Accepted Manuscript Determination of Total Mercury in Fish and Sea Products by Direct Thermal Decomposition Atomic Absorption Spectrometry N.A. Panichev, S.E. Panicheva PII: DOI: Reference:
S0308-8146(14)00918-2 http://dx.doi.org/10.1016/j.foodchem.2014.06.032 FOCH 15974
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Food Chemistry
Received Date: Revised Date: Accepted Date:
20 July 2013 1 March 2014 8 June 2014
Please cite this article as: Panichev, N.A., Panicheva, S.E., Determination of Total Mercury in Fish and Sea Products by Direct Thermal Decomposition Atomic Absorption Spectrometry, Food Chemistry (2014), doi: http://dx.doi.org/ 10.1016/j.foodchem.2014.06.032
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1
Determination of Total Mercury in Fish and Sea Products by Direct Thermal
2
Decomposition Atomic Absorption Spectrometry
3 4
N. A. Panichev*, S. E. Panicheva
5
Department of Chemistry, Tshwane University of Technology, P.O. Box 56208, Arcadia
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0007, Pretoria, South Africa
7
*Corresponding author. Tel: +27 12 382 6233; Cell: +27 73 661 6061;
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Fax: +27 12 382 6286
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E–mail: [email protected]
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1. Introduction
12
According to the Food and Agriculture Organization of the United Nations (FAO), it is
13
estimated that the world consumed about 154 million tonnes of fish in 2011 and that about
14
50 percent of this originated from aquaculture. Although precise data are lacking, it is
15
acknowledged that, with growth in volume and value of fish production in the past decade,
16
aquaculture has made a positive contribution to national, regional and global economies,
17
poverty reduction and food security (FAO, 2012).
18
Other factors driving the market include a growing trend towards healthy eating. Due to the
19
high protein content of fish, it is becoming an ever-more popular choice for health conscious
20
consumers wishing to avoid the health drawbacks of meat. Despite fish and seafood
21
containing key nutrients, including omega-3 polyunsaturated fatty acids, essential amino
22
acids, trace elements and vitamins, they are also accumulators of mercury, posing a potential
23
threat to human health.
24
In aquatic environments, mercury is transformed by microorganisms into methylmercury
25
(MeHg), which bioaccumulates and biomagnifies through the food chain, which leads to high 1
26
concentrations at the top of aquatic food chain (WHO, 1990). Concerns over the toxicity and
27
human health risk of Hg deposited in ecosystems and bioaccumulating as MeHg in fish, has
28
prompted efforts to regulate anthropogenic emissions of that element (Mergler et al., 2007). It
29
is important for the public to be sufficiently informed prior to choosing what species of fish
30
to eat, as well as what size and how often it may be consumed without risk. The
31
concentration limit for Hg in fish for human consumption is set at 0.5 µg g-1( 500 ng g-1) wet
32
weight, (ww), (CCME, 1999; EEC, 2001; US EPA, 1997;); and 0.2 µg g-1 (200 ng g-1) (ww)
33
(WHO, 1990) for vulnerable groups, such as pregnant women, individuals under 15 years or
34
frequent fish consumers.
35
Although, in most cases, fish is consumed cooked, the majority of studies related to the
36
presence and the daily intake of mercury from the consumption of sea food provide data from
37
uncooked/raw products. To establish the maximum permissible Hg concentration in fish for
38
human consumption, the FAO (FAO/WHO, 1991) initiated a surveying of Hg concentration
39
in natural fish populations. Levels of Hg in fish from local markets have been reported by
40
many authors, covering different countries (Burger & Gochfeld, 2006; Kojadinovich et al.,
41
2006; Chien et al., 2007; Ruelas-Inzunza et al., 2008; Hajeb et al., 2009; Katner et al., 2010;
42
Burger &Gochfeld, 2011; Obeid et al., 2011; Focardi, 2012; Bonsignore et al., 2013; Wang et
43
al., 2013). However, information from African countries is limited to a publication
44
(Voegborlo & Akagi, 2007) about the Hg concentrations in fish from the Atlantic coast of
45
Ghana. The lack of specific information about the levels of Hg in fish and sea products from
46
South African market does not permit consumers to intelligently control their intake of fish.
47
Currently, the total Hg concentration in fish samples is usually performed by cold vapour
48
atomic absorption spectrometry (CVAAS). In this method, the sample is digested with hot,
49
concentrated mineral acids such as nitric, sulfuric, perchloric together with hydrogen
50
peroxide (Inhant, 2003). The sample pretreatment step, using wet digestion, requires about
2
51
30-60 min for oxidation of 0.5-1.0 g of fish. The method is time consuming and complicated
52
by the possibility of losses through volatilization or incomplete digestion as well as
53
contamination of the samples ( Kuboyama et al., 2005).
54
Measurements of Hg in solids matrices without chemical treatment of samples recently
55
become available by integration of thermal decomposition of samples, collections of Hg
56
vapour by amalgamation on gold wires or sponge, followed by atomic absorption
57
determination after thermal release of Hg from golden wire (US EPA method 7473).
58
Analysis of fish by method of solid sampling thermal decomposition, in which Hg is
59
selectively trapped on a gold amalgamator have been described (Torres et al., 2012). Upon
60
heating, Hg is desorbed from the amalgamator for an atomic absorption measurement. The
61
limit of quantification (LOQ) was 0.3 ng g-1. The only disadvantage of such systems could be
62
connected with Hg measurements in two steps: collection of Hg on golden sorbent and
63
evaporation of Hg into measuring cuvette. Each step should be properly optimized.
64
This study was undertaken with two main objectives. The first was to develop a method for
65
the determination of Hg in fish and sea products by direct thermal decomposition of the
66
samples, without preliminary collection of Hg vapour on golden amalgamator. The second
67
objective was to create a database for total Hg in fish and sea products purchased from the
68
local Tshwane fish market in Pretoria, South Africa. This would enable the evaluation of
69
fresh, frozen, salted, dried and canned fish of South African origin or obtained from her
70
trading partners.
71 72
2. Experimental
73 74
2.1 Materials, reagents and solutions 3
75 76
Nitric acid, HNO3, (Superpure, 60%, Merck, Germany) , hydrochloric acid, HCl (superpure,
77
30%, Merk, Germany) and hydrogen peroxide, H2O2, analytical grade (Merck) were used in
78
the digestion of fish samples. SnCl2 ·2 H2O (Rochelle Chemicals) was used to prepare the
79
reducing solution. For the dilution of digested fish samples 7% v/v HCl was used.
80
Hg standard solutions were prepared in the range 5-100 µg L-1 from 1001 ± 0.002 mg L-1 Hg
81
standard SS-1232 (Spectroscan) by dilution in 7% HCl. High-purity water (resistivity 18.2
82
MΩ cm) was used to prepare all aqueous solutions.
83 84
2.2 Certified Reference Materials
85
Several standard and certified reference materials (CRMs) for the determination of Hg were
86
used in this study: SRM 1515, apple leaves, (National institute of Standards and Technology,
87
U.S. Department of commerce), certified value 44 ± 4 ng g-1 , CRM 7002, light sandy soil
88
(Analytika Co Ltd, Czech Republic), certified value 90 ± 12 ng g-1 and CRM 024-050, metals
89
on soil, (Resource Technology Corporation, USA), certified value 710 ± 110 ng g-1 were
90
used for calibrating the RA-915+ Zeeman Mercury analyzer. CRM MESS-3, marine
91
sediment, certified value 90 ± 6 ng g-1 and CRM TORT-2 lobster hepatopancreas (National
92
Research Council of Canada), certified value 270 ± 60 ng g-1 were used for validation of
93
results.
94 95
2.3 Apparatus
96
An analytical balance RADWAG model AS 220/C/2AY was used to weigh the samples using
97
units of milligrams For Hg determination by CVAAS, the microwave MARS 6 (CEM
98
Corporation, USA) was used for fish samples digestion. The equipment contains 16
99
digestions Teflon vessels with a capacity of approximately 100 ml each. A Model RA-915+ 4
100
Zeeman Mercury analyzer (Lumex, St. Petersburg, Russia) with a PYRO-915 and CVAAS
101
attachments were used for Hg measurements. The working principle of the instrument (Fig.1)
102
is based on the thermal evaporation of Hg from a sample (1) placed in two-stage pyrolysis
103
tubes (2 and 3) heated to 750 and 800 oC. After inserting a known amount of sample into the
104
preheated tube, Hg vapour, together with smoke, formed by the combustion of the matrix’s
105
organic matter, are transported into the analytical cell (4), with a total optical length of 0.4 m,
106
and is also heated to 800oC. Background absorption is eliminated by the high-frequency
107
Zeeman correction system. The content of Hg in the sample is determined from the
108
calibration curve of the absolute amount of Hg (ng) versus the integrated analytical signal.
109
Finally, Hg0 vapour were absorbed in Hg trap (6)to prevent laboratory air contamination.
110
The PYRO-915 attachment enables Hg determination in samples with complex-matrices,
111
such as soils, sediments, oil products, foodstuff, etc., using pyrolysis technique without
112
chemical pretreatment of samples (Sholupov et al., 2004, Huang et al., 2005). Real-time
113
measurements are made with visualization of the process on a computer display. Without any
114
chemical pretreatment or the addition of chemical modifiers the risk of sample contamination
115
is minimized.
116
117
2.4. Fish samples collection and preparation
118 119
Using the Tshwane market for obtaining samples, 39 of the most popular species of fish and
120
sea food were collected as fresh (wet) and/or frozen for the determination of total Hg. Frozen
121
fish were thawed and analyzed as “wet”. The samples for analysis were cut from the fillet of
122
the fish and divided into several portions. Some were analyses as “wet” samples, the others
123
were air dried at room temperature (about 25 oC) for a week and then grounded in a mill. The
5
124
homogenized material was analyzed as “dry” samples. To prevent the samples from
125
degrading and losses Hg prior to analysis they were kept in small plastic bags in a refrigerator
126
(Schmidt et al., 2013).
127
2.5 Analytical procedure of Hg determination in fish
128
Each sample taken from the refrigerator was weighed on an analytical balance in milligrams.
