INTRODUCTION
Persistent organic pollutants (POPs) are toxic chemicals that are
resistant to degradation in the environment. Due to their fat solubility and
resistance to biological degradation, ingestion of certain classes of POPs by
animals leads to bioaccumulation throughout their livers, generally in the fatty
tissues, and to biomagnifications in the food chain (Gioia et al., 2013; Safe,
1994). Among the POPs, organochlorine pesticides (OCPs) and polychlorinated
biphenyls (PCBs) are highly prevalent in vertebrates. The majority of POPs,
such as PCBs and OCPs, are currently banned from use and are no longer
produced or used around the world; therefore, their levels have been constantly
declining through the years (Addison et al., 2013; Ryan et al., 2013; Schuster et
al., 2011). Nevertheless, relevant amounts of these pollutants still persist in the
environment, and certain species, specially those top predators, are especially
contaminated (Bourgeon et al., 2013), and it has been described that these
pollutants lead to adverse health effects on living beings (Hamlin and Guillette,
2010).
Pesticides reach aquatic ecosystems by direct application, spray drift,
aerial spraying, erosion and runoff from factories and sewage. The
contamination of water sources is a major source of concern since it is the
habitat of fish and other aquatic organisms such as mussels, oysters, prawns
and lobsters. Pesticides end up in the tissue of aquatic organisms and bioaccumulates
with time (Jiries et al., 2002). Fish consumption could be therefore
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considered as one of the major sources of human exposure to all environmental
contaminants (EFSA, 2005; Storelli, 2008). OC pesticides are ubiquitous
anthropogenic contaminants that are persist in the environment; accumulate in
fatty tissues and increase in concentration as they move up the food chain
(WHO, 1999). Due to their lipophilic nature they accumulate along trophic levels
and induce multiple adverse effects in many organisms (Fleming et al., 2006).
Although the production and use of many types of OCs have been severely
limited in many countries including Egypt, they are, nevertheless, still being
used unofficially in large quantities in many parts of the world, and in other
developing countries because of their effectiveness as pesticides and their
relatively low cost. OCs were detected in fresh water fish in previous studies in
Egypt by Salah El-Dien and Nasr (2004). The probable sources of this pesticide
group originated from previous or illegal use.
Polychlorinated biphenyls (PCBs) were commercially produced as
complex mixtures containing multiple isomers at different degrees of
chlorination. Today, PCBs can still be released into the environment from poorly
maintained hazardous waste sites that contain PCBs; illegal or improper
dumping of PCB wastes; leaks or releases from electrical transformers
containing PCBs (ATSDR, 2002). Some PCB congeners elicit a divers spectrum
of toxic and biochemical responses including body weight loss, immunotoxicity
(Sormo et al., 2009) and induction of gene expression (El Nemr et al., 2003).
Because of the dangerous effect of the presence of OC and PCBs in fish
tissues on human health a simple method for the determination of these
compounds residues was evaluated.
MATERIALS AND METHODS
Standards and reagents
Organochlorine pesticides, fenopropathrin and PCBs reference
standards were purchased from Dr. Ehrensdorfer (Augsburg, Germany), with
purity of >95% and they were used to prepare stock and diluted solutions. Stock
solutions of reference standard of concentration 1000 μg/ml were prepared in
toluene and kept at 4 ±2 oC. Spiking mixture standard solutions were prepared
for fatty food analysis at concentration levels as shown in Tables(1 and 2).
Spiking mixture solutions of PCB’s and organochlorine pesticides can be
prepared once a year.
Working standard of Aldrin with concentration of 0.1 μg/ml was prepared
in n-hexane/ acetone (9:1) solution and it was used as an injection standard for
GC- ECD.
The solvents used were aceton, hexane, petroleum ether, benzene, ethyl
acetate and acetonitrile. Florsil, anhydrous sodium sulphate and sodium
chloride were also used. All the reagents were of analytical (HPLC) grade
supplied by BDH, London, UK. Before use; sodium sulphate was heated at 650
°C for 4 h and kept in a desiccator. Distilled water was obtained with a Milli-Q
system (Millipore, Bedford, MA, USA).
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Table 1: The concentration levels of organochlorines and fenopropathrin
insecticides spiked to fatty food samples
Organochlorines and fenopropathern pesticides
Mixture 1 Mixture 2
Compound
Spike
Conc.
