Accepted Manuscript Mercury speciation in fish muscles from major Czech rivers and assessment of health risks Lenka Sedlá čková, Kamila Kruž íková, Zdeňka Svobodová PII: DOI: Reference:
S0308-8146(13)01475-1 http://dx.doi.org/10.1016/j.foodchem.2013.10.041 FOCH 14827
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
21 March 2013 22 July 2013 8 October 2013
Please cite this article as: Sedlá čková, L., Kružíková, K., Svobodová, Z., Mercury speciation in fish muscles from major Czech rivers and assessment of health risks, Food Chemistry (2013), doi: http://dx.doi.org/10.1016/ j.foodchem.2013.10.041
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mercury speciation in fish muscles from major Czech rivers
1
and assessment of health risks
2 3 1
4
Lenka Sedláčková*, Kamila Kružíková, Zdeňka Svobodová
5 6
Department of Veterinary Public Health and Toxicology, Faculty of Veterinary Hygiene and
7
Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Palackého 1/3, Brno, 612
8
42, Czech Republic. [email protected], [email protected], [email protected]
9 10
1
Corresponding author: E-mail address: [email protected] (L. Sedláčková)
11 12
ABSTRACT
13
The aim of this work was to determine the mercury and methyl mercury content in
14
muscle tissue of chub (Leuciscus cephalus L.), to assess the health risks of eating the fish
15
and to determine the number of fish meat servings that are suitable for weekly
16
consumption. Total mercury concentrations were determined using a single-purpose
17
atomic absorption spectrophotometer AMA 254. Methylmercury concentrations were
18
determined by gas chromatography. The location where the highest total mercury
19
concentrations in fish muscle tissues were found was the Vltava – Vraňany (0.236 ±
20
0.1001 mg/kg-1), and the highest methylmercury concentration was found at the Labe –
21
Obříství (0.231 ± 0.1056 mg/kg-1). The conclusion based on the data ascertained is that
22
the locations from which the lowest number of fish meat servings can be eaten are the
23
Vltava – Vraňany and the Labe – Obříství. The results of this study helped evaluate
24
contamination levels of rivers that flow out of the Czech Republic.
25 26
1
27
Keywords
28
Total mercury, methylmercury, Leuciscus cephalus L., hazard index
29 30
1. Introduction
31
Mercury is one of the major pollutants of the aquatic environment. Mercury
32
bioaccumulates in aquatic organisms, persists in bottom sediments for long
33
periods of time and is released into the environment from both natural and
34
anthropogenic
35
Svobodová, 2008). An important indicator of mercury pollution in the aquatic
36
environment is fish, where the highest mercury concentrations are found mainly in
37
muscle tissue (Čelechovská, Svobodová, Žlábek & Macharáčková, 2007). The
38
most common entry route for mercury into the human body is the consumption
39
of fish from mercury-polluted areas, with the greatest health hazard posed by its
40
organic form, i.e. methylmercury (Clarkson, 1990; Shimshack, Ward & Beatty,
41
2007; WHO, 1990). For that reason, attention has been focused on fish as the
42
main source of human exposure to methylmercury. The effects of methylmercury
43
in the body adversely impact the nervous, cardiovascular and immune systems
44
(Miero, Pacheco, Pereira & Duarte, 2009). As fish is an important part of the
45
human diet, it is necessary to monitor the levels of total mercury and its organic
46
form in fish muscle tissues.
47
In 2011, attention was given to outlet section of several major rivers in the Czech
48
Republic where elevated levels of mercury contamination of fish were observed,
49
and also to pollution in the rivers before they flow out of the Czech Republic.
50
The aim of the study was to determine the total mercury and methylmercury
51
levels
sources
(Havelková,
Dušek,
Némethová,
Poleszczuk
&
2
52
in muscle tissues of the indicator species, i.e. the chub (Leuciscus cephalus L.) at
53
12 locations on major rivers in the Czech Republic, and to assess health risks
54
associated with eating fish from the locations studied.