129
The mass of samples ranged between 30 and 300 mg. The exact weight of the fish sample
130
was imported to the analyzer software. Using a quartz weighing boat, the sample was inserted
131
into the furnace of the analyzer. The software displays a curve of Hg absorption, the area of
132
the peak, the maximum value of the absorption and the calculated concentration of mercury
133
in the sample. A baseline check was performed periodically on the instrument to insure that
134
zero line did not shift.
135
Direct analysis of fish samples affords many benefits. Eliminating wet chemistry greatly
136
reduces waste generation, systematic errors and technician exposure due to the volatilization
137
of chemicals and Hg during sample handling. Direct analysis of a wet fish typically provides
138
an answer in approximately three minutes after the sample had been introduced into the
139
instrument. For sea products and samples of fish with low Hg concentrations, the absorption
140
signal is usually low and extends for several minutes. It was decided that the analysis of dry
141
fish samples could improve accuracy. For this purpose, fish was analyzed as wet and dry
142
samples.
143
3. Results and discussions
144
3.1. Analytical performance of mercury analyzer
145 146
3.1.1. Calibration graph
147
6
148
The calibration curve (Fig 2) for the Hg determination has been plotted as absolute mass of
149
Hg (ng) versus absorption peak area (arbitrary units). The absolute mass of mercury (m
150
was calculated from the following relationship between the certified value of the Hg
151
concentration (C Hg) and CRMs mass, (m CRM ), taken for the analysis: m Hg (ng) = C Hg (ng mg-1) x m CRM (mg)
152
Hg)
(1)
153 154
The calibration curve, described by the equation: y = 372.1x+ 39.0, exhibited excellent
155
linearity (R2 value of 0.998) from 2.5 ng up to 300 ng Hg mass. These amounts are
156
equivalent to Hg concentration of 10 to 1200 ng g-1 in a wet fish sample, assuming a sample
157
weight of 0.250 g.
158 159
3.1.2. The Limit of Detection
160
161
Due to the absence of fish samples without Hg, which could be used as blanks, the limit of
162
detection (LOD = 3 Sa/b) and limit of quantification (LOQ = 10 Sa/b) were calculated from
163
the data of the calibration curve presented in general form as y =a +bx where Sa is the
164
standard deviation of the response y and b is the slope of calibration curve. This method is the
165
most applicable when the method of analyses does not involve background noise (
166
Shrivastava, 2011).
167
ANOVA (ANOVA Statistics), and were found to be 0.15 ng and 0.50 ng of absolute mass of
168
Hg or 0.6 ng g-1 and 2.0 ng g-1 for 250 mg of a wet fish sample. The results indicate that it is
169
possible to measure mercury in fish at concentrations much lower than the 0.5 µg g-1 (500 ng
170
g-1) limit.
Numerical calculations of LOD and LOQ were performed using
171
7
172
3.1.3. Validation of the method
173
The accuracy of the direct thermal decomposition atomic absorption spectrometry (DTD
174
AAS) method
175
materials and by the analysis of selected fish samples by an CVAAS, which is the most-used
176
technique for Hg measurements in fish (?) and is US EPA accepted method (US EPA method
177
245.6).
178
The results of Hg determination in CRMs are presented in Table 1. Although the standards
179
used to test the accuracy of the DTD method had different origins, the results of
180
measurements show good correlation between certified and found values. The recovery of Hg
181
in all analyzed CRMs was in the range of 95-106 % and the total Hg content appear to be
182
independent of the nature of the reference materials.
183
For a comparative determination of Hg in fish by CVAAS, five samples of fish covering a
184
range of Hg concentration between 50 and 300 ng g-1 were analyzed. For this, approximately
185
0.5 g of each fish, were digested in 5.0 mL HNO3 + 2.0 mL H2O2 in microwave MARS 6.
186
After digestion, all samples were diluted to 25 mL with 7% solution of HCl.. The Hg
187
measurement was performed by Lumex Zeeman mercury analyzer RA-915+ with an
188
attachment for CVAAS analysis. The calibration graph (y=194.47x – 21.63, R2=0.999) was
189
plotted using a set of 6 concentrations of diluted Hg aqueous standards. The LOD of Hg
190
determination was found to be 5.8 ng g-1 and LOQ 19.2 ng g-1 in wet fish, when 0.5 g of fish
191
tissue had been digested and diluted to 25.0 mL after decomposition. These results are in a
192
good correspondence with LOD = 4.9 ng g-1 and LOQ = 15.7 ng g-1 obtained during CVAAS
193
method validation for the determination of Hg in fish (Nascimento et al, 2012).
194
The results of Hg determination by the new DTD and a reference CVAAS method are
195
presented in Table 2. The comparison of the means was carried out on the basis of a null
196
hypothesis (Miller and Miller, 2005). The result for the comparison of the means do not differ
was validated by analyzing three different types of certified reference
8
197
significantly at the 95% confidence level, and therefore, the concentration of Hg found by
198
both methods in the analyzed fish samples were found to be statistically equivalent.
199
It should be noted that the results for the determination of Hg in fish using the DTD method
200
are available in 6 min, while using CVASS the results were only known after at least 6 h, due
201
to the time required for samples decomposition.
202 203
3.1.4. Determination of moisture loses
204 205
Due to the high moisture content of the fish, the appearance of the analytical signal was
206
shifted, in time, until all the water evaporated and the sample reached the temperature of
207
thermal decomposition. As a result, the time of analysis was 3.0 - 3.5 min. For some samples
208
with low Hg concentration the value of the analytical signal was slightly higher than the zero
209
line, causing the accuracy of determination to be questioned, while for dry fish samples the
210
analytical signal was much higher and perfectly symmetrical.
211
To increase the sensitivity of the determination in fish and sea products with low Hg
212
concentrations, it was decided to analyse dry as well as wet samples. Wet fish samples were
213
dried in the open air, at ambient temperature (about 25 oC), for a week. For each species of
214
fish the a moisture correction factor , Rw/d, which represents the ratio of wet fish weight (w)
215
to its dry weight (d), has been calculated (Table 3 and 4).
216
Knowledge of R
217
results of analysis of dry fish samples. The moisture content (M) in wet fish samples can be
218
calculated by the following equation:
219
w/d
permits the calculation of the Hg content in wet fish samples, using the
1 M = 1 − × 100% Rw d
(2)
9
220
It was found that the moisture content varied from 69.7 to 79.0% in fresh fish and in frozen
221
fish from 68.5 to 76.9%.
222 223
3.2. Results of Hg determination in fish
224 225
Total mercury concentrations of all analyzed samples of fish and selected sea products have
226
been summarized in Table 3. The data are sorted according to alphabetical order of common
227
fish names, starting with angelfish and ending with tuna. Since information regarding the
228
origin of the fish is usually unavailable, understand the reasons for differences in Hg
229
concentration in commercial fish is difficult, except in generalities, e.g., connected to species,
230
size and ecology of habitat (Burger & Gochfeld, 2011). Not unexpectedly, the top-level
231
predator, like tuna, was found to have the highest mercury concentration (534 ±23 ng g-1),
232
while Norwegian salmon the lowest (9.8 ± 0.4 ng g-1). The variation of Hg concentration in
233
fish of the same species is mostly associated with the size (weight) of the fish. Larger fish
234
usually have higher concentration than their smaller counterparts. In this study a positive
235
correlation in this regard was found for angelfish (R2 =0.943, n=4), rock cod (R2 = 0.917, n=3
236
), hake (R2 = 0.987, n=5) and silver flesh spincheek (R2 = 0.995, n=4).
237
The mean value of RSD% for all measurements of dry fish samples was found to be 6.1%,
238
for fresh fish samples 7.4%, and frozen fish 9.5%. Mercury concentrations in most fish
239
species, except angelfish, some species of tuna and skate, were below the 0.5 µg g-1 (500 ng
240
g-1) ,(ww), recommended by FAO/WHO. In general, the mean concentrations of Hg in fish
241
species reported in this study are close to those reported by US/FDA. The analysis of dry fish
242
had some advantages over the direct analysis of wet samples, such as shorter time of analysis,
243
improved analytical signal and long time stability of the samples. Since to the requirements
244
that only the Hg concentration of wet fish is accepted by FAO/WHO as legislative, this value
10
245
can be calculated using the data of the Hg concentration of dry samples and the moisture
246
correction factor, R w/d .
247
From data presented in Table 3, the measured Hg concentrations of wet fish and the
248
calculated values are very close Considering that the R
249
gravimetrically, the good correlation of measured (by AAS) and calculated Hg concentrations
250
clearly demonstrate that such a method can be applied for the analysis of wet samples. The
251
results confirmed that there are no Hg losses during sample preparation by air drying. They
252
also demonstrate that solid CRMs can be used for the calibration of the mercury analyzer for
253
analysis of wet fish samples. The moisture correction factor, R
254
from results of the determination of Hg by AAS on dry and wet fish samples.
w/d
values were determined
w/d,
can also be calculated
255 256
3.3 Results of Hg determination in seafood products
257 258
Shrimp (prawns) are promoted as a ‘low-mercury” seafood (Burger et al., 2005), but local
259
geological and ecological conditions may influence Hg levels in shrimp. Hence the Hg
260
concentration in shrimp may vary. The results, presented in Table 4 confirm that a level of
261
Hg in shrimp of different origin is not uniform. The lowest Hg concentration was found in
262
prawns Penaeus Monodon (4.6 ±0.3 ng g-1), the highest in prawns Nalporolders triarthus
263
(from 106 ± 5.0 to 254 ±10 ng g-1) .These concentration are comparable to those for fish
264
species.
265
Relatively high concentrations of Hg (126±14 ng g-1) were found in samples of octopus
266
(Octopoda) tentacles and its absence (≤ LOD) in shrimps (Caridea) and scallop (Pectinidae).
267 268
3.4 Results of Hg determination in canned fish
269
11
270
The samples of canned fish were also analyzed, because this product is popular and is the
271
form of fish most commonly eaten. Thus, assessing the levels of Hg in canned goods is
272
important from a public health perspective.
273
The results of Hg determination in canned fish are presented in Table 5. It can be seen that
274
the Hg concentration in all samples of canned fish analyzed did not exceed the FAO/ WHO
275
limit of 0.5 mg kg
276
was analyzed. Prior to analysis, the liquid samples were filtrated though a 0.45 µm filter.
