, respectively. This indicates clearly



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246

that with increasing incubation period, the amount of radioactivity found in the eluate of not

247

extracted soil became less extractable (under Soxhlet conditions); after 56 days the difference of

248

the portions of 14C in the eluate between non-extracted (ESBE) and extracted soil (EExS) was only

249

3% of the applied radioactivity. The amino acid hydrolysate may in addition also contain other

250

biogenic and xenobiotic residues that bind to the cation exchanger during purification. Our

251

analyses, however, focussed only on the amino acids formed.

252 253

Table 1. Radioactivity detected in the AA eluates of non-extracted (ESBE) and extracted (EExS)

254

soil, as well as in the Soxhlet extracted soil (NER). % of ARa in fraction

% of SBEc Incubation days

Incubation days 14 days

28 days

56 days

14 days

28 days

56 days

a) ESBE

46.2

16.4

9.9

49.1

21.3

21.1

b) NER

25.3

48.1

41.8

26.9

62.5

89.0

c) EExS b

6.0 (23.7)

11.3 (23.5)

7.1 (17.1)

6.4

14.7

15.2

255

a

AR, applied radioactivity

256

b

Percentages in brackets (EExS) are based on amounts of radioactivity found as NER.

257

c

SBE, radioactivity in soil before extraction

258

The AA eluates of all samples were analysed by two-dimensional TLC. As an example, the

259

TLC analysis of the sample derived from the AA eluate of Soxhlet-extracted soil incubated for

260

56 days (EExS) is shown in Figure 3. Besides radioactivity detected at the start point (R1, 6.9% of

261

14

262

the extracted soils (in % of total radioactivity on plates) are given as Supporting Information,

263

Table S1.

C on the plate), Figure 3 shows 15 radioactive spots. The percentages of the separated AA of


Page 15 of 27


264

265 266

mensional radio-TLC annalysis of thhe AA eluatee derived from Soxhlett-extracted Figure 3. Two dim

267

soil (lefft) and not eextracted soiil (right) inccubated for 56 days. Phhenylalaninee (R14), valine (R13),

268

leucine and isoleuucine (R155), proline (R10), alaanine (R9),, glutamic acid (R3) and the

269

bromoxxynil-byprodduct (R16, formed f undder the hyddrolytic condditions) weere identifieed by LC-

270

S, the other spots were analysed bby GC-MS: R1 Start, R R2 aspartic acid, R4 unnidentified MS/MS

271

and gluttamic acid, R5 unidenttified, R6 gllycine, R7 unidentified u d, R8 serinee, R11 threoonine, R12

272

tyrosinee, R17 unideentified.

273

Durinng the time course of inncubation thhe radioactiivity of a byyproduct off bromoxyniil (formed

274

during hhydrolysis, spot R16 inn Figure 3, lleft) decreaased rapidly from 44.3% % of the raddioactivity

275

in the AA A eluate of the extrracted soil after 14 ddays of incuubation to 6.9% after 56 days,

276

correspoonding to 22.7% and 0.5% 0 of thee applied raadioactivityy, respectiveely. Due too this, the

277

relative portions off the assumed AA and the startingg area increeased. Spot R7, correspponding to

278

S or LC-MS S/MS. The lysine bby means off co-chromaatography ccould not bee identified by GC-MS



Page 16 of 27

279

acidic hydrolysis led to a chemical modification of bromoxynil, which was still present in the

280

soil after incubation and after exhaustive extraction (Soxhlet). This byproduct (containing one or

281

more products) was subsequently also found to some extent in the AA eluates. Preliminary

282

experiments showed that after applying

283

hydrolysis, about 42% of applied 14C was found in the corresponding AA eluate, corresponding

284

to this byproduct of bromoxynil. Radio-TLC and -HPLC analysis of the eluate demonstrated that

285

the entire radioactivity of spot R16 co-chromatographed with bromoxynil acid. Using LC-MS

286

(negative mode), a weak signal of bromoxynil acid was detected (m/z = 294.84294 (100%),

287

292.84534 and 296.84110 (65 and 71%); identified by the corresponding reference compound).

288

Further signals of any bromoxynil byproducts, formed by acidic hydrolysis, were not detected.

289

We conclude that especially the AA eluates of the non-extracted soil samples contained the

290

hydrolytic reaction products of bromoxynil, unlike in the extracted soils in which most of the

291

bromoxynil has been removed.