(μg/ml)
Expected
Spike Level
(mg/kg)
on 25 g
Compound Spike Conc.
(μg/ml)
Expected Spike
Level (mg/kg)
on 25 g
HCH-alpha 1 0.04 HCB 0.5 0.02
HCH-g (Lindane) 1 0.04 HCH-beta 1 0.04
Heptachlor 1 0.04 HCH-delta 1 0.04
Heptachlor-exo-
Epoxide
1 0.04 Endouslfan-alpha 1 0.04
DDE-p,p` 1 0.04 Dieldrin 1 0.04
Endrin 1 0.04 Endouslfan-beta 1 0.04
DDT-p,p` 5 0.2 Endosulfan Sulfate* 5 0.2
Fenpropathrin 2 0.08 Heptachlor-endoepoxide
1 0.04
Esfenvalerate 5 0.2
Deltamethrin 5 0.2
Table 2: The concentration levels of PCBs compounds spiked to fatty food
samples
Name Spike conc. (μg/ml)
Expected Spike
Level on 2.5 g
(mg/kg)
Expected Spike Level on
25 g (mg/kg)
PCB's 28 0.05 0.02 0.002
PCB's 52 0.05 0.02 0.002
PCB's 101 0.05 0.02 0.002
PCB's 118 0.05 0.02 0.002
PCB's 153 0.05 0.02 0.002
PCB's 138 0.05 0.02 0.002
PCB's 180 0.05 0.02 0.002
Sample extraction
Twenty five grams (W) of edible fish or animal tissue was placed into
blender jar with 50 g sodium sulphate, blended and mixed with spatula until
sample and sodium sulphate were well mixed. The sides of blender jar were
scraped down and broke up caked material with spatula. For spike sample, 1 ml
spike solution was added on 25 g sample that was proved to be free of PCB’s
and organochlorine pesticides.150 ml petroleum ether was added and blended
at high speed for 2 min. Petroleum ether supernatant decanted and filtered
under suction through buchner fitted with filter paper. The sides were scraped
down of blender jar and broke up caked material with spatula.
The residue was re-extracted in blender jar with two 100 ml portions of
petroleum ether, blended for 2 min each time. After 1 min blending, bender was
stopped material was scraped from sides of blending jar and broke up caked
material with spatula. Sides of blender jar were scraped down and broke up
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caked material between extractions. Petroleum ether was decanted through
buchner funnel and combined with first extract. After last blending, residue from
blender jar was transferred into the buchner funnel, blender jar and material
were rinsed in buchner with three 25 ml portions of petroleum ether and filtration
was continued through the same buchner.
Combined extracts were poured the buchner funnel was rinsed with 25
ml petroleum ether and the filtrate was collected through anhydrous sodium
sulphate in pre-weighed 500 ml flask. Petroleum ether from combined extracts
was evaporated at 35-40 oC on rotary evaporator. The 500 ml flask after
evaporation was weighed and the weight of the extracted fat was calculated
(W1). If the total amount of fat is more than 3 g, 2.5 g (W2) fat is taken only for
liquid-liquid partitioning. If the extracted fat weight is less than 3 g, all the fat
was taken for liquid-liquid partitioning should follow the adjusted formula
(W1/W2=1) (Pesticide Analytical Manual, 1994) (PAM).
Modifications to the PAM method
Rotary evaporator and air blow are used instead of Kurdana- Danish.
Mixture of hexane/benzene/ethyl acetate is used to elute organochlorine
pesticides instead of petroleum ether/ethyl ether mixture.