277
In two samples of canned light tuna (John West and Lucky Star, Thailand) Hg concentration
278
of 68±5 ng g-1 and 199±40 ng g-1 were measured. These values for Hg in canned tuna fish are
279
in close agreement with those found by Burger for light tuna (Burger & Gochveld, 2004). In
280
the case of canned tuna, the amount of Hg obtained by the consumption of one can was 39 µg
281
kg-1 body weight (60 kg). International agencies (WHO, 2008) indicate a provisional
282
tolerable daily intake of methylmercury as 0.1 µg kg-1 body weight. The implication is that a
283
single can of tuna, with Hg concentration 199 ng g-1 should be shared by four persons and a
284
can of Pacific salmon or marinated herring by two persons. The other tested canned fish and
285
sea products: anchovies, brisling sardines, mackerel fillet, murky octopus, smoked oysters,
286
South African and Portuguese sardines may be consumed without limitation.
-1
(500 ng g-1). In some canned fish products, the liquid part (oil /water)
287 288
4. Conclusions
289
Total mercury in fish and sea products purchased from the Tshwane market (South Africa)
290
has been determined by DTD AAS method in wet and dry samples using a RA-915+ mercury
291
analyzer. Limit of detection (LOD) and limit of quantification (LOQ) values were 0.58 ng g -1
292
and 1.93 ng g
293
respectively. The method of DTD does not require any chemical pretreatment of fish and
294
gives precise, reliable values of for a wide range of Hg concentrations. The obtained results
-1
for DTD AAS method and 5.8 µg kg
-1
and 19.2 µg kg
-1
for CVAAS,
12
295
demonstrate that solid CRMs can be used for calibration of mercury analyzer for Hg
296
determination in wet fish samples.
297
It was shown that the analysis of dry fish samples and sea products has some advantages over
298
wet samples, such as shorter time of analysis, improved analytical signal and long time
299
stability of the samples. This method was found to be very efficient for the analysis of
300
seafood samples with low Hg concentrations. Therefore it can be used for studies linked to
301
early stages of biological accumulation of Hg by marine species. The Hg concentration in wet
302
samples can be calculated with high accuracy using the moisture correction factor- R w/d.
303
From the results follows that the Hg concentration in all fresh fish species obtained from the
304
Tshwane fish market were below 0.5 mg kg-1 (500 ng g-1), wet weight, as recommended by
305
the FAO/WHO 2008, with the exceptions of frozen angelfish, tuna and skate, in which Hg
306
concentrations were found to be above this limit.
307
The information on mercury concentration in marine fishes and sea products, commonly
308
marketed in South Africa, can be used by fish consumers for the choice of fish and the
309
amount consumed.
310
311
Acknowledgments
312 313
The authors wish to thank South African National Research Foundation for financial support
314
(Grants No 77135:2011 and No 81298:2012) and members of the Research and Innovation
315
Department of Tshwane University of Technology, Ingrid Botha and Rita Raseleka, for their
316
support of this study.
317 318
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Obeid, P.J., El-Khoury, B., Burger, J., Aouad, S., Younis, M., Aoun, A. & El-Nakat, J.H.
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368
dietary importance from the coast of Sinaloa (SE Gulf of California). J. Food Compos. Anal.,
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drying conditions of fish tissues for inorganic mercury and methylmercury speciation
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373
Sholupov, S., Pogarev, S., Ryzhov, V., Mashyanov, N., Stroganov, A. (2004). Zeeman
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atomic absorption spectrometer RA-915+ for direct determination of mercury in air and
375
complex matrix samples. Fuel Process Technology, 84, 473-485.
376
Shrivastava, A., Gupta, V. (2011). Methods for the determination of limit of detection and
377
limit of quantification of the analytical methods. Chronicles of Young Scientists, 2, 21.
378
Torres, D. P., Martins-Teixeira, M. B., Silvia, E.F., Queeiroz, H.M. (2012). Method
379
development for the control determination of mercury in seafood by solid-sampling thermal
380
decomposition amalgamation atomic absorption spectrometry (TDA AAS). Food Addit.
381
Contam. Part A, 29, 625-632.
382
US Enviromental Protection Agency (US EPA). Mercury in solids and solutions by thermal
383
decomposition, amalgamation, and atomic absorption spectrophotometry. Method 7473.
384
Available at: http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/7473.pdf
385
US Environmental Protection Agency (US EPA). (1997).
386
Congress. Volume 1: Executive Summary. EPA 452/R-97-003. Washington, DC.
387
US Enviromental Protection Agency (US EPA). Fish Consumption Advisories. Available at:
388
http://www.epa.gov/hg/advisories.htm
Mercury Study Report to
16
389
United States Food and Drug Administration (US FDA). Mercury concentrations in
390
commercial
391
http://www.fda.gov/food/foodborneillnesscontaminants/metals/ucm115644.htm
392
Voegborlo, R.B., Akagi, H. (2007). Determination of mercury in fish by cold vapour atomic
393
absorption spectrometry using an automatic mercury analyzer. Food Chemistry, 100, 853-
394
858.
395
Wang, H.-Sh., Xu, W.-F., Chen, Zh.-J., Cheng, Zh., Ge, L.-Ch., Man, Y.-B., Giesy, J.P., Du,
396
J., Wong, Chr.K.C., Wong, M.-H. (2013). In vitro estimation of exposure of Hong Kong
397
residents to mercury and methylmercury via consumption of market fishes. J. Hazard. Mater.,
398
248-249, 387-393.
399
WHO.(1990). Environmental Health
400
Switzerland. Available at: http://www.inchem.org/documents/ehc/ehc/ehc101.htm
401
United Nations Environment Programme (UNEP), World Health Organization. (2008)
402
Guidance for identifying populations at risk from mercury exposure. Issued by UNEP DTIE
403
Chemicals Branch and WHO Department of Food Safety, Zoonoses and Foodborne Diseases,
404
Geneva, Switzerland.
fish
and
shellfish
Criteria.
(1990-2010).
Available
at:
Methylmercury. vol. 101, Geneva,
405 406
407
17
408
Figure captions
409
Fig.1. Schematic diagram of the Lumex RA-915+ mercury analyzer: Fish solid sample (1);
410
First tube heated to 700 oC (2); Second tube heated to 800 oC (3); Analytical cell heated to 800
411
o
412
lamp (9);Computer data base (10).
C (4); Mirror (5); Mercury trap sorbent (6); Air outlet (7); Body of RA-915+ (8); Mercury
413
414
Fig.2. Calibration curve for Hg determination
415 416
18
417 418
419 420 421 422
Fig.1
423 424 425 426 427 428 429
430
19
431 432 433 434
120000
Peak area, arbitrary unit
100000 y = 372,1x + 39,0 R² = 0,9984
80000 60000 40000 20000 0 0
50
100
150
200
250
300
350
Absolute mass of Hg, ng
435 436 437 438 439 440 441 442
Fig. 2.
443
444
445
20
446 447 448
Table 1 The results of Hg determination in Certified Reference Materials (CRMs) Certified Reference Material CRM 1515, Apple leaves MESS-3, marine sediments TORT-2, Lobster Hepatopancreas,
449 450 451
a
Certified value (ng g-1) 40 ± 4 90 ± 6 270 ± 60
Found value (ng g-1) a 38 ± 6 91 ± 4 283 ± 45
Recovery % 95.0 101.1 104.8
Average of six determinations at 95% level of confidence: mean ± t 0.05 ×(s/√n).
452
453
21
454 455
Table 2. Comparison of total Hg determination in CRM TORT-2 and fish samples by direct thermal decomposition (DTD) and cold vapour atomic absorption spectrometry (CVAAS) Samples CRM Tort-2
456 457
Hg concentration, C ± SD, ng g-1 Cold Vapour Thermo Generation decomposition 272 ± 12 272 ± 6.2 *n=8 n=6
Kariba Kapenta, sun- dried, salted
315 ± 16 n=5
324 ±14 n=4
Mackerel, frozen, 0.79 kg
48 ± 7.5 n=7
47 ± 3.0 n=3
Skate wings, sample1, frozen, chunk
963 ± 30 n=6
942 ± 80 n=4
Skate wings, sample 3 frozen, chunk
242 ± 16 n=6
227 ± 14 n=3
Slinger, fresh, 1.28 kg
86 ± 8.0 n=6
93 ± 9.0 n=3
Soldier, fresh 0.98 kg
171 ± 10 n=6
178 ± 10 n=4
* number of measurements
458
459
460
22
461 462 463
Table 3 Total Hg concentrations in dry, wet and frozen fish samples from the Tshwane fish market Fish name
Fish weight (kg)
Hg concentration Mean ± SD, (ng g-1), n or f Dry weight Cdr
Angelfish (Pomacanthus semicirculatus)
0.46 0.53 1.12 * 1.56 *
Codfish(Gadus morhua) Hake (Merluccius capensis)
1.32 salted, dry 0.76 0.78 1.00 1.25 1.82
Jacopever (Helicolenus dactylopterus)
0.78 * 0.96 *
John Dory (Zeus faber)
Chunk*
Kabeljou (Argyrosomus hololepidotus)
1.18
Kariba Kapenta (Stolothrissa tanganical)
Kingklip
1.38 mixture salted, sundried 1.60
∆ % **
Wet weight
Ratio of wet/dry fish weight Mean ± CL n or f
measured Cm 35 ±3.5 n=3 83.3 ±2.9 n=3 372±8.5 n=4 505±24 n=5 -
calculated Ccalc 35
0
85.3
2.4
374
0.54
505
0
-
-
229±13 n=6 598± 7.0 n=7 298±8. 9 n=3 358 ± 16 f=4 682±56 n=3 1458±33 f=4 1804±171 f=5 1912±94 n=3
47.8±1.6 n=5 112±2.1 n=2 58.3±4.0 n=3 76.7±3.8 n=3 147±3.6 n=3 389±39 n=3 478±36 n=4 452±10 n=3
48.2
0.8
126
12
62.7
7.5
75.4
1.7
144
2.0
362
6.9
448
6.3
469
3.8
4.08±0.04 n=3
360±1.7 n=3 235±13 n=3 301 ±7.3 f=7
98.7±3.5 n=3 -
81.1
18
4.44±0.36 n=3
52.9
-
-
-
-
-
886±66
178±11
205
14.6
4.33±0.08
119± 5.9 n=3 290±15 f=4 1270±26 n=3 1510±38 f=6 115±7.8 n=3
3.40±0.15 f=9
2.99±0.22 n=6 -
4.75±0.17 f=7
4.03±0.18 n=4
23
(Xiphiurus capensis)
1.99 2.18
Mackerel (Scombe japonicas)
0.31 *
0.33* 0.79* Monkfish tail
chunk*
Panga (Pterogymnus laniarius)
0.36 *
steak
1.85
575±38
0.37 *
chunk 1* chunk 2* chunk 3 * 0.25 0.26 0.32* 0.33* 0.35* 0.99 1.86
Salmon (Canadian salmon, Salmonidae, Oncorhynchus keta) Salmon (Cape
283±30 n=3 447±41 n=3 159±23 n=7 414±48 n=3 1113±12 n=3 1773±65 n=3 651±40 n=3 711±46 n=3 756±48 n=3 334±6.4 n=3 436±9.0 n=3 217±5.0 n=3 238±4.6 n=3 238±6.0 n=3 798±60 n=3 529±15 f=4 87.7±11 n=3 -
0.39 *
Rockcod (Epinephelus chlorostigma)
n=4 104± 6.0 n=3 166±13 n=3 -
n=3 107
2.9
139
16.3
67.6
-
3.15±0.07 n=5
0.32*
Ribbonfish (Trichiurus lepturus)
f=4 464±8.5 n=3 603±12 n=3 213±12 n=4
2.10
-
89.8
-
-
142
-
47.0±3.0 n=3 277±26 n=4 101±7.4 n=3 272±15 n=3 440±20 n=3 183±15 n=5 201±15 n=4 225±21 n=3 73.7±1.2 n=3 86.7±1.5 n=3 -
50.5
7.4
-
-
-
96
5.0
4.33±0.08 f=6
257
5.5
409
7.0
156
15
170
15
181
20
72.6
1.5
94.8
9.3
47
-
-
52
-
-
52
-
172±2.6 n=3 109±2.5 n=3 -
173
0.6
115
5.5
24.1
-
25.3±1.2 n=3
-
-
-
143
-
4.18±0.14 n=3
4.60±0.16 f=7
3.64±0.06 f=9
4.02±0.07 24
salmon, Atractoscion aequidens) Salmon (Chinese chum salmon, Oncorhynchus keta) Salmon (Norwegian salmon, Salmonidae, Salmor salar)
n=12
1.73
-
33±4.2 n=3
-
-
3.64±0.06 n=3
1.82
153±12 n=3 75.0±10 n=3 42.0±2.0 n=3 48.0±4.4 n=3 34.7±3.5 n=3 39.7±1.5 n=3 110±27 n=3 -
-
46.4
-
3.30±0.12 f=6
-
22.7
-
11.0±1.0 n=3 -
12.7
15.
