PESTICIDES EXTRACTION METHODS

1. A method for the simultaneous extraction of seven pesticides from soil and sediment

Pesticide standards were purchased as solids, from Sigma-Aldrich with a purity of >98%. Compounds
used in the analysis were: 2,4,6,8-tetramethyl-1,3,5,7-tetraoxocanemetacetaldehyde (metaldehyde), 3-
(3-chloro-4-methylphenyl)-1,1-dimethylurea (chlorotoluron), 3-(4-isopropylphenyl)-1,1-dimethylurea
(isoproturon), (R,S)-2-(4-chloro-2-methylphenoxy)propanoic acid (mecoprop), 4-(4- chloro-2-
methylphenoxy)butanoic acid (MCPB), 1-chloro-3-ethylamino-5-isopropylamino-2,4,6-triazine (atrazine)
and 2-chloro-N- (2,6-dimethylphenyl)-N-(1H-pyrazol-1-ylmethyl)acetamide (metazachlor). Internal
standards d5-atrazine and methyl palmitate were purchased from LGC Standards and Sigma-Aldrich,
respectively. Stock pesticide standard solutions were prepared at a concentration of 1 mg ml1 using
methanol and refrigerated at <5 C for no more than one year. All pesticide standards in powder form
and trimethylsilyldiazomethane (TMSDAM) were stored at 20 C. Solvents (methanol, acetone, hexane
and toluene) used in the process were HPLC grade and purchased from Rathburn Chemicals Ltd.
TMSDAM and dimethyldichlorosilane (DMDCS)at 5% in toluene were purchased from Sigma-Aldrich

Experimental preparation Prior to extraction the soils were prepared for analysis as follows:
wet soil was frozen and dried using a Thermo Scienti c Heto PowerDry LL3000 freeze dryer for
up to 5 days depending upon soil moisture content. Soil was then ground using a pestle and
mortar and passed through a 2 mm sieve to remove stones, vegetation and other extraneous
objects. The resulting soil or sediment sample was then stored, frozen, in sealed glass vials prior
to extraction. Soil and sediment samples can be stored frozen for up 450 days without major
degradation.38 A silylation procedure was undertaken for all glassware used in the experiments
to prevent adsorption of compounds during the extraction and methylation processes thereby
maximising recovery of all compounds. All glassware was furnaced for 4 hours at 450 C and le
to cool before being treated. DMDCS in 5% toluene was applied to the glassware (including
pipette tips, vials and asks) for 2 minutes. The glassware was then double rinsed in toluene
and then methanol before being le in a fume cupboard to air dry overnight. Tests showed a
consistently higher average recovery for all compounds from silylated (81% 22) rather than
nonsilylated (45% 3) glassware used in the experiments. The results indicate that a considerable
quantity of pesticide is adsorbed to glassware if it is not deactivated before being used.
Extraction and methylation A solvent extraction was undertaken which enables the separation
of the bound constituents from the soil particles without chemically altering the target
compounds. Previous methods have used a range of individual or mixed solvent systems for
extraction including methanol, acetonitrile, acetone and sulfuric acid.37,39,40 In the method
reported herein, pesticide extraction was carried out by ultrasonication with methanol or
acetone as these solvents have been previously proven to efficiently extract pesticides from soil
particles. Approximately 2 g of soil was weighed into a 28 ml silylated vial and 8 ml of HPLC
grade solvent added. The vial was then sonicated for 20 minutes. Extracts were separated from
soil by centrifugation for 5 minutes at 3000 rpm and the solution removed using a silylated
glass pipette and stored in another silylated vial. The ultrasonication step was repeated twice
more resulting in a nal volume of solution of 24 ml. This was then dried under a gentle stream
of nitrogen for a minimum time period and stored ready for analysis.

Ultrasonic extraction uses a lower volume of solvent than Soxhlet extractions and, although it is
not as exhaustive a process, it is substantially less costly.13 Pesticides are also less likely to
degrade due to the lower temperatures used. Extraction tests using Soxhlet apparatus and
rotary evaporation showed high losses of the more volatile compounds (average loss:
metaldehyde 33%, chlorotoluron 30%, mecoprop 18%, atrazine 43%, MCPB 35%, isoproturon
21% and metazachlor 3%). These losses could have occurred due to the high temperatures and
long time period of extraction using Soxhlet extractors and, subsequently, rotary evaporation at
the end of the procedure to remove large volumes (250 ml) of solvent. Tests between drying
using rotary evaporation and nitrogen blow-down apparatus showed losses were on average
5% less using the nitrogen blow-down. A er extraction a test was conducted whereby the
samples were subjected to a clean-up procedure using Supelclean ENVI-Carb SPE tubes. These
were used to increase pesticide recovery by avoiding losses using drying techniques alone. The
tests showed percentage recovery to range between 76% and 106% which was similar to those
not subjected to this procedure (70% to 114% using acetone). In addition, the chlorotoluron
was not detected on the GC-MS a er the SPE phase. Therefore this was deemed an
unnecessary step and abandoned, thereby reducing overall cost and time for analysis.