Liquid-liquid partitioning
The extracted fat was quantitavely transferred 3 times with 5 ml
petroleum ether in 100 ml separatory funnel. Thirty ml acetonitrile saturated with
petroleum ether were added , shaked vigorously for 1 min till the layers are
separated, and drain acetonitrile (lower layer) into one liter separatory funnel
containing 600 ml de-ionized water, 40 ml saturated sodium chloride solution,
and 100 ml petroleum ether. Petroleum ether solution was extracted in 100 ml
separatory funnel with three additional 30 ml portions of acetonitrile saturated
with petroleum ether, shaked vigorously for 1 min each time, and all acetonitrile
extracts were combined in the one liter separatory funnel. The separatory funnel
was shaked gently for 1 min, let layers to separate and drain the aqueous layer
(lower layer) into second one liter separatory funnel. A hundrad ml petroleum
ether was added to second one liter separatory funnel, shaked vigorously for 15
seconds and let layers to separate. Aqueous layer was discarded and the
petroleum ether layer was combined with that in the original one liter separatory
funnel. Petroleum ether layer was washed with two 100 ml portions de-ionized
water. Washings were discarded and petroleum ether layer was drained
through anhydrous sodium sulphate supported on washed cotton with
petroleum ether in funnel on receiving flask. Evaporation was done on rotary
evaporator to dryness at 35-40 oC. The residue was dissolved in 10 ml
hexane/acetone (9:1) (V1); aliquot (V2) of 2 ml for organochlorine pesticide
clean up. For PCB’s, clean up aliquot of 5 ml (V2) was taken, evaporated, and
re-dissolved in 5 ml petroleum ether.
Florisil Column
Organochlorine pesticides and PCB’s clean up
The chromatographic column was placed (length 40cm and internal
diameter 16mm) in the following order, glass wool plug, 10 g activated florisil
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and 2cm height of anhydrous sodium sulphate on the top of florisil. Florisil and
sodium sulphate were settled by tapping the column. The column was pre-wet
with 50 ml hexane. 250 ml receiving flask was placed under the column to
receive elutes. Sample extract solution was transferred to the column and the
flow was adjusted to about 2.5 ml/min. The column was eluted with 50 ml (elute
I) and 25 ml (elute II). The two elutes were combined in 250 ml flask,
evaporated on rotary evaporator to about 2 ml at 35-40 oC. Evaporation was
continued by air just to dryness. The residues were re-dissolved in 2 ml (V3) and
the injection standard was immediately done after the evaporation is completed
and 1 μl was injected into GC-ECD system. The same steps were done for
PCB’s clean up except of using petroleum ether for Pre-wet and the residues
were dissolved in 2 ml (V3) hexane/acetone (9:1) and sonnicated for 1 minute
before injection.
GC conditions
Gas Chromatograph HP 6890 equipped with two electron capture detectors
was used and the instrument was conditioned as follow:
- Injector temp = 225 °C Detector temp = 300 °C
- Flow rate of nitrogen: 1.3 ml/min carrier, total flow (carrier + makeup):
55 ml/min.
- Septum purge: 3 ml/min, purge flow 50 ml/min, purge time 0.7 min.
Oven program
The oven was programmed as follows:
Initial temp: 90 °C Initial time: 2 min
Level Rate
(°C/min)
Temp
(°C)
Time
(min)
(1) 20 150 0
(2) 6 270 15
Capillary columns
Two different capillary columns were used and they were:
a) Agilent Technologies: HP-PAS5
Column ID: 0.32 mm, Film thickness: 0.52 um, Column length: 25 m
b) Agilent Technologies: DB-1701P
Column ID: 0.32 um, Film thickness: 0.25 um, Column length: 25m
Calculations
The analyte concentration in sample (Cs) (mg/kg) is calculated as follows:
2
3 1
2
1
W
W
W
V
V
Cs Ci V
Where:
Ci = Concentration in injection (μg/ml).
V1 = Dilution volume after partitioning.
V2 = Volume taken for clean up (ml).
V3 = Final dilution volume (ml).
W = Weight of original sample.
W1 = Weight of extracted fat (g).
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W2 = Weight of fat taken for liquid-liquid partitioning (g).
This equation is used when the weight of the extracted fat from the sample is
greater than 3 g.
Organochlorine and fenopropathrin pesticides calculations
Calculations of organochlorine pesticides were based on “injection
stranded calculations” method and one level calibration curve.
In case of the extracted weight of fat is less than 3 g, all the extracted fat
will be taken for liquid-liquid partitioning (W1/W2=1) and dilution volume after
partitioning is 10 ml (V1 = 10 ml), the volume was taken for clean up is 2 ml (V2
= 2 ml), the final dilution volume is 2 ml (V3 = 2 ml) and the weight of original
sample is 25 g (W= 25 g).
So, the equation used for calculations can be summarized as follows:
OCh. (Fish samples)
Cs Ci 0.4
Where; Ci = Concentration in injection (μg/ml).