14.5
-
9.8±0.41 n=3 -
10.5
7.1
12.0
-
-
33.3
-
16.0±0.6 n=3 109±5.7 n=3 121±3.2 n=3 172±3.0 n=3 870 ±23 n=3 67.0±12 n=3 60.3±5.7 n=3 -
-
-
112
2.8
116
4.1
160
7.0
2.0 2.72 2.82 2.84
3.1 4.0 4.1 Silver flesh spinecheek (Scolopsis vosmeri)
0.64 0.66 0.92
Skate (Rajidae)
427±9.6 f=4 423±16 f=4 585±8.9 f=4
chunk 1* chunk 2
Slinger (Chrysoblephus puniceus)
n=6
0.24 0.4 0.45 0.57 0.58 0.59 0.7 * 0.73
237±9.5 n=3 288±12 n=3 542±9.0 n=3 209±7.3 n=4 430±34 n=8 704±24 n=6 414±8.5 f=4 280±9.9 n=3 323±19 n=6
3.80±0.08 n=3 3.66±0.12 n=3
4.00 ±0.094 f=10 59.2
12
61.0
1.2
115
-
48.7±2.5 n=3 79.0±1.4 n=2 120±5.7 n=2 97.0±2.6 n=3 -
44.3
9.0
91.1
15
149
24
87.7
10
59.3
-
69.0±1.0 n=3
68.4
0.9
4.72±0.08 n=3
25
0.75* 0.9 * 1.1 * 1.11 Snapper (Lutjanus sanguineus )
0.84 1.90 2.19 1.21*
Snoek (Thyrsites atun)
1.38 * 2.24 *
Soldierfish (Myripristinae)
0.35 0.69
Sole (Sole large, Bothidae)
0.5 0.5 0.8 0.8 0.8 1.12
Trout (freshwater, Salmoninae)
0.54 0.81 * 0.84
Tuna (Yellowtail tuna, Seriola lalandi)
0.58 1.74 3.6
steak *
380±30 n=3 420±131 n=3 749±16 n=3 474±12 n=3 155±12 n=3 113±4.2 n=3 159±25 n=3 240±9.5 n=3 303±19 f=9 601±26 f=4 515±9.0 n=3 483±13 n=5 210±10 n=3 160±7.1 n=3 210±12 n=4 78.7±1.5 n=3 260±3.0 n=3 103±4.6 n=3 170±6.1 n=3 128±8.7 n=4 167±2.5 n=3 309±17 n=3 461±23 n=3 794±23 n=3 1697±100
-
80.5
-
-
89.0
-
-
159
-
98.2±6.1 n=4 37.0±5.6 n=3 30.7±2.2 n=3 51.7±7.8 n=3 -
100
0.8
37.4
1.1
27.3
11
38.4
26
71
-
97.6±7.2 n=5 169±17 n=4 -
89.6
8.2
178
5.3
-
-
90.0±1.0 n=3 -
-
-
44.9
-
-
34.2
-
44.7±0.58 n=3 16±2.5 n=3 47.0±2.6 n=3 15±2.6 n=3 52.3±3.8 n=3 55±10 n=3 54.7±4.5 n=3 74.0±9.6 n=3 -
44.9
0.4
16.8
5.0
55.6
18
22.0
47
50.3
3.8
37.9
31
49.4
9.7
73.2
1.1
109
-
-
188
-
515±10
517
0.4
4.14±0.05 n=7
3.38±0.22 f=7
4.68±0.18 f=6
3.38±0.17 f=7
4.22±0.22 n=3
3.28±0.07 26
n=3 Tuna (Sashimi tuna steaks) Tuna (Tuna loin)
steak* steak*
Tuna (Big eye tuna, Scombridae)
steak*
476±33 n=5 1507±24 n=6
n=3 534±23 n=3 158±9.2 f=6 457±16 n=6
f=6
149
5.7
471
3.0
3.20±0.04 f=6
464 465
- data not available
466 467
*-frozen fish
468
**- ∆ % =
C m − C Calc Cm
× 100 %
469 470
471
472
27
473
Table 4 Total Hg concentrations in selected sea products Sea food name
Calamari tubes (Loliginidae)
Weight of a product, (g) or weight per unit (g/unit) 86 90* 95 110 150 steak*
Clams (Whole clams, Veneridae) Clam meat ( Venerupis Variegata) Crab (Alaskan red crab, Paralithodes camtschaticus)
Mussel (Mytilus edulis)
Mussel(Mytilus Chilensis)
Octopus tentacle (Octopoda)
mixture * mixture *, 0.7/unit section I* section* II mixture * 4/unit, I, II, III
mixture * 3.35/unit 500 piece
Prawns pink, tails, jumbo (Nalporoldes triarthus)
Hg concentration Mean ± SD ( ng g-1) n or f Dry weight, Cdr
∆% **
Wet weight
Ratio of wet/dry fish weight Mean±CL n or f
measured, Cm -
calculated Ccalc 8.6
-
-
4.2
-
12±2.0 n=3 -
10.1
16
6.0
-
-
4.7
-
-
27.8
-
≤LOD
-
-
-
23±4.5 n=5
-
-
-
111±11 n=3
27.6±4.6 n=5
33.2
20
3.34±0.07 1 n=4
128±12 n=6 33.0±4.0 n=3 21±9.3 n=6 74±18 n=3 19±2.0 n=3
37.0±2.0 n=3
38.3
3.5
-
5.6
-
923±63 f=4 818±16 n=3 465 ±19 n=3 743±12
-
137
-
126±14 n=3 106±5.0 n=3 157±12
121
4.0
102
3.8
163
3.8
75.3±7.3 n=6 37.3±3.1 n=3 89±10 n=5 52.7±1.5 n=3 41.3±2.3 n=3 244±9.0 n=3 14.8±1.9 n=4 81±9.7 n=4
9.8
8.78±0.54 n=3
3.37±0.29 n=3
6.2 22.0
6.75±0.43 n=3
4.55± 0.14 f=6 28
23/unit*
Prawns, 60/80 (Penaeus Monodon) Large pink prawn tails (Panadalus borealis) Scallop roe (Pectinidae) Shrimp (Caridea)
4.8/unit* 22/unit*
-* -*
n=3 1160±44 n=3 26.5±2.6 n=6 147±4.6 n=3 24±4.0 n=3 18.2±0.75 n=6
n=3 254±10
255
0.4
4.8
4.3
30.8
0.6
-
7.0
-
≤ LOD
-
-
4.6±0.17 n=3 31.0±2.6 n=3
5.55±0.52 n=3 4.78±0.31 n=3 3.42±0.22 n=4 -
474 475
- data not available
476 477
* -frozen product
478
** ∆ %
**-
=
C m − C Calc Cm
× 100 %
479
480
481
29
482
Table 5 Total Hg concentrations in canned fish Sample Product name, N weight and drained weight (g)
Analyzed sample state
1
Drained meat Sunflower oil Drained meat
3
10±1.0
3
1.1±0.12
3
Drained meat
Drained meat Liquid fraction, filtered Drained meat
2
3
4
5
6
7
8
9
10
Anchovies, vegetable oil added, 80, 56 Brisling sardines , vegetable oil added (Sprattus sprattus), smoked, 106, 80 Herring (marinated fillets), Matjes style, 200 Light meat Tuna chunks, 170, 120
Light meat Tuna chunks, 170, 119 Mackerel fillet in brine, 125, 90
Musky octopus in vegetable oil, 100, 60 Pink Salmon, 212
Portuguese sardines from Algarve in olive oil, 120 Smoked oysters,
Calculated Hg µg kg-1 body weight (60kg) 0.01
Producer, country of production
27.7±0.76
0.037
Latvia
4
57.2±6.0
0.19
Germany
3
68.0±4.6
0.14
JOHN WEST, Thailand
3
≤LOD
3
199±40
0.39
3
58.3±4.2
0.090
LUCKY STAR, Thailand JOHN WEST, Portugal
4
3.8±1.6
3
67.3±1.2
3
2.3±0.20
Drained meat
3
Drained meat Olive oil, filtered Drained meat
Drained meat Water fraction filtered Drained meat Soya oil
n*
Concentration of Hg C±SD (ng g-1 )