2. Development of a simple extraction and clean-up procedure for determination of

organochlorine pesticides in soil using gas chromatography–tandem mass spectrometry

Chemical and reagents - Certified pesticide reference standards of high purity (>98%) were
purchased from Qmx (Thaxted, UK) and LGC-Promochem (Teddington, UK). Acetonitrile and
water (HPLC grade) and nhexane (analytical reagent grade) were purchased from Fisher
Scientific (Loughborough, UK). Anhydrous magnesium sulfate and sodium acetate tri-hydrate
(analytical reagent grade) were purchased from York Glassware (York, UK). Individual stock
solutions for 19 OC pesticides were prepared in n-hexane at 1000 g ml−1. The working standard
solutions containing each of the 19 OCs were prepared in n-hexane at 10, 1.0 and 0.1 g ml−1.
The working standard solutions were used for spiking blank soil samples and for preparation of
matrix matched calibration solutions.

Soil samples - Pesticide free soil sample were collected from various parts of the UK for use
during method development and validation (samples 1–5). These samples were tested to show
absence of detectable OC residues (Fig. 1). Another six samples (samples 6–11) were collected
from different cotton growing regions of Pakistan with a history of widespread use of OC
pesticides. These samples were used to compare the proposed procedure (Section 2.8) with an
established procedure based on Soxtec extraction (US-EPA method 3541). The Soxtec
procedure involved extraction of these samples with methylene chloride using Tecator-HT2
(Tecator, Sweden). The extraction using Soxtec procedure involved boiling, extraction and
rinsing steps of 1 h each. Physico-chemical properties of the samples used in the study are
given in Table 2. Soil pH was determined in soil + water suspension (1:1), particle distribution
was determined using hydrometer method and textural class was determined using USDA
triangle. Organic matter content was measured by reduction of potassium dichromate
(K2Cr2O7) by organic carbon and subsequent determination of unreduced dichromate by
titration with ferrous ammonium sulfate

Preparation of soil samples -Soil samples were air-dried at room temperature, mixed to
homogenize, ground using pestle and mortar, passed through a 2 mm sieve, and stored at room
temperature.

Sample fortification -Pesticide free soil was spiked following the principle described by Tor et
al. [6]. A sieved soil sample (5 g) was weighed into a 40 ml polypropylene centrifuge tube and
spiked at the required fortification level by adding an appropriate volume of mixed standard
solution containing 19 OC pesticides. After spiking, acetone (10 ml) was added and suspension
was mixed thoroughly to achieve homogeneity of pesticides in soil. The tubes containing the
fortified soil samples were left open in the fume hood for a minimum of 4 days to allow
evaporation of excess solvent and aging before extraction.

Extraction

Two extraction procedures based on the QuEChERS method were assessed using aliquots of a
blank soil sample (n = 10) fortified with OC pesticides. Fortification levels used for this
assessment were 50 and 200 g kg−1. The two procedures were identical with the exception of
the hydration step.

1. The first procedure involved hydration of soil sample (5 g) with water (10 ml) for 30 min
before extraction (Section 2.7.3)

2. The second procedure involved hydration of soil sample (5 g) with 1.0 M aqueous Na2-EDTA
solution (10 ml) for 30 min before extraction (Section 2.7.3).

3. An aliquot (10 ml) of acetonitrile + acetic acid mixture (99:1, v/v) was added to the centrifuge
tube containing the hydrated sample. After 30 s vortex, 4 g anhydrous MgSO4 (4.0 g) and
NaAc·3H2O (1.7 g) were added. The contents were shaken vigorously and then centrifuged at
5000 × g for 5 min. The centrifuge step facilitated the separation of acetonitrile from the
aqueous layer.
 Simultaneous extraction and clean-up - An aliquot (5 g) of soil sample was extracted as
described above in Sections 2.7.1 and 2.7.3. The simultaneous back-extraction and clean-up
involved transfer of an aliquot (8 ml) of the upper acetonitrile layer into a 10 ml glass test tube.
The extract was concentrated to approximately 1 ml under a gentle stream of nitrogen on a dry
block at 30 ◦C. The concentrated extract was mixed with water (1 ml) and n-hexane (5 ml) and
swirled on a vortex mixer for 15 s. The mixture was allowed to stand and an aliquot (4 ml) of
the upper n-hexane layer was transferred into another glass tube. After addition of 50 l of 10 g
ml−1 -HCH solution as internal standard, the n-hexane extract was concentrated to near
dryness under a gentle stream of nitrogen on a dry block at 30 ◦C. The extract was
quantitatively transferred to a 1 ml volumetric flask using n-hexane and made up to volume
with n-hexane, prior to GC–MS/MS analysis

3.Assessment of two extraction methods to determine pesticides in soils, sediments and

sludges. Applicationto the Túria River Basin.