55 56
2. Materials and methods
57
2.1. Sampling sites
58
Samples were collected at 12 locations on 11 rivers in the Czech Republic.
59
To monitor the total mercury and methylmercury content, localities on rivers that
60
are leaving the Czech Republic were chosen: Labe – Děčín (506.7 m3/s-1),
61
Odra – Bohumín (38.9 m3/s-1), Morava – Lanžhot (120.0 m3/s-1). More localities
62
situated at tributaries to the main rivers, upstream of the first migration barrier;
63
tributaries of Labe: Vltava – Vraňany (161.2 m3/s-1) a Ohře – Louny (64.0 m3/s-1);
64
tributaries of Vltava: Berounka – Srbsko (36.0 m3/s-1), Otava – Topělec (26.0
65
m3/s-1), Lužnice – Bechyně (24.3 m3/s-1), Sázava – Nespeky (25.2 m3/s-1);
66
tributaries of Morava: Dyje – Pohansko (43.9 m3/s-1). Localities heavily populated
67
with chemical industries: Labe – Obříství (235.0 m3/s-1), where chlorine
68
production in which mercury is used, and Vltava – Vraňany (161.2 m3/s-1) and
69
Kaučuk Kralupy chemical industries. Localities situated downstream from big
70
urban agglomeration: Brno (384,277 inhibitants) Svratka – Židlochovice (27.2
71
m3/s-1), Praha (1,272,690 inhibitants) Vltava – Vraňany and Ostrava (302,456
72
inhibitants) Odra – Bohumín.
73 74
2.2. Sampling of sediment
3
75
Sampling of sediments (8 samples from each localities) were carried out
76
according to a methodology accepted and used in the Czech hydrometeorological
77
institute (CHMI) within state monitoring of water quality in compliance with EU
78
directives. Samples of so called ‘fresh sediments‘, that are altering in the course of
79
the year, were taken. Samples were taken from a surface layer of sediment to a
80
depth of 3 - 10 cm (depending on the thickness of deposited sediment) from at
81
least 3 sampling sites within one locality. In the case of deeper streams, a sampler
82
of raker type was used (constructed to collect a thin layer of fresh sediment). For
83
shallow streams with problematic quantity of sediment, a stainless steel flat spoon
84
was used. After sampling from several sites, sediments were mixed, homogenized
85
and stored in plastic, glass or stainless steel sample containers at up to 4 oC, and
86
transported to a workplace where they were immediately frozen. Once frozen
87
samples were transferred to laboratories.
88 89
2.3. Sample collection
90
A total of 130 indicator fish (Leuciscus cephalus L.) were caught. The fish were
91
caught during May and June in 2011 at 12 locations on eleven rivers in the Czech
92
Republic (Fig. 1). At each location, 9 to 12 chub were captured, except at the
93
Ohře – Louny where only 6 chub were captured. The fish were captured using
94
electrofishing gear. The fish were then weighed, and a scale sample collected for
95
age verification and muscle tissue sampled for mercury content. The fish muscle
96
samples were collected from the cranial area dorsal to the lateral line. Muscle
97
samples were placed in polyethylene bags, labelled and stored at – 18 °C until
98
analysis.
4
99
2.4. Determination of total mercury and methylmercury concentrations
100
Total mercury (THg) concentrations in fish muscle (wet weight) and sediment
101
(dry weight) samples were determined using a single-purpose atomic absorption
102
spectrophotometer AMA 254 (Advanced Mercury Analyzer), where mercury can
103
be determined without sample pretreatment (detection limit: 1 μg/kg-1 recovery:
104
82 ± 6%) (Kružíková, Svobodová, Valentová, Randák & Velíšek, 2008a).