Morocco
0.069
TRATA, Greece
48±5.0
0.17
5
48.0±2.0
0.096
WILD ALASKA SALMON, USA Estovil, Portugal
3
≤LOD
4
25.8±1.5
0.037
China 30
cottonseed oil added, 85 11
Snails in brine
12
South Africa sardines, vegetable oil added (Sardinops sagax), 120
Cottonseed oil Drained meat Dried meat Drained meat Sunflower oil, filtered
3
≤LOD
3
≤LOD
-
5 3
6.3±1.2 18±2.0
0.036
3
29±4.0
Indonesia
LUCKY STAR, South Africa
483 484
n*- Number of measurements
485
486 487
31
488
Highlights
489 490
1. A method of direct thermal decomposition for Hg measurement in raw samples of fish was developed.
491
2. Samples of wet fish were analyzed without any chemical pretreatment.
492
3. There were no losses of Hg during analysis of wet and dry fish samples.
493 494
4. The concentration of Hg in most fishes from Tshwane market (Pretoria, South SAfrica) was found to be within safety limits.
495 496
32
S0308-8146(14)00918-2 http://dx.doi.org/10.1016/j.foodchem.2014.06.032 FOCH 15974
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
20 July 2013 1 March 2014 8 June 2014
Please cite this article as: Panichev, N.A., Panicheva, S.E., Determination of Total Mercury in Fish and Sea Products by Direct Thermal Decomposition Atomic Absorption Spectrometry, Food Chemistry (2014), doi: http://dx.doi.org/ 10.1016/j.foodchem.2014.06.032
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1
Determination of Total Mercury in Fish and Sea Products by Direct Thermal
2
Decomposition Atomic Absorption Spectrometry
3 4
N. A. Panichev*, S. E. Panicheva
5
Department of Chemistry, Tshwane University of Technology, P.O. Box 56208, Arcadia
6
0007, Pretoria, South Africa
7
*Corresponding author. Tel: +27 12 382 6233; Cell: +27 73 661 6061;
8
Fax: +27 12 382 6286
9
E–mail: [email protected]
10
11
1. Introduction
12
According to the Food and Agriculture Organization of the United Nations (FAO), it is
13
estimated that the world consumed about 154 million tonnes of fish in 2011 and that about
14
50 percent of this originated from aquaculture. Although precise data are lacking, it is
15
acknowledged that, with growth in volume and value of fish production in the past decade,
16
aquaculture has made a positive contribution to national, regional and global economies,
17
poverty reduction and food security (FAO, 2012).
18
Other factors driving the market include a growing trend towards healthy eating. Due to the
19
high protein content of fish, it is becoming an ever-more popular choice for health conscious
20
consumers wishing to avoid the health drawbacks of meat. Despite fish and seafood
21
containing key nutrients, including omega-3 polyunsaturated fatty acids, essential amino
22
acids, trace elements and vitamins, they are also accumulators of mercury, posing a potential
23
threat to human health.
24
In aquatic environments, mercury is transformed by microorganisms into methylmercury
25
(MeHg), which bioaccumulates and biomagnifies through the food chain, which leads to high 1
26
concentrations at the top of aquatic food chain (WHO, 1990). Concerns over the toxicity and
27
human health risk of Hg deposited in ecosystems and bioaccumulating as MeHg in fish, has
28
prompted efforts to regulate anthropogenic emissions of that element (Mergler et al., 2007). It
29
is important for the public to be sufficiently informed prior to choosing what species of fish
30
to eat, as well as what size and how often it may be consumed without risk. The
31
concentration limit for Hg in fish for human consumption is set at 0.5 µg g-1( 500 ng g-1) wet
32
weight, (ww), (CCME, 1999; EEC, 2001; US EPA, 1997;); and 0.2 µg g-1 (200 ng g-1) (ww)
33
(WHO, 1990) for vulnerable groups, such as pregnant women, individuals under 15 years or
34
frequent fish consumers.
35
Although, in most cases, fish is consumed cooked, the majority of studies related to the
36
presence and the daily intake of mercury from the consumption of sea food provide data from
37
uncooked/raw products. To establish the maximum permissible Hg concentration in fish for
38
human consumption, the FAO (FAO/WHO, 1991) initiated a surveying of Hg concentration
39
in natural fish populations. Levels of Hg in fish from local markets have been reported by
40
many authors, covering different countries (Burger & Gochfeld, 2006; Kojadinovich et al.,
41
2006; Chien et al., 2007; Ruelas-Inzunza et al., 2008; Hajeb et al., 2009; Katner et al., 2010;
42
Burger &Gochfeld, 2011; Obeid et al., 2011; Focardi, 2012; Bonsignore et al., 2013; Wang et
43
al., 2013). However, information from African countries is limited to a publication
44
(Voegborlo & Akagi, 2007) about the Hg concentrations in fish from the Atlantic coast of
45
Ghana. The lack of specific information about the levels of Hg in fish and sea products from
46
South African market does not permit consumers to intelligently control their intake of fish.
47
Currently, the total Hg concentration in fish samples is usually performed by cold vapour
48
atomic absorption spectrometry (CVAAS). In this method, the sample is digested with hot,
49
concentrated mineral acids such as nitric, sulfuric, perchloric together with hydrogen
50
peroxide (Inhant, 2003). The sample pretreatment step, using wet digestion, requires about
2
51
30-60 min for oxidation of 0.5-1.0 g of fish. The method is time consuming and complicated
52
by the possibility of losses through volatilization or incomplete digestion as well as
53
contamination of the samples ( Kuboyama et al., 2005).
54
Measurements of Hg in solids matrices without chemical treatment of samples recently
55
become available by integration of thermal decomposition of samples, collections of Hg
56
vapour by amalgamation on gold wires or sponge, followed by atomic absorption
57
determination after thermal release of Hg from golden wire (US EPA method 7473).
58
Analysis of fish by method of solid sampling thermal decomposition, in which Hg is
59
selectively trapped on a gold amalgamator have been described (Torres et al., 2012). Upon
60
heating, Hg is desorbed from the amalgamator for an atomic absorption measurement. The
61
limit of quantification (LOQ) was 0.3 ng g-1. The only disadvantage of such systems could be
62
connected with Hg measurements in two steps: collection of Hg on golden sorbent and
63
evaporation of Hg into measuring cuvette. Each step should be properly optimized.
64
This study was undertaken with two main objectives. The first was to develop a method for
65
the determination of Hg in fish and sea products by direct thermal decomposition of the
66
samples, without preliminary collection of Hg vapour on golden amalgamator. The second
67
objective was to create a database for total Hg in fish and sea products purchased from the
68
local Tshwane fish market in Pretoria, South Africa. This would enable the evaluation of
69
fresh, frozen, salted, dried and canned fish of South African origin or obtained from her
70
trading partners.
71 72
2. Experimental
73 74
2.1 Materials, reagents and solutions 3
75 76
Nitric acid, HNO3, (Superpure, 60%, Merck, Germany) , hydrochloric acid, HCl (superpure,
77
30%, Merk, Germany) and hydrogen peroxide, H2O2, analytical grade (Merck) were used in
78
the digestion of fish samples. SnCl2 ·2 H2O (Rochelle Chemicals) was used to prepare the
79
reducing solution. For the dilution of digested fish samples 7% v/v HCl was used.
80
Hg standard solutions were prepared in the range 5-100 µg L-1 from 1001 ± 0.002 mg L-1 Hg
81
standard SS-1232 (Spectroscan) by dilution in 7% HCl. High-purity water (resistivity 18.2
82
MΩ cm) was used to prepare all aqueous solutions.
83 84
2.2 Certified Reference Materials
85
Several standard and certified reference materials (CRMs) for the determination of Hg were
86
used in this study: SRM 1515, apple leaves, (National institute of Standards and Technology,
87
U.S. Department of commerce), certified value 44 ± 4 ng g-1 , CRM 7002, light sandy soil
88
(Analytika Co Ltd, Czech Republic), certified value 90 ± 12 ng g-1 and CRM 024-050, metals
89
on soil, (Resource Technology Corporation, USA), certified value 710 ± 110 ng g-1 were
90
used for calibrating the RA-915+ Zeeman Mercury analyzer. CRM MESS-3, marine
91
sediment, certified value 90 ± 6 ng g-1 and CRM TORT-2 lobster hepatopancreas (National
92
Research Council of Canada), certified value 270 ± 60 ng g-1 were used for validation of
93
results.
94 95
2.3 Apparatus
96
An analytical balance RADWAG model AS 220/C/2AY was used to weigh the samples using
97
units of milligrams For Hg determination by CVAAS, the microwave MARS 6 (CEM
98
Corporation, USA) was used for fish samples digestion. The equipment contains 16
99
digestions Teflon vessels with a capacity of approximately 100 ml each. A Model RA-915+ 4
100
Zeeman Mercury analyzer (Lumex, St. Petersburg, Russia) with a PYRO-915 and CVAAS
101
attachments were used for Hg measurements. The working principle of the instrument (Fig.1)
102
is based on the thermal evaporation of Hg from a sample (1) placed in two-stage pyrolysis
103
tubes (2 and 3) heated to 750 and 800 oC. After inserting a known amount of sample into the
104
preheated tube, Hg vapour, together with smoke, formed by the combustion of the matrix’s
105
organic matter, are transported into the analytical cell (4), with a total optical length of 0.4 m,
106
and is also heated to 800oC. Background absorption is eliminated by the high-frequency
107
Zeeman correction system. The content of Hg in the sample is determined from the
108
calibration curve of the absolute amount of Hg (ng) versus the integrated analytical signal.
109
Finally, Hg0 vapour were absorbed in Hg trap (6)to prevent laboratory air contamination.