Reagent- Standards of acetochlor, alachlor, atrazine, azinphos-ethyl, azinphos-methyl,

buprofezin, carbofuran, chlorfenvinphos, chlorpyriphos, deisopropylatrazine, deethylatrazine,
diazinon, dichlofenthion, dimethoate, diuron, ethion, fenitrothion, fenthion, fenthion-sulfoxide,
fenthion-sulfone, hexythiazox, 3-hydroxycarbofuran,imazalil, imidacloprid, isoproturon,
malathion, methiocarb, molinate, metolachlor, omethoate, parathion-ethyl, parathion-methyl,
prochloraz, propanil, propazine, pyriproxyfen, simazine, terbutryn, tolclophos-methyl were
purchased from Sigma-Aldrich (Steinheim, Germany) with a purity of at least 99%. Fenoxon,
fenoxon sulfoxide, fenoxon-sulfone were obtained from Dr. Ehrenstorfer (Augsburg,
Germany)as 1-mL acetonitrile solutions at 10 μg/mL.

For PLE procedure, Florisil® from Sigma-Aldrich, Silica gel from Scharlau (Barcelona Spain) and
acidic, neutral and basic alumina (Al 2 O3 ) from Merk (Darmstadt, Germany) were tested as
sorbents. Acetone, ethyl acetate, dichloromethane and acetonitrile were also obtained from
Merck.

For QuEChERS, MgSO4 , sodium acetate and sodium citrate dibasic sesquihydrate [HOC(COOH)
(CH2 COONa) 2 · 1.5 H2 O] were from Alfa Aesar GmbH & Co KG (Karlsruhe, Germany), NaCl,
trisodium citrate dehydrate (Na 3 C 6 H 5 O 7 · 2H2 O) and acetic acid from Prolabovwr (Leuven,
Belgium) and primary-secondary amine (PSA), graphitized black carbon (GBC) and C 18 from
AnálisisVínicos (Tomelloso,Spain)
Extraction procedures/Sample preparation

Pressurized Liquid Extraction

PLE was performed with an ASE 200 system. A sample of 1 g of lyophilized sample was weighed
and homogenized into a mortar using a pestle and then, was introduced into a stainless steel 11
mL extraction cell. The cell was prepared placing a Whatman glass fiber filters at the bottom of
the extraction cell to avoid the obstruction of the end caps by particles, and 5 g of acidic
alumina to clean-up the extract. Then, a glass filter was also place on the top, the cell was
closed and positioned in the PLE system connected to a four-bottle solvent controller. Nitrogen,
at a pressure of 10 bar, was supplied to assist the pneumatic system and to purge the
extraction cells. The extraction cells were preheated for 2 min. Then, the analytes were heat for
5 min and extracted using acetonitrile at 100 ºC and 1.500 psi for 7 minutes of static time, in
one cycle, at 100 % flush. Then, extraction cells were purged for 60 s with nitrogen to eliminate
any trace of extraction solvent. The total volume extract obtained under those conditions was
ca 10 mL. Approximately 1 mL of the extract was filtered through a 0.45 μm PTFE filter into the
autosampler vials for LC–MS/MS175analysis.

QuEChERS

1 g of lyophilized sample, which was weighed in a 50 mL falcon tube,and homogenized with 7.5
mL water and 10 mL acetonitrile. Then, MgSO4 (6 g), NaCl (1.5 g), tri-sodium citrate dehydrate
[Na 3 C 6 H 5 O7 + 2H 2 O (1.5 g)] and disodium hydrogen citrate sesquihydrate
[HOC(COOH)(CH2 COONa) 2 ·1.5H 2 O (0.75 g)] were added for phase-separation adjustment.
The mixture was agitated intensively in a vortex for 1 min and centrifuged at 3000 rpm for 5
min. An aliquot (1 mL) of the upper organic phase was cleaned up by dispersive solidphase
extraction (d-SPE) using PSA (50 mg), MgSO4 (150 mg) and C 18 (50 mg). This mixture was
shaken in a vortex for 1 min and centrifuged at 3000 rpm for 5 min. The supernatant was
filtered through a 0.45 μm PTFE filter and introduced in a vial for LC-MS/MS analysis

Conclusion The simultaneous extraction of 50 pesticides from soil, sediments and sludges

was difficult, because of their great structural variability. The efficiency and efficacy of
optimized QuEChERS demonstrated to be superior to PLE.

4. Ultrasonic solvent extraction of organochlorine pesticides from soil

Reagents, solvents and adsorbents

All chemicals used were of analytical grade. Single pesticide standard, -HCH, -HCH,
-HCH, -HCH, heptachlor,aldrin, o,p′-DDE, dieldrin, p,p′-DDE, p,p′-
DDT, methoxychlor, mirex, was from Promochem Co. Solvents used, acetone, n-hexane, ethyl
acetate and petroleum ether were from Merck Co. Alumina 90 active, neutral, [(0.063–0.200
mm), (70–230 mesh ASTM)], were also from Merck Co.