PCB's calculations
Calculations of resulted concentrations of residues are based on
multilevel calibration curve.
If we assume that the extracted fat is less than 3 g, so all the extracted
fat will be taken for liquid-liquid partitioning must meet the equation (W1/W2=1)
and dilution volume after partitioning is 10 ml (V1 = 10 ml), the volume taken for
clean up is 5 ml (V1 = 5 ml), the final dilution volume is 2 ml (V3 = 2 ml) and the
weight of original sample is 25 g (W= 25 g).
So the equation used for calculations can be summarized as follows;
PCB’s (Fish samples)
Cs Ci 0.16
Where; Ci = Concentration in injection (μg/ml).
RESULTS AND DISCUSSIONS
The validation study was carried out using the blackcurrant samples that
were previously checked to be free of the pesticides of interest. The recoveries
were determined in six repetition and the three spiking levels ranged between
0.01 to 0.4mg/kg. The samples were spiked before proceeding with the sample
preparation. Average recovery and relative standard deviation (RSD), values
per spiking level and the overall value were calculated for each pesticide. The
results were assessed for compliance with the European Union guidelines
SANCO/12495/2011, according to which the average recovery should be in the
range of 70–120% with RSD less or equal to 20% (SANCO/12495, 2011). The
limit of quantification (LOQ) was set at the least spiking concentration that has
been validated with satisfactory recovery and precision parameters.
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Recovery tests
The recovery of organochlorine pesticides and PCB’s were tested by
performing repeated spike fish samples (Repeatability) at different
concentration levels. The recovery percentage and relative standard deviation
on each level were calculated. The results of the recovery percentages of tested
pesticides and PCBs are presented in Table 3.
Table 3: The recovery percentages of the tested pesticides and PCBs
spiked and analyzed from fish samples
Group
Compound
Level
1 2 3
Exp.
(mg/kg)
Rec.
%
CV
%
Exp.
(mg/kg)
Rec.
%
CV
%
Exp.
(mg/kg)
Rec.
%
CV
%
OCP-Mix1
HCH-alpha
HCH-g
(Lindane)
Heptachlor
Epox.
DDE-p,p`
Endrin
Fenpropathrin
0.2
0.2
0.2
0.2
0.2
0.4
94
116
82
86
112
94
5.7
3.8
11.1
10.6
11.0
16.9
0.04
0.04
0.04
0.04
0.04
0.08
86
94
88
99
102
95
7.0
9.0
7.0
7.0
7.0
6.0
0.01
0.01
0.01
0.01
0.01
0.02
77
94
92
81
94
102
13.5
11.4
13.5
19.0
15.6
15.1
OCP-Mix2
HCH-beta
HCH-delta
Endouslfanalpha
Dieldrin
0.2
0.2
0.2
0.2
83
93
77
80
8.7
7.7
11.2
12.1
0.04
0.04
0.04
0.04
96
75
83
84
8.5
6.5
9.5
10.3
0.01
0.01
0.01
0.01
107
83
85
95
9.5
17.6
18.2
16.6
PCB's
PCB's 28
PCB's 52
PCB's 101
PCB's 118
PCB's 153
PCB's 138
PCB's 180
0.1
0.1
0.1
0.1
0.1
0.1
0.1
109
103
95
99
103
98
93
12.0
10.3
9.5
11.6
8.3
9.2
8.2
0.02
0.02
0.02
0.02
0.02
0.02
0.02
83
84
70
96
82
83
77
14
12
6.0
12
10
12
11
0.005
0.005
0.005
0.005
0.005
0.005
0.005
96
91
80
80
88
86
82
12.8
17.8
20.0
15.8
16.9
13.0
13.5
Limit of detection (LOD)
Limit of detection is the minimum concentration of analyte in the test
sample that can be measured with a stated probability that the analyte is
present at a concentration above that in the blank sample. The limit of detection
is estimated as 3s of sample blanks fortified at the least acceptable
concentration level. The limit of detection (LD) ranged between 0.003-0.009
mg/kg for organochlorine and fenopropathrin pesticides and 0.002 mg/kg for
PCB’s (Table 4).