110
The PYRO-915 attachment enables Hg determination in samples with complex-matrices,
111
such as soils, sediments, oil products, foodstuff, etc., using pyrolysis technique without
112
chemical pretreatment of samples (Sholupov et al., 2004, Huang et al., 2005). Real-time
113
measurements are made with visualization of the process on a computer display. Without any
114
chemical pretreatment or the addition of chemical modifiers the risk of sample contamination
115
is minimized.
116
117
2.4. Fish samples collection and preparation
118 119
Using the Tshwane market for obtaining samples, 39 of the most popular species of fish and
120
sea food were collected as fresh (wet) and/or frozen for the determination of total Hg. Frozen
121
fish were thawed and analyzed as “wet”. The samples for analysis were cut from the fillet of
122
the fish and divided into several portions. Some were analyses as “wet” samples, the others
123
were air dried at room temperature (about 25 oC) for a week and then grounded in a mill. The
5
124
homogenized material was analyzed as “dry” samples. To prevent the samples from
125
degrading and losses Hg prior to analysis they were kept in small plastic bags in a refrigerator
126
(Schmidt et al., 2013).
127
2.5 Analytical procedure of Hg determination in fish
128
Each sample taken from the refrigerator was weighed on an analytical balance in milligrams.
129
The mass of samples ranged between 30 and 300 mg. The exact weight of the fish sample
130
was imported to the analyzer software. Using a quartz weighing boat, the sample was inserted
131
into the furnace of the analyzer. The software displays a curve of Hg absorption, the area of
132
the peak, the maximum value of the absorption and the calculated concentration of mercury
133
in the sample. A baseline check was performed periodically on the instrument to insure that
134
zero line did not shift.
135
Direct analysis of fish samples affords many benefits. Eliminating wet chemistry greatly
136
reduces waste generation, systematic errors and technician exposure due to the volatilization
137
of chemicals and Hg during sample handling. Direct analysis of a wet fish typically provides
138
an answer in approximately three minutes after the sample had been introduced into the
139
instrument. For sea products and samples of fish with low Hg concentrations, the absorption
140
signal is usually low and extends for several minutes. It was decided that the analysis of dry
141
fish samples could improve accuracy. For this purpose, fish was analyzed as wet and dry
142
samples.
143
3. Results and discussions
144
3.1. Analytical performance of mercury analyzer
145 146
3.1.1. Calibration graph
147
6
148
The calibration curve (Fig 2) for the Hg determination has been plotted as absolute mass of
149
Hg (ng) versus absorption peak area (arbitrary units). The absolute mass of mercury (m
150
was calculated from the following relationship between the certified value of the Hg
151
concentration (C Hg) and CRMs mass, (m CRM ), taken for the analysis: m Hg (ng) = C Hg (ng mg-1) x m CRM (mg)
152
Hg)
(1)
153 154
The calibration curve, described by the equation: y = 372.1x+ 39.0, exhibited excellent
155
linearity (R2 value of 0.998) from 2.5 ng up to 300 ng Hg mass. These amounts are
156
equivalent to Hg concentration of 10 to 1200 ng g-1 in a wet fish sample, assuming a sample
157
weight of 0.250 g.
158 159
3.1.2. The Limit of Detection
160
161
Due to the absence of fish samples without Hg, which could be used as blanks, the limit of
162
detection (LOD = 3 Sa/b) and limit of quantification (LOQ = 10 Sa/b) were calculated from
163
the data of the calibration curve presented in general form as y =a +bx where Sa is the
164
standard deviation of the response y and b is the slope of calibration curve. This method is the
165
most applicable when the method of analyses does not involve background noise (
166
Shrivastava, 2011).
167
ANOVA (ANOVA Statistics), and were found to be 0.15 ng and 0.50 ng of absolute mass of
168
Hg or 0.6 ng g-1 and 2.0 ng g-1 for 250 mg of a wet fish sample. The results indicate that it is
169
possible to measure mercury in fish at concentrations much lower than the 0.5 µg g-1 (500 ng
170
g-1) limit.
Numerical calculations of LOD and LOQ were performed using
171
7
172
3.1.3. Validation of the method
173
The accuracy of the direct thermal decomposition atomic absorption spectrometry (DTD
174
AAS) method
175
materials and by the analysis of selected fish samples by an CVAAS, which is the most-used
176
technique for Hg measurements in fish (?) and is US EPA accepted method (US EPA method
177
245.6).
178
The results of Hg determination in CRMs are presented in Table 1. Although the standards
179
used to test the accuracy of the DTD method had different origins, the results of
180
measurements show good correlation between certified and found values. The recovery of Hg
181
in all analyzed CRMs was in the range of 95-106 % and the total Hg content appear to be
182
independent of the nature of the reference materials.
183
For a comparative determination of Hg in fish by CVAAS, five samples of fish covering a
184
range of Hg concentration between 50 and 300 ng g-1 were analyzed. For this, approximately
185
0.5 g of each fish, were digested in 5.0 mL HNO3 + 2.0 mL H2O2 in microwave MARS 6.
186
After digestion, all samples were diluted to 25 mL with 7% solution of HCl.. The Hg
187
measurement was performed by Lumex Zeeman mercury analyzer RA-915+ with an
188
attachment for CVAAS analysis. The calibration graph (y=194.47x – 21.63, R2=0.999) was
189
plotted using a set of 6 concentrations of diluted Hg aqueous standards. The LOD of Hg
190
determination was found to be 5.8 ng g-1 and LOQ 19.2 ng g-1 in wet fish, when 0.5 g of fish
191
tissue had been digested and diluted to 25.0 mL after decomposition. These results are in a
192
good correspondence with LOD = 4.9 ng g-1 and LOQ = 15.7 ng g-1 obtained during CVAAS
193
method validation for the determination of Hg in fish (Nascimento et al, 2012).
194
The results of Hg determination by the new DTD and a reference CVAAS method are
195
presented in Table 2. The comparison of the means was carried out on the basis of a null
196
hypothesis (Miller and Miller, 2005). The result for the comparison of the means do not differ
was validated by analyzing three different types of certified reference
8
197
significantly at the 95% confidence level, and therefore, the concentration of Hg found by
198
both methods in the analyzed fish samples were found to be statistically equivalent.
199
It should be noted that the results for the determination of Hg in fish using the DTD method
200
are available in 6 min, while using CVASS the results were only known after at least 6 h, due
201
to the time required for samples decomposition.
202 203
3.1.4. Determination of moisture loses
204 205
Due to the high moisture content of the fish, the appearance of the analytical signal was
206
shifted, in time, until all the water evaporated and the sample reached the temperature of
207
thermal decomposition. As a result, the time of analysis was 3.0 - 3.5 min. For some samples
208
with low Hg concentration the value of the analytical signal was slightly higher than the zero
209
line, causing the accuracy of determination to be questioned, while for dry fish samples the
210
analytical signal was much higher and perfectly symmetrical.
211
To increase the sensitivity of the determination in fish and sea products with low Hg
212
concentrations, it was decided to analyse dry as well as wet samples. Wet fish samples were
213
dried in the open air, at ambient temperature (about 25 oC), for a week. For each species of
214
fish the a moisture correction factor , Rw/d, which represents the ratio of wet fish weight (w)
215
to its dry weight (d), has been calculated (Table 3 and 4).
216
Knowledge of R
217
results of analysis of dry fish samples. The moisture content (M) in wet fish samples can be
218
calculated by the following equation:
219
w/d
permits the calculation of the Hg content in wet fish samples, using the
1 M = 1 − × 100% Rw d
(2)
9
220
It was found that the moisture content varied from 69.7 to 79.0% in fresh fish and in frozen
221
fish from 68.5 to 76.9%.
222 223
3.2. Results of Hg determination in fish
224 225
Total mercury concentrations of all analyzed samples of fish and selected sea products have
226
been summarized in Table 3. The data are sorted according to alphabetical order of common
227
fish names, starting with angelfish and ending with tuna. Since information regarding the
228
origin of the fish is usually unavailable, understand the reasons for differences in Hg
229
concentration in commercial fish is difficult, except in generalities, e.g., connected to species,
230
size and ecology of habitat (Burger & Gochfeld, 2011). Not unexpectedly, the top-level
231
predator, like tuna, was found to have the highest mercury concentration (534 ±23 ng g-1),
232
while Norwegian salmon the lowest (9.8 ± 0.4 ng g-1). The variation of Hg concentration in
233
fish of the same species is mostly associated with the size (weight) of the fish. Larger fish
234
usually have higher concentration than their smaller counterparts. In this study a positive
235
correlation in this regard was found for angelfish (R2 =0.943, n=4), rock cod (R2 = 0.917, n=3
236
), hake (R2 = 0.987, n=5) and silver flesh spincheek (R2 = 0.995, n=4).
237
The mean value of RSD% for all measurements of dry fish samples was found to be 6.1%,
238
for fresh fish samples 7.4%, and frozen fish 9.5%. Mercury concentrations in most fish
239
species, except angelfish, some species of tuna and skate, were below the 0.5 µg g-1 (500 ng
240
g-1) ,(ww), recommended by FAO/WHO. In general, the mean concentrations of Hg in fish
241
species reported in this study are close to those reported by US/FDA. The analysis of dry fish
242
had some advantages over the direct analysis of wet samples, such as shorter time of analysis,
243
improved analytical signal and long time stability of the samples. Since to the requirements
244
that only the Hg concentration of wet fish is accepted by FAO/WHO as legislative, this value
10
245
can be calculated using the data of the Hg concentration of dry samples and the moisture
246
correction factor, R w/d .
247
From data presented in Table 3, the measured Hg concentrations of wet fish and the
248
calculated values are very close Considering that the R
249
gravimetrically, the good correlation of measured (by AAS) and calculated Hg concentrations
250
clearly demonstrate that such a method can be applied for the analysis of wet samples. The
251
results confirmed that there are no Hg losses during sample preparation by air drying. They
252
also demonstrate that solid CRMs can be used for the calibration of the mercury analyzer for
253
analysis of wet fish samples. The moisture correction factor, R
254
from results of the determination of Hg by AAS on dry and wet fish samples.
w/d
values were determined
w/d,
can also be calculated
255 256
3.3 Results of Hg determination in seafood products
257 258
Shrimp (prawns) are promoted as a ‘low-mercury” seafood (Burger et al., 2005), but local
259
geological and ecological conditions may influence Hg levels in shrimp. Hence the Hg
260
concentration in shrimp may vary. The results, presented in Table 4 confirm that a level of
261
Hg in shrimp of different origin is not uniform. The lowest Hg concentration was found in
262
prawns Penaeus Monodon (4.6 ±0.3 ng g-1), the highest in prawns Nalporolders triarthus
263
(from 106 ± 5.0 to 254 ±10 ng g-1) .These concentration are comparable to those for fish
264
species.