Preparation of spiked samples

Soil, which was not treated with any organochlorine pesticide collected .The texture of soil was
56.1% sand, 22.9% silt, 21.0% clay,organic matter: 1.68%, pH (0.01 M CaCl2 ): 7.1 and maximum
water capacity: 21.2%. Soil samples were sieved to <2 mm and stored at room temperature
until spiking procedure. Spiked soil samples were prepared by adding 200 uL of standard
mixture of pesticides (the concentration is 10 ng uL−1 for each compound) to 10 g of soil. This
spike level corresponds to 200 ug kg−1 .Then, 10 mL of acetone was added and suspension was
mixed for 30 min with a mechanical shaker. After the bulk of the solvent was evaporated at
room temperature, the samples were stored at 4 ◦C in stoppered conical flask for 3 days. Then,
the extractions were carried out

Extraction

Optimization of ultrasonic extraction

The recovery experiments were carried out for the determination of efficiency of the extraction
procedure. At the beginning of the experiments, the extraction efficiency of n-hexane, ethyl
acetate, acetone and a mixture of petroleum ether and acetone (1/1 v/v) was compared. For
that, 10 g of spiked soil was sonicated 20 min with 20 mL of solvents in an ultrasonic bath
(frequency 35 kHz, 320 W, Super RK 510, Sonorex, Bandelin, Germany). The extracts were
filtered by using Whatman filter paper. The filtrates were reduced to 2 mL with rotary evapora-
tor (Buchi B-160 Vacobox, Switzerland) at 40 ◦C and adjusted to exactly 1 mL with a gentle
nitrogen stream. The concentrated extract was transfered onto the clean-up column and
elution was performed as described in Section 2.4. The amount of extracted pesticides was
determined by GC-ECD and the recoveries (%) were calculated. In the second set of
experiments, the optimum volume of solvent, optimum sonication time and optimum
repetition of extraction were determined. These optimization experiments were carried out by
using of a mixture of petroleum ether and acetone (1/1 v/v), which gave the highest recovery
for the pesticides studied. In order to determine optimum volume of petroleum ether–acetone
mixture (1/1 v/v), 10 g of spiked soil was sonicated for 20 min with 10, 15, 20, 25, 30 and 35 Ml
of petroleum ether–acetone mixture. In order to determine the optimum sonication time, 10 g
of spiked soil was sonicated with 25 mL of petroleum ether–acetone mixture giving highest
recoveries for 10, 15, 20, 25, 30 min. The extraction of 10 g of spiked soil with 25 mL of
petroleum ether–acetone mixture (1/1 v/v) for 20 min was repeated four times. The cumulative
recovery was calculated by adding recoveries of compounds determined in each extract. Three
different fortification levels for soil (level 1,15 g kg−1 ; level 2, 45 g kg−1 ; level 3, 200 g kg−1)
were performed and fortified samples were stored at 4 ◦C for 3 days and extractions were
carried out according to optimum ultrasonic extraction conditions

Shake-flask extraction

Ten grams of real soil sample was suspended in 50 mL of petroleum ether–acetone mixture
(1/1 v/v) and shaken on a horizontal shaker (GFL Type 3020) for 3 h. The extract was filtered
and concentrated to exactly 1 mL by using rotary evaporator and nitrogen stream, respectively.
Then clean-up procedure and GC-ECD analysis were performed

Soxhlet extraction

Ten grams of real soil sample was weighed into the extraction thimble and extracted with 150
mL of petroleum ether–acetone mixture (1/1 v/v) for 4 h. The extract was filtered and
concentrated to exactly 1 mL, then clean-up and GC-ECD analyses were performed

Conclusion- The results obtained indicated that the ultrasonic solvent extraction method could
be efficiently applied in order to extract OCP from soils. Recoveries of pesticides from fortified
soil samples are over 92%, 88% and 91% for fortification level 1, 2 and 3, respectively, with the
relative standard deviations generally below 6%. ultrasonic solvent extraction is more rapid
than conventional shake-flask and soxhlet extraction methods

5. QuEChERS and solid phase extraction methods for the determination of energy crop
pesticides in soil, plant and runoff water matrices