14

C-bromoxynil to soil followed by immediate

292

Some radioactive TLC-spots were further identified by LC-MS/MS in combination with radio

293

analysis (proline, alanine, phenylalanine, valine and leucine/isoleucine). The masses of this AA,

294

measured in positive mode and in negative mode and the main fragment of the MS/MS

295

experiment in positive mode are given as Supporting Information, Table S2. As an example,

296

Figure 4 shows the chromatogram of spot R15 (leucine/isoleucine) for m/z 132 – 133. The two

297

corresponding peaks are not well separated (RT 4.17 and 4.47 min) and revealed m/z of

298

132.10193 in positive mode and 130.08589 in negative mode, respectively. MS/MS analysis of

299

the m/z 132 signal (positive mode) resulted in one fragment, m/z 86.09695 assumed to represent

300

the loss of formic acid (HCOOH) from the carboxyl group of the AA. For further

301

characterization the samples were fractionated and the radioactivity of these fractions was


Page 17 of 27


302

matogram of spot R15 iss shown in measureed using a pplate counterr. As an exaample, the rradio chrom

303

Figure 44. Two incoompletely seeparated peaks of radiooactivity occur at retenntion times of o 3.9 and

304

4.1 min.

A

B

  305 306

Figure 4. LC-MS//MS-chrom matogram (A A) and radioo HPLC anaalysis of 2D D-TLC spott R15 (B),

307

leucine and isoleucine (see Figgure 3).

308

Quanttitation of thhe total 14C--biogenic reesidues from m 14C- bromooxynil in sooil

309

On thhe TLC plaates, the sppots of leuccine/isoleuccine and phhenylalaninee/valine weere clearly

310

separateed from inteerfering com mpounds annd they madde up nearlyy 20% of thhe AA in sooils. Thus,

311

both couuples were suitable to calculate thhe total amoount of labeelled aminoo acids in thhe sample.

312

The relaative distribution of am mino acids inn soil is quitte constant, even for veery differentt soils and

313

for diffeerent absoluute contentss of AA. Frriedel and S Scheller meaasured in 8 soils 9.5 ± 0.6% for

314

phenylaalanine + vaaline and 9.8 ± 0.6% fo for leucine + isoleucinee, each from m hydrolysaable AA.26

315

Therefoore the relatiive distributtion of the AA A on the T TLC plate annd the conteent of radioactivity in

316

the eluaate was usedd to calculatte the perceentage of radiolabelled AA in relattion to the amount a of

317

NER orr in relation to the amoount of appllied radioactivity. The contents off phenylalannine/valine

318

from ann extracted ssoil was calcculated usinng



319

% %

, ,

∗

%

Page 18 of 27

(1) 14

320

with %(P,V in eluate) as relative content of

321

soil) as relative percentage of hydrolysable phenylalanine + valine in soil (according to Friedel

322

and Scheller26), EExS as radioactivity in the eluate in relation to the amount of NER in percent and

323

%AANER as percentage of NER composed of AA. This procedure was repeated with

324

leucine/isoleucine and the results were averaged. Table 2 gives the content of labelled AA from

325

extracted and not extracted soil relative to applied radioactivity; in addition, the content of

326

labelled AA of extracted soils are given relative to NER.

327

Table 2. Percentages of labelled P+V (phenylalanine + valine) and L+I (leucine + isoleucine)

328

obtained from TLC (% on TLC) and calculated total amino acids in relation to the amount of

329

NER and to the applied radioactivity (AR) using formula (1). P+Va

L+Ib

14d NER % on TLC

5,4

5.9

% of NER

13.5

14.3

13.9

3.4

3.6

3.5

28d NER % on TLC

7.8

8.9

% of NER

19.3

21.4

20.3

9.3

10.3

9.8

56d NER % on TLC

8.0

8.4

% of NER

14.4

14.7

14.5

% of AR

6.1

6.2

6.1

% on TLC

5.3

6.0

% of AR

5.6

6.1

% of AR

% of AR

56d SBE

C phenylalanine + valine in the eluate, %(P,V in

Mean

5.9


Page 19 of 27

330 331


a

P+V phenylalanine + valine, 9.5% of hydrolysable AA in soil according to Friedel and Scheller.26

332

b

L+I leucine + isoleucine, 9.8% of hydrolysable AA in soil (dito).26

333

c

SBE = Soil before extraction

334

It is apparent that amounts of radiolabelled AA did not increase continuously with incubation

335

period but showed a maximum after 28 days (9.8% of AR and 20.3% of NER). After 56 days of

336

incubation, portions of radiolabelled AA in both, extracted and not extracted soil amounted to

337

6% of the applied radioactivity. This means that in exhaustively extracted soil, 14.5% of the not

338

extracted radioactivity was incorporated into AA.