265
Relatively high concentrations of Hg (126±14 ng g-1) were found in samples of octopus
266
(Octopoda) tentacles and its absence (≤ LOD) in shrimps (Caridea) and scallop (Pectinidae).
267 268
3.4 Results of Hg determination in canned fish
269
11
270
The samples of canned fish were also analyzed, because this product is popular and is the
271
form of fish most commonly eaten. Thus, assessing the levels of Hg in canned goods is
272
important from a public health perspective.
273
The results of Hg determination in canned fish are presented in Table 5. It can be seen that
274
the Hg concentration in all samples of canned fish analyzed did not exceed the FAO/ WHO
275
limit of 0.5 mg kg
276
was analyzed. Prior to analysis, the liquid samples were filtrated though a 0.45 µm filter.
277
In two samples of canned light tuna (John West and Lucky Star, Thailand) Hg concentration
278
of 68±5 ng g-1 and 199±40 ng g-1 were measured. These values for Hg in canned tuna fish are
279
in close agreement with those found by Burger for light tuna (Burger & Gochveld, 2004). In
280
the case of canned tuna, the amount of Hg obtained by the consumption of one can was 39 µg
281
kg-1 body weight (60 kg). International agencies (WHO, 2008) indicate a provisional
282
tolerable daily intake of methylmercury as 0.1 µg kg-1 body weight. The implication is that a
283
single can of tuna, with Hg concentration 199 ng g-1 should be shared by four persons and a
284
can of Pacific salmon or marinated herring by two persons. The other tested canned fish and
285
sea products: anchovies, brisling sardines, mackerel fillet, murky octopus, smoked oysters,
286
South African and Portuguese sardines may be consumed without limitation.
-1
(500 ng g-1). In some canned fish products, the liquid part (oil /water)
287 288
4. Conclusions
289
Total mercury in fish and sea products purchased from the Tshwane market (South Africa)
290
has been determined by DTD AAS method in wet and dry samples using a RA-915+ mercury
291
analyzer. Limit of detection (LOD) and limit of quantification (LOQ) values were 0.58 ng g -1
292
and 1.93 ng g
293
respectively. The method of DTD does not require any chemical pretreatment of fish and
294
gives precise, reliable values of for a wide range of Hg concentrations. The obtained results
-1
for DTD AAS method and 5.8 µg kg
-1
and 19.2 µg kg
-1
for CVAAS,
12
295
demonstrate that solid CRMs can be used for calibration of mercury analyzer for Hg
296
determination in wet fish samples.
297
It was shown that the analysis of dry fish samples and sea products has some advantages over
298
wet samples, such as shorter time of analysis, improved analytical signal and long time
299
stability of the samples. This method was found to be very efficient for the analysis of
300
seafood samples with low Hg concentrations. Therefore it can be used for studies linked to
301
early stages of biological accumulation of Hg by marine species. The Hg concentration in wet
302
samples can be calculated with high accuracy using the moisture correction factor- R w/d.
303
From the results follows that the Hg concentration in all fresh fish species obtained from the
304
Tshwane fish market were below 0.5 mg kg-1 (500 ng g-1), wet weight, as recommended by
305
the FAO/WHO 2008, with the exceptions of frozen angelfish, tuna and skate, in which Hg
306
concentrations were found to be above this limit.
307
The information on mercury concentration in marine fishes and sea products, commonly
308
marketed in South Africa, can be used by fish consumers for the choice of fish and the
309
amount consumed.
310
311
Acknowledgments
312 313
The authors wish to thank South African National Research Foundation for financial support
314
(Grants No 77135:2011 and No 81298:2012) and members of the Research and Innovation
315
Department of Tshwane University of Technology, Ingrid Botha and Rita Raseleka, for their
316
support of this study.
317 318
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17
408
Figure captions
409
Fig.1. Schematic diagram of the Lumex RA-915+ mercury analyzer: Fish solid sample (1);
410
First tube heated to 700 oC (2); Second tube heated to 800 oC (3); Analytical cell heated to 800
411
o
412
lamp (9);Computer data base (10).
C (4); Mirror (5); Mercury trap sorbent (6); Air outlet (7); Body of RA-915+ (8); Mercury
413
414
Fig.2. Calibration curve for Hg determination
415 416
18
417 418
419 420 421 422
Fig.1
423 424 425 426 427 428 429
430
19
431 432 433 434
120000
Peak area, arbitrary unit
100000 y = 372,1x + 39,0 R² = 0,9984
80000 60000 40000 20000 0 0
50
100
150
200
250
300
350
Absolute mass of Hg, ng
435 436 437 438 439 440 441 442
Fig. 2.
443
444
445
20
446 447 448
Table 1 The results of Hg determination in Certified Reference Materials (CRMs) Certified Reference Material CRM 1515, Apple leaves MESS-3, marine sediments TORT-2, Lobster Hepatopancreas,
449 450 451
a
Certified value (ng g-1) 40 ± 4 90 ± 6 270 ± 60
Found value (ng g-1) a 38 ± 6 91 ± 4 283 ± 45
Recovery % 95.0 101.1 104.8
Average of six determinations at 95% level of confidence: mean ± t 0.05 ×(s/√n).
452
453
21
454 455
Table 2. Comparison of total Hg determination in CRM TORT-2 and fish samples by direct thermal decomposition (DTD) and cold vapour atomic absorption spectrometry (CVAAS) Samples CRM Tort-2
456 457
Hg concentration, C ± SD, ng g-1 Cold Vapour Thermo Generation decomposition 272 ± 12 272 ± 6.2 *n=8 n=6
Kariba Kapenta, sun- dried, salted
315 ± 16 n=5
324 ±14 n=4
Mackerel, frozen, 0.79 kg
48 ± 7.5 n=7
47 ± 3.0 n=3
Skate wings, sample1, frozen, chunk
963 ± 30 n=6
942 ± 80 n=4
Skate wings, sample 3 frozen, chunk
242 ± 16 n=6
227 ± 14 n=3
Slinger, fresh, 1.28 kg
86 ± 8.0 n=6
93 ± 9.0 n=3
Soldier, fresh 0.98 kg
171 ± 10 n=6
178 ± 10 n=4
* number of measurements
458
459
460
22
461 462 463
Table 3 Total Hg concentrations in dry, wet and frozen fish samples from the Tshwane fish market Fish name
Fish weight (kg)
Hg concentration Mean ± SD, (ng g-1), n or f Dry weight Cdr
Angelfish (Pomacanthus semicirculatus)
0.46 0.53 1.12 * 1.56 *
Codfish(Gadus morhua) Hake (Merluccius capensis)
1.32 salted, dry 0.76 0.78 1.00 1.25 1.82
Jacopever (Helicolenus dactylopterus)
0.78 * 0.96 *
John Dory (Zeus faber)
Chunk*
Kabeljou (Argyrosomus hololepidotus)
1.18
Kariba Kapenta (Stolothrissa tanganical)
Kingklip
1.38 mixture salted, sundried 1.60
∆ % **
Wet weight
Ratio of wet/dry fish weight Mean ± CL n or f
measured Cm 35 ±3.5 n=3 83.3 ±2.9 n=3 372±8.5 n=4 505±24 n=5 -
calculated Ccalc 35
0
85.3
2.4
374
0.54
505
0
-
-
229±13 n=6 598± 7.0 n=7 298±8. 9 n=3 358 ± 16 f=4 682±56 n=3 1458±33 f=4 1804±171 f=5 1912±94 n=3
47.8±1.6 n=5 112±2.1 n=2 58.3±4.0 n=3 76.7±3.8 n=3 147±3.6 n=3 389±39 n=3 478±36 n=4 452±10 n=3
48.2
0.8
126
12
62.7
7.5
75.4
1.7
144
2.0
362
6.9
448
6.3
469
3.8
4.08±0.04 n=3
360±1.7 n=3 235±13 n=3 301 ±7.3 f=7
98.7±3.5 n=3 -
81.1
18
4.44±0.36 n=3
52.9
-
-
-
-
-
886±66
178±11
205
14.6
4.33±0.08
119± 5.9 n=3 290±15 f=4 1270±26 n=3 1510±38 f=6 115±7.8 n=3
3.40±0.15 f=9
2.99±0.22 n=6 -
4.75±0.17 f=7
4.03±0.18 n=4
23
(Xiphiurus capensis)
1.99 2.18
Mackerel (Scombe japonicas)
0.31 *
0.33* 0.79* Monkfish tail
chunk*
Panga (Pterogymnus laniarius)
0.36 *
steak
1.85
575±38
0.37 *
chunk 1* chunk 2* chunk 3 * 0.25 0.26 0.32* 0.33* 0.35* 0.99 1.86
Salmon (Canadian salmon, Salmonidae, Oncorhynchus keta) Salmon (Cape
283±30 n=3 447±41 n=3 159±23 n=7 414±48 n=3 1113±12 n=3 1773±65 n=3 651±40 n=3 711±46 n=3 756±48 n=3 334±6.4 n=3 436±9.0 n=3 217±5.0 n=3 238±4.6 n=3 238±6.0 n=3 798±60 n=3 529±15 f=4 87.7±11 n=3 -
0.39 *
Rockcod (Epinephelus chlorostigma)
n=4 104± 6.0 n=3 166±13 n=3 -
n=3 107
2.9
139
16.3
67.6
-
3.15±0.07 n=5
0.32*
Ribbonfish (Trichiurus lepturus)
f=4 464±8.5 n=3 603±12 n=3 213±12 n=4
2.10
-
89.8
-
-
142
-
47.0±3.0 n=3 277±26 n=4 101±7.4 n=3 272±15 n=3 440±20 n=3 183±15 n=5 201±15 n=4 225±21 n=3 73.7±1.2 n=3 86.7±1.5 n=3 -
50.5
7.4
-
-
-
96
5.0
4.33±0.08 f=6
257
5.5
409
7.0
156
15
170
15
181
20
72.6
1.5
94.8
9.3
47
-
-
52
-
-
52
-
172±2.6 n=3 109±2.5 n=3 -
173
0.6
115
5.5
24.1
-
25.3±1.2 n=3
-
-
-
143
-
4.18±0.14 n=3
4.60±0.16 f=7
3.64±0.06 f=9
4.02±0.07 24
salmon, Atractoscion aequidens) Salmon (Chinese chum salmon, Oncorhynchus keta) Salmon (Norwegian salmon, Salmonidae, Salmor salar)
n=12
1.73
-
33±4.2 n=3
-
-
3.64±0.06 n=3
1.82
153±12 n=3 75.0±10 n=3 42.0±2.0 n=3 48.0±4.4 n=3 34.7±3.5 n=3 39.7±1.5 n=3 110±27 n=3 -
-
46.4
-
3.30±0.12 f=6
-
22.7
-
11.0±1.0 n=3 -
12.7
15.