Experimental

Chemicals and solvents- Standards of metazachlor (99.9% purity), quinmerac (98.2% purity),
oxyfluorfen (99.8% purity), quizalofop-p-ethyl (98.5% purity), α(±)-cypermethrin (99.8%, a
racemic mixture (1:1) of two stereo-isomers) and s-metolachlor (98.2%) were obtained from
Riedel-de-Haën (Pestanal) (Seelze, Germany). HPLC-grade acetonitrile (AcN), methanol (MeOH),
ethyl acetate (EtOAc) and water, anhydrous magnesium sulfate (MgSO 4 ), sodium chloride
(NaCl), sodium sulphate (Na 2 SO4 ), sodium citrate tribasic dehydrate and sodium citrate
dibasic sesquihydrate were purchased from Sigma–Aldrich (Steinheim, Germany). Primary
secondary amine (PSA, 40–60 μm in size), graphitised carbon black (GCB, 40–60 μm in size) and
octadecyl silica (C 18 , 40–60 μm in size) sorbents were supplied by Supelco (Bellefonte, PA,
USA). Phosphoric acid (H 3PO 4 ) and acetic acid (analytical grade) were from Merck
(Darmstadt, Germany). Oasis HLB solid phase cartridges (6 mL, 200 mg) were from Waters
(Mildford, MA, USA). Stock standard solutions of the pesticides (100 mg L−1 ) in acetonitrile
were prepared and stored at 4°C. Working standard solutions were prepared by dilution of the
corresponding stock standard solution with acetonitrile or matrix extract and stored at 4°C
Sampling and solid phase extraction of run-off water samples

The experimental field area was approximately 500 m2 and it was divided in two groups of six
plots each with a plot dimension of 4 × 10 m 2 . One group of plots was used for the cultivation
of energy crops, and the other was the group of control plots, where the pesticide was applied
without cultivation (bare soil plots). Two different slopes (1% and 5%) were formed in each
group. The three sides of the plots were protected by soil boundaries and metal sheet boarders
to a level of ~40 cm height, whereas in the fourth, a surface runoff collection reservoir was
established. Composite soil samples were taken as follows: each plot was divided in three
subplots and three soil cores (0–10 cm depth) were randomly taken and pooled together in
each subplot. Runoff water samples were collected, after every rainfall or irrigation event. For
sunflower cultivation, oxyfluorfen and quizalofop-p-ethyl were applied as a water emulsion of
the commercial EC formulations LAPAFARM 24 EC (24% w/v) and TARGA 5 EC (5% w/v) at the
rates of 233 g ha−1 and 125 g ha−1 , respectively. For oilseed rape cultivation, metazachlorand
quinmerac were applied together as a water emulsion of the commercial SC formulation
Butisan 40/10 SC (40% and 10% w/v) at a rate of 940 and 245 g ha−1 respectively. Α (±)-
cypermethrin was applied as a water emulsion of the commercial EC formulation FASTAC 10 EC
(10% w/v) at a rate of 15 g ha−1 . Runoff water samples were collected, after a rainfall event,
before any pesticide application, and filtered using 0.45 μm Millipore filters (Millipore, Bedford,
MA, USA) and for quinmerac analysis were acidified with H 3PO 4 at pH 3. Pesticide extraction
was carried out using Oasis HLB solid phase cartridges (6 mL, 200 mg). The cartridges were
conditioned with 5 mL EtOAc, 5 mL MeOH and 5 mL HPLC water (acidified with H 3 PO 4 at pH 3
for quinmerac analysis) at 1 mL min−1 . The water samples (0.5 L) were passed through the
cartridges under vacuum using Visiprep large volume samplers (Supelco, Bellefonte, PA, USA) at
a flow-rate of 10 mL min−1 . After that the cartridges were dried for approximately 30 min. The
pesticides were eluted with 3 × 4 mL of EtOAc (2 × 5 mL of acidified MeOH for quinmerac) at 1
mL min−1 [14]. The final extract was dried over anhydrous Na 2 SO 4 and evaporated to a final
volume of 0.5 mL under a gentle stream of N2 , finally it was transferred to an autosampler vial.

QuEChERS extraction for soil and plant tissue samples

Soil samples - The procedure followed in this work was based on QuEChERS method . Before
use, soil samples collected from the experimental field were homogenised, sieved (2 mm mesh)
and air-dried at room temperature. A 10 g portion of soil was weighed into a 50 mL
polypropylene tube, then 5 mL of HPLC water was added and the mixture was energetically
shaken for 1 min with a vortex device. After that 10 mL of AcN was added (acidified with acetic
acid 1%, v/v for quinmerac analysis) and the mixture was shaken for 1 min by hand and for 1
min with a vortex device. Four grams of anhydrous magnesium sulfate, 1 g sodium chloride, 1 g
sodium citrate tribasic dehydrate and 0.5 g sodium citrate dibasic sesquihydrate were added,
and the mixture was immediately hand-shaken for 30 s, sonicated for 5 min in an ultrasonic
bath from Bandelin and centrifuged for 5 min at 4000 rpm in a Sigma 2–5 centrifuge. Then, a
clean-up dispersive solid phase extraction step was carried out by adding the supernatant (7.5
mL, i.e. 1.33 g of soil per mL), to a 15 mL polypropylene tube that contained 1.125 g of MgSO 4
(150 mg MgSO 4 per mL of extract) and 0.225 g of C 18 (30 mg C18 per mL of extract), hand-
shaken for 30 s, sonicated for 1 min and centrifuged for 5 min at 4000 rpm .The final extract (5
mL) was evaporated to 0.5 mL under a gentle stream of N 2 and was transferred to an
autosampler vial

6. Extraction of Pesticides from Sediments Using a Microwave Technique

Experimental Standards - Analytical reference standards of the organochlorine pesticides were

obtained from the U.S,-EPA, Pesticides and Industrial Chemicals Repository, Research Triangle
Park, NC, USA and Ultra Scientific, North Kingstown, RI, USA. Purities were claimed to be
greater than 98 %. Stock solutions (1 mg/mL) of each pesticide were prepared in iso-octane.
Working calibration solutions were prepared in toluene by serial dilution of a composite stock
solution prepared from the individual stock solutions.