339

To calculate the total amount of anabolized bioNER we use the fact, that 55% of the

340

anabolized biomass of microorganisms represent proteins and 45% other biomolecules. Thus, the

341

percentages of Table 2 have to be multiplied by a factor 1.82 (= 0.55-1) to consider the complete

342

radiolabelled anabolized bioNER. This results in bioNER amounts of 25.3%, 36.9% and 26.4%

343

with respect to the NER amounts in extracted soils after 14, 28 and 56 days, respectively, and

344

6.4%, 17.8%, 11.1% and 10.2% with respect to the applied radioactivity in extracted soils after

345

14, 28 and 56 days and after 56 days of not extracted soil, respectively.

346

DISCUSSION

347

Bromoxynil rapidly dissipated in soil with a DT50 of 2.0 days comparable to dissipation times

348

already reported: Zablotowicz27 determined half-lives for bromoxynil dissipation at either 2 or 10

349

mg kg-1 of bromoxynil of less than 1 day. Other authors found a 50% reduction of the

350

concentration after approx. 7 days at concentrations of 10 and at 50 mg kg-1.20 However, for the

351

second and third application at high rates (50 mg kg-1), DT50 increased to 19 and 28 days,

352

respectively, probably because of the toxic impact of the high concentrations on biodegrading

353

microorganisms. After several days of our incubation experiment only small amounts of the



Page 20 of 27

354

parent compound or metabolites are extractable, similar to findings by Smith et al.28,

355

Zablotowicz et al.27 and

356

amounts of NER in soil: around 70% of the applied radioactivity between 7 and 56 days in our

357

fate experiment and 42% after 56 days in the hydrolysis experiment. Similar or even higher NER

358

formation was reported before.17, 27, 29 In our study, a fast decline in extractable 14C residues in

359

the beginning corresponded to a concurrent increase in non-extractable

360

high initial mineralisation. Afterwards NER slowly declined by mineralisation, indicating that

361

parts are becoming bioavailable. The rate of mineralisation is correlated to microbial activity and

362

therefore to water content and temperature28 and to the concentration of the compound20, 27, 30 as

363

well as the content of SOM.27

Baxter and Cummings20. We determined, correspondingly, high

14

C-residues27 and to a

364

We separated and quantified the relative distribution of radiolabelled amino acids formed

365

during the metabolism of bromoxynil. In principle, there are two possible ways to calculate the

366

total amount of radiolabelled amino acids in soil. One possibility is to sum up all radiolabelled

367

spots of amino acids separated by TLC. However, although using two-dimensional separation of

368

the cation exchange eluate, it is possible that unknown compounds co-chromatograph with

369

amino acids and remain undetected. We calculated the contents of amino acids using four amino

370

acids, i.e., leucine, isoleucine, valine and phenylalanine, which all show high recoveries of 82 –

371

86% during hydrolysis and all purification steps (data are given as Supporting Information, Table

372

S3). Retention factors of these spots were about 0.5 and they are well separated from other AAs

373

and possible impurities. Some studies have analysed the pattern of AA in acid hydrolysates of

374

arable, fen and forest soils as well as soil from grassland and barren land. Extensive

375

investigations were carried out by Bremner31 (10 soils), Gupta and Reuszer32 (9 soils), Senwo

376

and Tabatabai33 (10 soils) as well as Friedel and Scheller26 (8 soils). Important for our calculation


Page 21 of 27


377

reasoning, all these studies described a similar AA pattern. The results of Bremner and Gupta /

378

Reuszer are not completely comparable with those using current analytical methods. Senwo and

379

Tabatabai33 as well as Friedel and Scheller26 quantified 15 and 16 AA, respectively. Senwo and

380

Tabatabai33 found slightly higher relative amounts of leucine + isoleucine (10.8 ± 0.5%,

381

compared to 9.8 ± 0.6%) and lower amounts of phenylalanine + valine (8.1 ± 0.6% compared to

382

9.5 ± 0.6%), however the differences are minor.