14.5
-
9.8±0.41 n=3 -
10.5
7.1
12.0
-
-
33.3
-
16.0±0.6 n=3 109±5.7 n=3 121±3.2 n=3 172±3.0 n=3 870 ±23 n=3 67.0±12 n=3 60.3±5.7 n=3 -
-
-
112
2.8
116
4.1
160
7.0
2.0 2.72 2.82 2.84
3.1 4.0 4.1 Silver flesh spinecheek (Scolopsis vosmeri)
0.64 0.66 0.92
Skate (Rajidae)
427±9.6 f=4 423±16 f=4 585±8.9 f=4
chunk 1* chunk 2
Slinger (Chrysoblephus puniceus)
n=6
0.24 0.4 0.45 0.57 0.58 0.59 0.7 * 0.73
237±9.5 n=3 288±12 n=3 542±9.0 n=3 209±7.3 n=4 430±34 n=8 704±24 n=6 414±8.5 f=4 280±9.9 n=3 323±19 n=6
3.80±0.08 n=3 3.66±0.12 n=3
4.00 ±0.094 f=10 59.2
12
61.0
1.2
115
-
48.7±2.5 n=3 79.0±1.4 n=2 120±5.7 n=2 97.0±2.6 n=3 -
44.3
9.0
91.1
15
149
24
87.7
10
59.3
-
69.0±1.0 n=3
68.4
0.9
4.72±0.08 n=3
25
0.75* 0.9 * 1.1 * 1.11 Snapper (Lutjanus sanguineus )
0.84 1.90 2.19 1.21*
Snoek (Thyrsites atun)
1.38 * 2.24 *
Soldierfish (Myripristinae)
0.35 0.69
Sole (Sole large, Bothidae)
0.5 0.5 0.8 0.8 0.8 1.12
Trout (freshwater, Salmoninae)
0.54 0.81 * 0.84
Tuna (Yellowtail tuna, Seriola lalandi)
0.58 1.74 3.6
steak *
380±30 n=3 420±131 n=3 749±16 n=3 474±12 n=3 155±12 n=3 113±4.2 n=3 159±25 n=3 240±9.5 n=3 303±19 f=9 601±26 f=4 515±9.0 n=3 483±13 n=5 210±10 n=3 160±7.1 n=3 210±12 n=4 78.7±1.5 n=3 260±3.0 n=3 103±4.6 n=3 170±6.1 n=3 128±8.7 n=4 167±2.5 n=3 309±17 n=3 461±23 n=3 794±23 n=3 1697±100
-
80.5
-
-
89.0
-
-
159
-
98.2±6.1 n=4 37.0±5.6 n=3 30.7±2.2 n=3 51.7±7.8 n=3 -
100
0.8
37.4
1.1
27.3
11
38.4
26
71
-
97.6±7.2 n=5 169±17 n=4 -
89.6
8.2
178
5.3
-
-
90.0±1.0 n=3 -
-
-
44.9
-
-
34.2
-
44.7±0.58 n=3 16±2.5 n=3 47.0±2.6 n=3 15±2.6 n=3 52.3±3.8 n=3 55±10 n=3 54.7±4.5 n=3 74.0±9.6 n=3 -
44.9
0.4
16.8
5.0
55.6
18
22.0
47
50.3
3.8
37.9
31
49.4
9.7
73.2
1.1
109
-
-
188
-
515±10
517
0.4
4.14±0.05 n=7
3.38±0.22 f=7
4.68±0.18 f=6
3.38±0.17 f=7
4.22±0.22 n=3
3.28±0.07 26
n=3 Tuna (Sashimi tuna steaks) Tuna (Tuna loin)
steak* steak*
Tuna (Big eye tuna, Scombridae)
steak*
476±33 n=5 1507±24 n=6
n=3 534±23 n=3 158±9.2 f=6 457±16 n=6
f=6
149
5.7
471
3.0
3.20±0.04 f=6
464 465
- data not available
466 467
*-frozen fish
468
**- ∆ % =
C m − C Calc Cm
× 100 %
469 470
471
472
27
473
Table 4 Total Hg concentrations in selected sea products Sea food name
Calamari tubes (Loliginidae)
Weight of a product, (g) or weight per unit (g/unit) 86 90* 95 110 150 steak*
Clams (Whole clams, Veneridae) Clam meat ( Venerupis Variegata) Crab (Alaskan red crab, Paralithodes camtschaticus)
Mussel (Mytilus edulis)
Mussel(Mytilus Chilensis)
Octopus tentacle (Octopoda)
mixture * mixture *, 0.7/unit section I* section* II mixture * 4/unit, I, II, III
mixture * 3.35/unit 500 piece
Prawns pink, tails, jumbo (Nalporoldes triarthus)
Hg concentration Mean ± SD ( ng g-1) n or f Dry weight, Cdr
∆% **
Wet weight
Ratio of wet/dry fish weight Mean±CL n or f
measured, Cm -
calculated Ccalc 8.6
-
-
4.2
-
12±2.0 n=3 -
10.1
16
6.0
-
-
4.7
-
-
27.8
-
≤LOD
-
-
-
23±4.5 n=5
-
-
-
111±11 n=3
27.6±4.6 n=5
33.2
20
3.34±0.07 1 n=4
128±12 n=6 33.0±4.0 n=3 21±9.3 n=6 74±18 n=3 19±2.0 n=3
37.0±2.0 n=3
38.3
3.5
-
5.6
-
923±63 f=4 818±16 n=3 465 ±19 n=3 743±12
-
137
-
126±14 n=3 106±5.0 n=3 157±12
121
4.0
102
3.8
163
3.8
75.3±7.3 n=6 37.3±3.1 n=3 89±10 n=5 52.7±1.5 n=3 41.3±2.3 n=3 244±9.0 n=3 14.8±1.9 n=4 81±9.7 n=4
9.8
8.78±0.54 n=3
3.37±0.29 n=3
6.2 22.0
6.75±0.43 n=3
4.55± 0.14 f=6 28
23/unit*
Prawns, 60/80 (Penaeus Monodon) Large pink prawn tails (Panadalus borealis) Scallop roe (Pectinidae) Shrimp (Caridea)
4.8/unit* 22/unit*
-* -*
n=3 1160±44 n=3 26.5±2.6 n=6 147±4.6 n=3 24±4.0 n=3 18.2±0.75 n=6
n=3 254±10
255
0.4
4.8
4.3
30.8
0.6
-
7.0
-
≤ LOD
-
-
4.6±0.17 n=3 31.0±2.6 n=3
5.55±0.52 n=3 4.78±0.31 n=3 3.42±0.22 n=4 -
474 475
- data not available
476 477
* -frozen product
478
** ∆ %
**-
=
C m − C Calc Cm
× 100 %
479
480
481
29
482
Table 5 Total Hg concentrations in canned fish Sample Product name, N weight and drained weight (g)
Analyzed sample state
1
Drained meat Sunflower oil Drained meat
3
10±1.0
3
1.1±0.12
3
Drained meat
Drained meat Liquid fraction, filtered Drained meat
2
3
4
5
6
7
8
9
10
Anchovies, vegetable oil added, 80, 56 Brisling sardines , vegetable oil added (Sprattus sprattus), smoked, 106, 80 Herring (marinated fillets), Matjes style, 200 Light meat Tuna chunks, 170, 120
Light meat Tuna chunks, 170, 119 Mackerel fillet in brine, 125, 90
Musky octopus in vegetable oil, 100, 60 Pink Salmon, 212
Portuguese sardines from Algarve in olive oil, 120 Smoked oysters,
Calculated Hg µg kg-1 body weight (60kg) 0.01
Producer, country of production
27.7±0.76
0.037
Latvia
4
57.2±6.0
0.19
Germany
3
68.0±4.6
0.14
JOHN WEST, Thailand
3
≤LOD
3
199±40
0.39
3
58.3±4.2
0.090
LUCKY STAR, Thailand JOHN WEST, Portugal
4
3.8±1.6
3
67.3±1.2
3
2.3±0.20
Drained meat
3
Drained meat Olive oil, filtered Drained meat
Drained meat Water fraction filtered Drained meat Soya oil
n*
Concentration of Hg C±SD (ng g-1 )
Morocco
0.069
TRATA, Greece
48±5.0
0.17
5
48.0±2.0
0.096
WILD ALASKA SALMON, USA Estovil, Portugal
3
≤LOD
4
25.8±1.5
0.037
China 30
cottonseed oil added, 85 11
Snails in brine
12
South Africa sardines, vegetable oil added (Sardinops sagax), 120
Cottonseed oil Drained meat Dried meat Drained meat Sunflower oil, filtered
3
≤LOD
3
≤LOD
-
5 3
6.3±1.2 18±2.0
0.036
3
29±4.0
Indonesia
LUCKY STAR, South Africa
483 484
n*- Number of measurements
485
486 487
31
488
Highlights
489 490
1. A method of direct thermal decomposition for Hg measurement in raw samples of fish was developed.
491
2. Samples of wet fish were analyzed without any chemical pretreatment.
492
3. There were no losses of Hg during analysis of wet and dry fish samples.
493 494
4. The concentration of Hg in most fishes from Tshwane market (Pretoria, South SAfrica) was found to be within safety limits.
495 496
32