Preparation of Sediment Samples - The air-dried sediment samples (50.0 g oven-dried basis) in
500 mL square bottles were treated individually by pipeting onto the sediment surface 10 mL of
an acetone solution of the pesticide mixture. The bottle then was rotated to mix the sediment.
The sediment used was spiked with a 100 ~tg amount of both pesticides (endrin and
heptachlor) unless stated other-wise and the sediment was aged for at least 1 month before
extraction. Their concentration i n t h e sample was 2 mg/kg. The second standard mixture
composition is given in Using this mixture a series of sediment
standards were prepared containing pesticides at 50 gg/kg per component Solvents All solvents
used were redistilled in glass. The primary extraction solvent was iso-octane; others used were
n-hexane-acetone, b e n z e n e - a c e t o n e (2:1 v/v), methanol-acetic acid, methanol and n-
hexane.

ExtractionProcedure

The sediment samples after aging were extracted using K E N M O R E Microwave / Convection
oven, Mode l85962. It should be noted that only a commercially available explosion-proof oven
must be used, if more than a couple of samples are extracted simultaneously. Approximately
1.0 g of a sample was accurately weighed into a 5 m L Reacti-Vial. Prior to extraction, the
sediment samples were saturated with distilled water and 2 m L solvent was added. The sample
was extracted inside the microwave oven cavity and the microwave frequency used was
matched to setup a resonant pattern at 2.45 G H z frequency as is common for all home
microwave ovens. The sediment sample was extracted for 30 sec using maximum power but
the content of the vial was not allowed to boil. After 30 sec, the vial was immersed into an ice
bath for 2 to 5minutes. The extraction step and cooling were repeated up to 5 times. It is
recommended to establish a number of repeated extractions to obtain optimal extraction fora
particular type of samples.

7. Multiresidue Determination of Pesticides in Soil by Gas Chromatography−Mass

Spectrometry Detection

Materials and Standards. Pesticide standards were obtained from Reidel-de Hae¨n (Seelze,
Germany), and all compounds were of 99% purity. Ethyl acetate, residue analysis grade, was
purchased from Scharlau (Barcelona, Spain), and anhydrous sodium sulfate, reagent grade, was
obtained from Merck (Darmstadt, Germany). Stock solutions (500 µg/mL) of each pesticide
standard were prepared by dissolving 0.050 g of the pesticide in 100 mL of ethyl acetate and
stored at 4 °C. A pesticide intermediate standard solution (5 µg/mL) was prepared by
transferring 1 mL from each pesticide solution to a 100 mL volumetric flask and diluting to
volume with ethyl acetate to obtain a concentration of 5 µg/mL. A set of calibration standard
solutions of 5.0, 1.0, and 0.5 µg/mL was prepared by dilution. The solutions containing 5.0, 1.0,
and 0.5 µg/mL of each pesticide were used to fortify soil samples. The internal standards were
prepared by dissolving lindane and hexazinone in ethyl acetate to make a 500 µg/mL solution.

Apparatus. Extraction Equipment. - Polypropylene columns (20 mL) of 10 cm × 20 mm i.d.,

Becton-Dickinson, Spain, with Whatman no. 1 filter paper circles of 2 cm diameter (Whatman,
Maidstone, U.K.) were used. One-way stopcocks were employed to close the columns. An
ultrasonic water bath (Raypa, Barcelona, Spain) was used in the extraction step. The generator
of this ultrasonic bath has an output of 150 W and a frequency of 35 kHz. A vacuum manifold
(Supelco Visiprep, Madrid, Spain) was employed to remove the extraction solvent.

Sample Preparation. - Soil Samples. Soil used in the recovery assay was collected from the plow
layer (0-10 cm) of an experimental plot located in the region of Madrid (Spain). Soil samples
were sieved (2 mm) and stored at room temperature until fortified. The characteristics of the
selected soil were as follows: pH, 7.69; organic matter content, 0.97%; sand, 44.34%; silt,
37.44%; and clay, 18.22%. Real samples were collected from several Spanish regions: 16
samples from tomato fields in Badajoz, 8 samples from forested fields in Badajoz, and 18
samples from corn fields in Badajoz and Albacete. Samples were collected from the plow layer
(0-10 cm), sieved (2 mm), and stored at -18 °C until analysis.