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Limit of quantitation (LOQ)
The limit of quantitation is the minimum concentration of analyte in the
test sample that can be determined with acceptable precision (repeatability) and
recovery under the stated conditions of the test. The lowest practical limit of
quantitation was estimated by using repeated spiked samples at about the
expected lowest quantitation level on fish samples. The limit of quantitation
(LOQ) was 0.005 mg/kg for PCB’s and in the range of 0.01-0.02 mg/kg for
organochlorine compounds.
Table 4: The limit of detection (LOD) of PCB’s and organochlorine
pesticides.
Group
Compound
Expected
(mg/kg)
Signal to
noise(s)
(mg/kg)
LD (3s)
(mg/kg)
OCP-Mix1
HCH-alpha
HCH-g (Lindane)
Heptachlor Epox.
DDE-p,p`
Endrin
Fenpropathrin
0.01
0.01
0.01
0.01
0.01
0.02
0.0010
0.0011
0.0012
0.0015
0.0015
0.0031
0.003
0.003
0.004
0.005
0.005
0.009
OCP-Mix2
HCH-beta
HCH-delta
Endouslfan-alpha
Dieldrin
0.01
0.01
0.01
0.01
0.0010
0.0015
0.0015
0.0016
0.003
0.004
0.005
0.005
PCB's
PCB's 28
PCB's 52
PCB's 101
PCB's 118
PCB's 138
PCB's 153
PCB's 180
0.005
0.005
0.005
0.005
0.005
0.005
0.005
0.0006
0.0008
0.0008
0.0006
0.0006
0.0007
0.0006
0.002
0.002
0.002
0.002
0.002
0.002
0.002
Linearity
Linear range
For quantitative analysis, the range of analyte concentrations over which
the method may apply was determined. For organochlorine pesticides, the
calculations are based on internal standard calculations method.
For PCB's the calculations are based on five levels calibration curve (0.01, 0.03,
0.05, 0.1 and 0.2 μg/ml). No internal standard was used. The correlation
coefficient was found to be greater than 0.999. The calibration curve must be
done with every set of samples.
Method Linearity
Method linearity was tested by performing recovery tests at different
three levels on fish samples. The method showed to be linear from the LOQ up
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to the maximum level; 0.1 mg/kg for PCB’s and in the range of 0.2-0.4 mg/kg for
organochlorine compounds. Figure 3 shows an example of each tested group
(mix.1, mix.2 and PCBs). It illustrate that the calculated correlations were
greater than 0.99 (Fig. 1).
(a) PCBs
(b) Mix.1
(c) Mix.2
Figure 1: The linear curves of certain compounds as an example of these
tested compounds (a: an example of mix.1, b: an example of mix.2 and c:
an example of PCBs).
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Accuracy
Accuracy expresses the closeness of a result to a true value. Accuracy is
expressed in terms of two components: “Trueness” and “Precision”
Trueness
The trueness of a method is an expression of how close the mean of a
set of results (produced by the method) is to the true value. To check trueness
of the method, spiked samples are used at different levels on fish samples. Bias
expressed as absolute relative difference percentage (RD%) was found to be
within the codex criteria.
Table 5: The results of trueness calculations.
Group
Compound
Level
1 2 3
Exp.
mg/kg
Found
mg/kg
Bias
%
Exp.
mg/kg
Found
mg/kg
Bias
%
Exp.
mg/kg
Found
mg/kg
Bias
%
OCP-Mix1
HCH-alpha
HCH-g
(Lindane)
Heptachlor
Epox.