383

Calculation of the total amount of AAs which were obtained in the eluate shows comparable

384

results. As an example, for extracted soil after 56 days of incubation the obtained results are

385

84.0% and 82.2% of the radioactivity of the eluate using the AA distribution of Senwo &

386

Tabatabai33 and Friedel & Scheller,26 respectively. We decided using the latter results, because

387

like in our study Friedel and Scheller26 hydrolyzed the whole soil. In contrast, Senwo and

388

Tabatabai hydrolyzed pre-extracted soil organic matter from soil.

389

Microbial biomass contains about 55% of protein of the dry weight of bacterial cells.14

390

Therefore we can assume that about 55% of the anabolized radiolabeled C was converted into to

391

amino acids and 45% of the radiolabel were anabolized into other microbial compounds like

392

fatty acids and DNA. If we extrapolate the amount of labelled AA based on the content of

393

protein in microbial cells, after 56 days of incubation a minimum of 26% of the initial measured

394

NER in the sample has become part of the microbial biomass derived SOM. Studies with stable

395

isotopes (13C) and high resolution GC-MS analyses showed that after incubation with the readily

396

biodegradable 2,4-D 44% of the initially added label were found in bioNER, i.e., making up

397

nearly all of the NER.8, 15 Similar studies with 13C-ibuprofen in soil showed that 2% of the label

398

was integrated into proteins, corresponding to a contribution of 54% of bioNER to NER.9 The

399

differences indicate that the relative importance of bioNER formation may be substance-specific:



Page 22 of 27

400

readily biodegradable substances like 2,4-D show high mineralization rates (almost 60% of the

401

applied isotope label after 64 days) and high amounts of bioNER (44%) as compared to less

402

biodegradable substances like bromoxynil with lower mineralization and correspondingly lower

403

amounts of bioNER.

404

We are aware that also xenobiotic degradation products can be accumulated in microbial cells

405

due to binding to structural components and natural polymers.16 When labelled with radioactive

406

isotopes the measured radioactivity of the microbially derived NER may comprise both biogenic

407

NER and xenobiotic derivatives. However in the present paper we focussed on amino acid

408

analysis: because 55 % of the anabolized biomass represents proteins, our conversion to 100%

409

considers only anabolized biogenic biomass. .

410

Proteins were shown to be very stable in the soil environment, a phenomenon which cannot be

411

explained by their chemical structure; rather their persistence is due to the adsorption to soil

412

components .34 Similarly, the mean residence time of hexoxes and pentoses in soil can be around

413

20 years, for proteins even around 40 years.34-36 The decrease of radioactive AA in the time

414

course of incubation (9.8% of the applied radioactivity after 28 days and 6.1% after 56 days)

415

might be explained by the biodegradability of AA freshly formed in the soil environment.

416

Accordingly, a decrease in the amounts of 13C-AA extracted from living biomass was observed

417

by Nowak et al. (2010); however, the total amount of labelled AA including those of non-living

418

SOM did not decrease during the incubation period of about nine weeks.8

419

BioNER formation can be expected for substances with a rapid mineralization and a high

420

formation rate of NER. Otherwise, xenobiotics may be degraded co-metabolically without

421

productive formation of microbial biomass leading only to minor amounts of bioNER.4 The

422

formation of NER has been considered as a process of irreversible binding to soil for decades;


Page 23 of 27


423

such residues were defined as not bioavailable. Thus, xenobiotic NER formation, i.e., entrapment

424

of residues in the voids of the organic and inorganic soil matrix,(type I), and covalent binding of

425

residues to humic material (type II) may be considered as long-term stabilization of a chemical in

426

soil. However, for the environmental risk assessment of NER the potential subsequent release of

427

non-extractable parent substances and xenobiotic metabolites especially from type I NER, should

428

be taken into account as recently discussed by Kästner et al. (2014).4 On the other hand, biogenic

429

(type III) NER, quantifiable for the first time in the here described radiolabelling method, are of

430

no environmental concern.

431

ASSOCIATED CONTENT

432

Supporting Information. Additional information comprises the methods for GC-MS and LC-

433

MS/MS analysis of amino acids, GC-MS chromatogram and mass spectra of proline, detailed

434

results of the Radio-TLC analysis of the AA eluates, data of the recovery of four AA during

435

hydrolysis and purification as well as results of LC-MS/MS analyses of amino acids. This

436

material is available free of charge via the Internet at http://pubs.acs.org.

437

AUTHOR INFORMATION

438

Corresponding Author

439

* E-mail: [email protected].