Procedure. Two filter paper circles were placed at the end of a plastic column, and anhydrous
sodium sulfate (2 g) was added; sieved soil (5 g) was then placed in the column. In the recovery
assays, soil samples were previously fortified with 0.5 mL of a mixture of the different
pesticides to reach final concentrations of 0.05, 0.1, and 0.2 µg/g, allowing 20 min for solvent
evaporation. Soil samples were extracted with 4 mL of ethyl acetate for 15 min in an ultrasonic water
bath at room temperature. Ethyl acetate was selected as extraction solvent due to the good results
obtained in previous works (9-11, 15). The water level in the bath was adjusted to equal the extraction
solvent level inside the columns, which were supported upright in a tube rack and closed with one-way
stopcocks. After extraction, the columns were placed on the multiport vacuum manifold, where the
solvent was filtered and collected in graduated tubes. Soil samples were extracted again with another 4
mL of ethyl acetate (15 min). The extracting solvent was filtered, and soil samples were washed with 1
mL of additional solvent. The total extracts collected in 10 mL graduated tubes were concentrated with a
gentle stream of air to an appropriate volume (10 mL for the highest and intermediate levels and 5 mL
for the lowest level and real samples) and stored at 4 °C until analyzed by GC-MS. A 0.5 mL of the
internal standard solution of 1 µg/mL (lindane and hexazinone) was added before GC analysis.
Chromatographic standards were prepared using blank sample extracts. These blank extracts were
fortified with 0.5 mL of the pesticide standard solution of 1 µg/mL and with 0.5 mL of the internal
standard solution (lindane and hexazinone) of 1 µg/mL.

8.Residues of organochlorinated pesticides in soils from the Czech Republic

Sample collection - Nine soil samples were collected from mountains along the Czech borders
with Poland, Germany and Slovakia. Five soil samples were taken from industrial area Mokra´ to
the east of Brno, the second largest city, and five soils were sampled from the background site
Kosˇetice. Kosˇetice, the observatory of the Czech Hydrometeorological Institute, serves as a
regional station of integrated background monitoring network of the United Nations
Environmental Programme (UNEP) project Global Environmental Monitoring System (GEMS)
since the end of the 1970s. All soil samples were air-dried, and the content of organic carbon
was determined. Mosses (Hypnum cuprresiforme L. ex Hedw.), and pine needles (Pinus
sylvestris L.) were collected in Kosˇetice observatory (five sampling sites for needles and three
sites for mosses). The moss samples were collected on the ground outside the crown protection
of the trees. Samples were wrapped in aluminum foil, transported to the laboratory, air-dried
at the room temperature and stored in paper sacks, and sealed polyethylene bags prior to
analysis.

Chemicals - Residue analysis solvents were used for sample preparation. Samples were
quantified using Pesticide Mix 13 (Dr. Ehrenstorfer) standard mixture.

Sample analysis - All samples (5 g of dry soil, needles or moss) were extracted with
dichloromethane in a Bu¨chi System B-811 automatic extractor. Surrogate recovery standards
(PCB 30 and PCB 185, 10 ng per sample) were spiked on each sample prior to extraction; PCB
155 was used as an internal standard. For the soil extracts rich in organic matter (C org > 20%),
and for needle extracts containing high amounts of waxes, a gel permeation chromatographic
column Bio-Beads SX-3 (50 0.8 cm i.d., 200e400 mesh) (Tessek Ltd., Prague, Czech Republic)
was used as a first clean-up step. One milliliter of extract was injected and eluted with
chloroform, and the volume was reduced under a gentle stream of nitrogen at ambient
temperature. Fractionation was achieved on a chromatographic column (1 cm i.d.) packed with
Florisil (5 g, activated at 130 C for 4 h, 10% water added) and pre-rinsed with 50 ml of n-hexane
before an application of the eluate. Samples were eluted in two fractions: 20 ml of 20% n-
hexane/dichloromethane (chlordane, aldrin, isodrin, heptachlor) and 50 ml of dichloromethane
(remaining OCPs). The volume of the samples was reduced to 5 ml using rotary evaporator,
then under gentle stream of nitrogen to the final volume of 0.2 ml. The organochlorinated
pesticides were analyzed by GC-MS on a Finnigan GCQ gas chromatograph coupled to an ion
trap mass spectrometer using EI/MS-MS mode. Chromatographic separation was achieved on a
DB-5MS fused-silica capillary column (J&W Scientific, 60 m 0.25 mm i.d., 0.25 mm film
thickness) with helium as a carrier gas (flow rate 30 cm s1 ). The column temperature was
programmed as follows: initial temperature 80 C for 1 min, 20 C min 1 to 200 C, 1.5 C min1 to
260 C, 15 C min 1 to 300 C, final hold for 10 min). Samples (1 ml, an autosampler A200S) were
injected in the splitless mode. Injector, transfer line, and ion source were kept at 250 C, 275 C,
200 C, respectively. Quantification was performed using the seven-point calibration curve.
Quantification limits for organochlorinated pesticides.