DDE-p,p`
Endrin
Fenpropathrin
0.2
0.2
0.2
0.2
0.2
0.4
0.187
0.231
0.163
0.172
0.225
0.375
7%
16%
19%
14%
13%
6%
0.04
0.04
0.04
0.04
0.04
0.08
0.034
0.038
0.035
0.04
0.041
0.076
15%
5%
13%
0%
3%
5%
0.01
0.01
0.01
0.01
0.01
0.02
0.0077
0.0094
0.0092
0.0081
0.0094
0.02
23%
6%
8%
19%
6%
0%
OCP-Mix2
HCH-beta
HCH-delta
Endouslfanalpha
Dieldrin
0.2
0.2
0.2
0.2
0.167
0.185
0.154
0.16
17%
8%
23%
20%
0.04
0.04
0.04
0.04
0.038
0.03
0.033
0.034
5%
25%
18%
15%
0.01
0.01
0.01
0.01
0.011
0.0083
0.0085
0.0095
10%
17%
15%
5%
PCB's
PCB's 28
PCB's 52
PCB's 101
PCB's 118
PCB's 153
PCB's 138
PCB's 180
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.109
0.103
0.095
0.099
0.103
0.098
0.093
9%
3%
5%
1%
3%
2%
7%
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.017
0.017
0.014
0.019
0.016
0.017
0.015
15%
15%
30%
5%
20%
15%
25%
0.005
0.005
0.005
0.005
0.005
0.005
0.005
0.0048
0.0045
0.004
0.004
0.0044
0.0043
0.0041
4%
10%
20%
20%
12%
14%
18%
Trueness was also tested by repeating FAPAS proficiency test which has
been analyzed by two different chemists for determination of certain selected
PCB’s and organochlorine pesticides as seen in Table 6.
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Table 6: The accepted recovery percentages of certain selected
compounds according to codex criteria.
Selected
Compound
Assigned
value
(mg/kg)
Chemist I Chemist II
Found
(mg/kg)
Trueness
(Recovery %)
Found
(mg/kg)
Trueness
(Recovery %)
alpha -HCH 0.0278 0.020 72 % 0.0270 97 %
Dieldrin 0.0544 0.0448 82 % 0.0570 105 %
PCB’s 28 0.0379 0.0385 102 % 0.0320 84 %
PCB’s 153 0.0803 0.0840 105 % 0.0642 80 %
Precision
Precision is a measure of how close results are to one another. The two
most common precision measures are (repeatability) and (reproducibility)
Repeatability
Qualitatively is the closeness of agreement between successive results
obtained with the same method on identical test material, under the same
conditions (same operator, apparatus and laboratory as well as short intervals
of time) (ISO 3534-1). Repeatability experiments were done by fortification on
fish samples at different levels.
The previous Table 3 shows the accepted recovery percentage and
CV%; except for fenpropathrin (CV%=16.9%) at level one which exceeded the
codex criteria (CV %<15% at 0.4 mg/kg level).
Reproducibility
Reproducibility is the precision under reproducibility conditions, i.e.
conditions where test results are obtained with the same method on identical
test items in different laboratories with different operators using different
equipment. In this study, intra-laboratory reproducibility has only be considered.
Spiking fish samples were analyzed by different analysts on several days.
Reproducibility results are shown in Table 7.
Measurement Uncertainty
Parameter associated with the result of a measurement that characterises the
dispersion of the values that could reasonably be attributed to the measurand.
The parameter may be, for example, a standard deviation (or a given multiple of
it), or the width of a confidence interval. For estimating the overall uncertainty, it
may be necessary to take each source of uncertainty and treat it separately to
obtain the contribution of each source. Each of the separate contributions to
uncertainty is referred to as an uncertainty component. When expressed as a
standard deviation an uncertainty component is known as standard uncertainty.
The total uncertainty, combined standard uncertainty, equal to the positive
square root of the sum of the squares of the individual uncertainty components.
For most purposes in analytical chemistry, an expanded uncertainty, should be
used. The expanded uncertainty provides an interval within which the value of
the measured is believed to lie in a higher level of confidence. Expanded
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uncertainty is obtained by multiplying the combined uncertainty, by a coverage
factor (k); for confidence level of 95% k is equal to 2 (EURACHEM, 2000).
Table 7: Reproducibility tests of fish samples analyzed by different
analysts on several days.
Group
Compound
Expected
(mg/kg)
Mean
Recovery
(mg/kg)
CV%
OCP-Mix1
HCH-alpha
HCH-g (Lindane)
Heptachlor Epox.
DDE-p,p`
Endrin
Fenpropathrin
0.04
0.04
0.04
0.04
0.04
0.08
83%
85%
72%
87%
98%
76%
7.6%
8.0%
8.2%
19.2%
14.2%
19.3%
OCP-Mix2
HCH-beta
HCH-delta
Endouslfan-alpha
Dieldrin
0.04
0.04
0.04
0.04
79%
92%
80%
80%
13.0%
12.4%
11.1%
11.7%
PCB's
PCB's 28
PCB's 52
PCB's 101
PCB's 118
PCB's 138
PCB's 153
PCB's 180
0.02
0.02
0.02
0.02
0.02
0.02
0.02
83%
83%
80%
79%
89%
93%
80%
8.7%
5.5%
8.6%
17.5%
13.8%
11.4%
15.1%
The ISO Guide defines the uncertainty in terms of type A and type B;
Type A evaluation of uncertainty: method of evaluation of uncertainty by
statistical analysis of series of observations.