440

Tel.: 00492418026678. Fax: 00492418022182

441

ACKNOWLEDGMENT

442

We gratefully acknowledge financial support from Bayer Crop Science Division, Monheim,

443

Germany, and in addition Mrs. Stefanie Schiecke for technical assistance (LC-MS analyses). We



Page 24 of 27

444

appreciate valuable discussions with Matthias Kästner and Anja Miltner (Helmholtz-Centre for

445

Environmental Research – UFZ, Leipzig, Germany).

446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485

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14. Madigan, M. T.; Martinko, J. M.; Parker, J., Brock Biology of Microorganisms. Pearson: San Francisco, CA, USA, 2000. 15. Girardi, C.; Nowak, K. M.; Carranza-Diaz, O.; Lewkow, B.; Miltner, A.; Gehre, M.; Schäffer, A.; Kästner, M., Microbial degradation of the pharmaceutical ibuprofen and the herbicide 2, 4-D in water and soil—Use and limits of data obtained from aqueous systems for predicting their fate in soil. Science of the Total Environment 2013, 444, 32-42. 16. Yassir, A.; Lagacherie, B.; Houot, S.; Soulas, G., Microbial aspects of atrazine biodegradation in relation to history of soil treatment. Pesticide Science 1999, 55, (8), 799-809. 17. Rosenbrock, P.; Munch, J. C.; Scheunert, I.; Dörfler, U., Biodegradation of the herbicide bromoxynil and its plant cell wall bound residues in an agricultural soil. Pesticide Biochemistry and Physiology 2004, 78, (1), 49-57. 18. Alef, K., Methodenhandbuch Bodenmikrobiologie. Aktivitaeten, Biomasse, Differenzierung. 1991. 19. Jaquet, J.; Weitzel, P.; Junge, T.; Schmidt, B., Metabolic fate of the 14C-labeled herbicide clodinafop-propargyl in soil. Journal of Environmental Science and Health, Part B 2014, 49, (4), 245-254. 20. Baxter, J.; Cummings, S. P., The degradation of the herbicide bromoxynil and its impact on bacterial diversity in a top soil. Journal of applied microbiology 2008, 104, (6), 1605-16. 21. Pillay, D.; Mehdi, R., Separation of amino acids by thin-layer chromatography. Journal of Chromatography A 1970, 47, 119-123. 22. Hušek, P., Chloroformates in gas chromatography as general purpose derivatizing agents. Journal of Chromatography B: Biomedical Sciences and Applications 1998, 717, (1), 57-91. 23. Hušek, P., Rapid derivatization and gas chromatographic determination of amino acids. Journal of Chromatography A 1991, 552, 289-299. 24. Chen, W.-P.; Yang, X.-Y.; Hegeman, A. D.; Gray, W. M.; Cohen, J. D., Microscale analysis of amino acids using gas chromatography–mass spectrometry after methyl chloroformate derivatization. Journal of Chromatography B 2010, 878, (24), 2199-2208. 25. Boesten, J.; Aden, K.; Beigel, C.; Beulke, S.; Dust, M.; Dyson, J.; Fomsgaard, I.; Jones, R.; Karlsson, S.; van der Linden, A., Guidance document on estimating persistence and degradation kinetics from environmental fate studies on pesticides in EU registration. Report of the FOCUS Work Group on Degradation Kinetics, EC Doc. Ref. Sanco/10058/2005, version 2005, 1. 26. Friedel, J. K.; Scheller, E., Composition of hydrolysable amino acids in soil organic matter and soil microbial biomass. Soil Biology and Biochemistry 2002, 34, (3), 315-325. 27. Zablotowicz, R. M.; Krutz, L. J.; Accinelli, C.; Reddy, K. N., Bromoxynil degradation in a Mississippi silt loam soil. Pest management science 2009, 65, (6), 658-64. 28. Smith, A. E., Degradation of Bromoxynil in Regina Heavy Clay. Weed Research 1971, 11, (4), 276-282. 29. Review report for the active substance bromoxynil - final. In European Commission, H. a. C. P., Ed. 2004. 30. Holtze, M. S.; Sorensen, S. R.; Sorensen, J.; Aamand, J., Microbial degradation of the benzonitrile herbicides dichlobenil, bromoxynil and ioxynil in soil and subsurface environments-insights into degradation pathways, persistent metabolites and involved degrader organisms. Environmental pollution 2008, 154, (2), 155-68. 31. Bremner, J., The amino-acid composition of the protein material in soil. Biochemical Journal 1950, 47, (5), 538.



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Page 27 of 27


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