Quality assurance/quality control - Recoveries were determined for all samples by spiking with
the surrogate standards prior to extraction. Recoveries were higher than 79% for all samples,
and the recovery factors were not applied to any of the data. Analytical method recoveries for
the given set of pesticides were also determined by repeating the whole analytical procedure
with the reference soil enriched by the standard mix of pesticides of the known concentration.
They ranged from 80% to 98% for all compounds except for endosulfan II, having lower
recovery of approximately 50%. Reproducibility was calculated based on the replicate analyses,
with RSD 10.3%. All laboratory blanks were below the detection limit for OCPs

9. Pharmaceuticals and organochlorine pesticides in sediments of an urban river in Florida,

USA

Study area and sampling locations - The Alafia River drains 1093 km2 of an urban watershed to
Tampa Bay estuary in Florida. The locations of sampling sites are shown in Fig. 1 and the
characteristics (location, land use, likely contaminant sources) at each of the sites .We collected
bed sediments from nine sites located in the Alafia River; these sites drain different areas of the
watershed representing agricultural, urban, and industrial areas (Table 1). Major land uses in
the Alafia River watershed are urban (20 %), agricultural (8 %), pasture (11 %), forest (18 %),
and phosphate mining (32 %) (Khare et al. 2012). North Prong and South Prong are two main
tributaries of the Alafia River that contributes about 60 % of total discharge. Three streams (Bell
Creek, Turkey Creek, and Fishhawk Creek) are the minor tributaries that discharge to the lower
reaches of the Alafia River. The area under residential land use in various subbasins ranged
from 3 to 64 %, and built-up area varied from 1 to 14 %. Other major land uses in sub-basins are
forest (12– 37 %), pasture (2–23 %), and agricultural (1–24 %). In two sub-basins, most of the
area is under phosphate mining (both abandoned and active), ranging from 39 % in North Prong
to 66 % in South Prong. The Alafia River is tidally influenced for 18 km from its mouth; total
length of the river is 80 km (Chen 2004). Lithia Springs, a second magnitude spring, and
Buckhorn Spring provide relatively steady freshwater flows to the river (Chen 2004). At each
sampling site, three transects of 1 m×1 m were selected. From each transect, five cores from 0
to 10 cm were collected in April 2009, and then, a composite sample was made to represent
one replication. This resulted in three replicate samples for each site.

Sediment analyses: - basic properties, pharmaceuticals, and organochlorine pesticides

Sediment samples were analyzed for various physical and chemical properties .

Twenty OCPs, as listed in Table, were extracted from sediments to investigate the
environmental persistence of legacy compounds using modified EPA method 1699 for
extraction (USEPA 2007a) and EPA method 8081 for analysis (USEPA 2007b). In brief, 10 g of
sediment was suspended in 25 ml of petroleum ether-acetone mixture (1:1v/v) and sonicated
for 20 min in an ultrasonic bath (35 kHz, 320 W, Super RK 510, Sonorex, Bandelin, Berlin,
Germany). The extraction procedure was repeated four times, and the extract from the same
sample was combined and filtered using Whatman filter paper. The extract was concentrated to
2 ml using rotary evaporator at 40 °C under a gentle nitrogen stream (Turbo Vap II, Zymark Inc.)
and then transferred onto a Resprep Florisil cartridge (3 ml, 250 mg). Prior to sample loading,
the cartridge was conditioned with 4 ml hexane. The sample was eluted with 100 ml of
hexane/ethyl acetate (7:3v/v) and concentrated to 1 ml prior to gas chromatography with an
electron capture Sediment pH was measured by equilibrating 10 g of sediment sample with 20
ml of deionized water (1:2) for 1 h with a digital meter (Accumet XL60, Dual channel
pH/ion/conductivity/dissolved oxygen meter, Fisher Scientific, Pandan Crescent, Singapore).
The electrical conductivity (EC) of was measured using a sediment to deionized water
suspension (1:1) with the same digital meter. Sediment samples were analyzed for sand, silt,
and clay using the hydrometer method (Day 1965). Sediment organic matter was determined
by the oxidation method of Walkley and Black. detector (GC-ECD; Perkin Elmer Clarus 500,
Waltham, MA, USA). During the analysis, verification of method performance was conducted by
using matrix spike recovery, blank, and duplicate samples in a set of each ten samples. Target
OCP analysis was performed using a GC-ECD coupled with a MultiPurpose sampler (MPS 2,
GERSTEL, Mülheim, Germany). An Rtx®-CLPesticides2 column (30 m× 0.1 mm id×0.25 μm,
Restek Corp, USA) was used for separation of OCPs. An aliquot of 1 μl sample was injected at
250 °C in splitless mode. Hydrogen was used as the carrier gas at a constant flow rate of 1 ml
min−1 . The oven temperature was programmed at 110 °C (hold 0.5 min), increased to 230 °C at
25 °C min−1 , and further to 330 °C (hold 1 min) at 15 °C min−1 .