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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
Environmental Science & Technology
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Quantitative identification of biogenic non-
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extractable pesticide residues in soil by 14C-analysis
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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
10
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,
15
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
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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
19
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
21
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
23
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).
27
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
31
by bioNER and thus is not environmentally relevant.
32
INTRODUCTION
33
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
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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
42
A variety of chromatographic, spectroscopic and spectrometric analyses have been applied to
43
study the nature of NER. In a recent review, these attempts have been summarized.4 As a
44
conclusion, NER can be differentiated into xenobiotic residues, either entrapped (type I NER) in
45
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
47
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
49
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,
51
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
53
proteins, protein-humic complexes or peptides.13 In microbial biomass, AA are the most
54
abundant components: microbial biomass contains about 55% of proteins of the dry weight of
55
bacterial cells.14
56
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.
59
Rather, such experiments, in which quantitative recovery of the fate of pesticides are needed,
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rely on the use of radiotracers (14C) but the method for ready 14C-analyses of biogenic residues is
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missing.
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Environmental Science & Technology
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
66
negligible low natural background and is therefore detectable in very low concentrations in
67
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
71
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
74
glucose as a substrate. The objective of the present investigation was to elaborate and establish a
75
method for ready
76
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
79
[UL-Ring14C-]-3,5-dibromo-4-benzonitrile ([14C]-bromoxynil; radiochemical purity >98%,
80
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
82
Bayer Crop Science Division (Monheim, Germany). Uniformly labelled AA (valine,
83
phenylalanine, leucine and isoleucin, radiochemical purity > 98.7%, specific radioactivity 10 to
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17 MBq µmol-1) were obtained from Hartmann Analytic, Germany. Non-labelled amino acids of
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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
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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).
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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)
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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,
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were developed using a bacterial culture of Rhodococcus wratislaviensis (Rhw) cultivated in a
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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
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prepared with DifcoTM Czapek Dox (Nordwald, Hamburg, Germany) medium in a 2 ml
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centrifugation tube and pre-incubated at 28°C. After one day of pre-incubation (during log-
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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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culture was frozen to stop the incubation. The cell suspension was thawed in a sonication bath
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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
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was examined for 14C by means of a liquid scintillation counting (Hidex 300 SL, Turku, Finland)
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and contained 15 % of the radioactivity (not analysed further). The pellet of cells and
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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,
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v/v) of bromoxynil (labelled and non-labelled) was applied to a 3 g soil aliquot resulting in a
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concentration of 20 µg g-1 soil (1 KBq). The concentration was chosen high enough to allow
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determination of bioNER and low enough to exert no toxic impact on microorganisms.17, 20 After
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evaporation of the ethanol at room temperature, the aliquot was stirred with a spatula, transferred
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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
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glass tube containing (15 g) soda lime for trapping 14CO2 and unlabelled CO2. The assays were
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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
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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).
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After evaporation of ethanol, the soil aliquot was stirred with a spatula and transferred to the
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incubation bottle and mixed with 9 g of fresh non-treated soil. The resulting spiked soil samples
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were then adjusted to 60% WHCmax and incubated for 14, 28 and 56 days in the dark. Soda lime
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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
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For both the fate study and the bioNERstudy, 1 g of the soil was subjected to combustion
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analysis in order to determine the total radioactivity remaining in the soil. For the bioNERstudy,
142
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
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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
150
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
156
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
161
Two ddimensionall TLC was eexecuted onn cellulose pplates (200 x 200 mm, 0.25 mm, MachereyM
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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
166
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
168
analysedd by LC--MS/MS inn combinaation with radioanalyytical quanntification or, after
169
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
174
was done with Optima 35 MS column (30 m length x 0.25 mm I.D. 0.25 µm film thickness)
175
purchased from Macherey-Nagel (Düren, Germany). Further information is given as Supporting
176
Information.
177
LC-MS and LC-MS/MS of amino acids
178
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.
181
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
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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
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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
203
with short incubation periods (data not shown). The DT50 of bromoxynil calculated from all data
204
determined by means of DFOP kinetics was 2.0 days ( 1.8 days). % of AR 100 80 60 40 20 0 0
205
20
40
60
Incubation period (days)
206
Figure 1. Fate of bromoxynil in soil in percentage of the applied radioactivity (AR).
207
Extractable radioactivity,
208
radioactivity: sum of 14CO2, NER and extractable radioactivity.
radioactivity mineralised to 14CO2,
NER,
recovery of
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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
229
Based on the results of the fate studies, incubation periods of 14, 28 and 56 days were selected
230
for the subsequent experiment targeting the quantitative analysis of radiolabelled AA presumably
231
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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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
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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
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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
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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
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319
% %
, ,
∗
%
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(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
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330 331
Environmental Science & Technology
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
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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
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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:
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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;
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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
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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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32. Gupta, U. C.; Reuszer, H. W., Effect of Plant Species on the Amino Acid Content and Nitrification of Soil Organic Matter. Soil Science 1967, 104, (6), 395-400. 33. Senwo, Z.; Tabatabai, M., Amino acid composition of soil organic matter. Biology and Fertility of Soils 1998, 26, (3), 235-242. 34. Schmidt, M. W.; Torn, M. S.; Abiven, S.; Dittmar, T.; Guggenberger, G.; Janssens, I. A.; Kleber, M.; Kogel-Knabner, I.; Lehmann, J.; Manning, D. A.; Nannipieri, P.; Rasse, D. P.; Weiner, S.; Trumbore, S. E., Persistence of soil organic matter as an ecosystem property. Nature 2011, 478, (7367), 49-56. 35. Stotzky, G., Persistence and biological activity in soil of the insecticidal proteins from Bacillus thuringiensis, especially from transgenic plants. Plant and soil 2005, 266, (1-2), 77-89. 36. Baldock, J.; Oades, J.; Vassallo, A.; Wilson, M., Incorporation of uniformly labeled 13C glucose carbon into the organic fraction of a soil-carbon balance and CP MAS 13C NMR Measurements. Soil Research 1989, 27, (4), 725-746.
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