Type B evaluation of uncertainty: method of evaluation of uncertainty by
means other than statistical analysis of series of observations.
Standard Uncertainty
Validation studies were used to quantify different uncertainty
components. The random effects were estimated as the relative standard
deviation of repeated spike samples. Standard uncertainty due to repeatability
experiments (Ur), expressed as relative standard deviation was found to be less
than 14 %.
Standard uncertainty due to bias (recovery) experiments (UR), was
estimated as relative standard deviation of the recovery of spike samples at
different concentration levels. The spike samples were run during several days
and by different analysts. Standard uncertainty due to recovery experiments
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expressed as relative standard deviation and that was found to be less than
20%.
Standard uncertainty due to pipettes and volumetric flasks are not
accounted for, since they are involved in recovery experiments.
The following equations were used for standard uncertainty
calculations (Type A);
1
2
n
x x
S i
% 100
x
RSd S
Where
S, is the standard deviation
RSd%, relative standard deviation x , the average of n samples
Combined Uncertainty (UC)
Combined uncertainty, is the positive square root of the sum of the
squares of different uncertainty components which was found to be less than
21%.
The following equation was used for combined uncertainty calculations
( )2 ( )2 C r R U U U
Expanded Uncertainty
Table 8 represent the obtained expanded uncertainty which has been
calculated by multiplying the combined uncertainty, by a coverage factor (k), for
confidence level of 95% k is equal to 2.
The expanded uncertainty (at 95 % confidence level) was found to be less than
40 %.
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Table 8: The standard uncertainty due to repeatability (Ur), standard
uncertainty due to reproducibility (UR), combined uncertainty
(Ucomb) and expanded uncertainty (Uexp).
Group
Compound
Ur UR Ucomb Uexp
OCP-Mix1
HCH-alpha
HCH-g
(Lindane)
Heptachlor
Epox.
DDE-p,p`
Endrin
Fenpropathrin
7.3%
9.2%
7.0%
6.8%
7.0%
6.0%
8.1%
8.9%
7.2%
6.8%
6.8%
19.3%
10.9%
12.8%
10.0%
9.6%
9.7%
20.2%
22%
26%
20%
19%
19%
40%
OCP-Mix2
HCH-beta
HCH-delta
Endouslfanalpha
Dieldrin
8.5%
6.5%
9.5%
10.3%
13.3%
12.4%
11.1%
11.7%
15.8%
14.0%
14.6%
15.6%
32%
28%
29%
31%
PCB's
PCB's 28
PCB's 52
PCB's 101
PCB's 118
PCB's 138
PCB's 153
PCB's 180
13.6%
12.3%
6.4%
12.4%
9.6%
12.0%
11.3%
10.5%
15.9%
5.6%
8.2%
3.3%
5.7%
8.0%
17.2%
20.1%
8.5%
14.8%
10.2%
13.3%
13.8%
34%
40%
17%
30%
20%
27%
28%
CONCLUSION
A multiresidue method was developed for rapid and simultaneous
determination of 9 organochlorines pesticides, one synthetic pyrithriod
(fenopropathrin) and 7 congeners of PCBs compounds in fish by a modified
procedure and GC-ECD analysis. The whole analytical procedure was validated
according to European Union SANCO/12495/2011 guidelines. Furthermore, the
method proved to be simple and gave quantitative results for the assayed
analytes, providing good validation parameters, such as linearity, limits of
detection and quantification and precision. The uncertainties values obtained for
each pesticide were below 50% at all the fortification levels, which complies with
the requirements of SANCO/12495/2011 document. The applicability of the
method was demonstrated by analysis of six fish samples. A good performance
of the method was observed, allowing the reliable determination of the target
compounds in real samples. Finally, the results presented in this investigation
demonstrate that the validated method is feasible to be applied in pesticide
routine analysis carried out.
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