105
Methylmercury (MeHg) was determined by gas chromatography with an electron
106
capture detector (GC/ECD). Samples were prepared by acidic digestion and
107
extraction with toluene (Maršálek & Svobodová, 2006). For the determination, a
108
gas chromatograph GC 2010A (Shimadzu GmbH, Czech Republic), a capillary
109
column DB 608 (30 m x 0.53 mm x 0.83 μm; J&W Scientific, Chromservis,
110
Czech Republic) and an electron capture detector (ECD) (Shimadzu GmbH,
111
Czech Republic) were used. Evaluation was made using GC Solution software
112
(Shimadzu GmbH, Czech Republic) and MS Excel software. The limit of
113
detection was 21 μg/kg-1 and the limit of quantification was 62 μg/kg-1 (recovery
114
89 ± 2.5%) (Kružíková et al., 2008a). The accuracy of the results
115
of THg and MeHg determinations were validated using standard reference
116
materials BCR-CRM 463 and 464 (IRMM, Belgium), respectively.
117
To express the portion of the organic form of mercury as a percentage of total
118
mercury (Hg-MeHg in THg), MeHg concentrations were converted to Hg
119
represented in that organic form.
120
In the sediment, dry matter was determined at 105°C until constant weight.
121 122
2.5. Health hazard assessment
5
123
Health hazards were assessed by a method according to Kannan et al. (1998)
124
using a THg reference dose (RfD) of 0.3 µg/kg-1 body weight per day as set forth
125
by US EPA (1989).
126
127 128 129 130 131 132 133 134
D c I W
Estimated dose Concentration of mercury in fish (µg/g wet weight) Fish intake rate: Moderate consumer: 1000 g/year – 2.74 g/day A fisher's family member: 10,000 g/year – 27.4 g/day Average body weight: 70 kg
RfD HI
Reference dose value: 3 x 10-4 mg/kg/day Hazard index
135 136 137 138 139 140
A hazard index (HI) less than 1 indicates that fish consumption poses no risk. The
141
HI was calculated from an average annual consumption of freshwater fish in the
142
Czech Republic, which is 1 kg by a moderate consumer and 10 kg by a fisher's
143
family member.
144
To determine the maximum number of fish meat servings that can be eaten in a
145
week, and which pose no health hazard to humans, the provisional tolerable
146
weekly intake (PTWI) limit of 1.6 µg MeHg per kg bodyweight per week was
147
used (WHO, 1990), with the portion size being 170 g.
148 149 150 151 152 153
D EL W
Estimated dose PTWI exposure limit: 1.6 µg/kg Consumer average weight: 70 kg
154
6
155 156 157
NTL c
Fish muscle weight for reaching the toxicological limit Level of pollutant in muscle tissue of fish from a given location
159
p
170 g serving
160
pp
Number of portions
158
161 162
2.6. Statistical evaluation
163
Weight, age of fish and THg and MeHg in fish muscle from each location was
164
tested for normal distribution using the Shapiro-Wilk test. Parameters that were
165
not normally distributed were analyzed with nonparametric statistical tests. The
166
Kruskal-Wallis test followed by multiple comparison was used to determine
167
differences among groups. To allow for comparison between individual locations,
168
THg and MeHg contents in muscle tissue were age-adjusted for correlation
169
analyzes due to the significant age difference among the groups.
170 171
3. Results and discussion
172
The weight of chub captured at individual locations together with their age and
173
the number of fish analyzed are given in Table 1.
174
Statistical evaluation showed significant differences between the age of fish from
175
the locations compared, and also between the weight of fish from those individual
176
locations. The location where the highest mean THg concentration in muscle
177
tissue of chub was found was the Vltava – Vraňany (0.236 ± 0.1001 mg/kg-1), and
178
the lowest mean THg concentration (0.071 ± 0.0249 mg/kg-1) was found at the
179
Berounka – Srbsko. The highest mean MeHg concentration (0.231 ± 0.1056
7
180
mg/kg-1) was at the Labe – Obříství, and the lowest (0.065 ± 0.0321 mg/kg-1) at
181
the Lužnice – Bechyně.
182
THg and MeHg tissue concentrations in fish increase with increasing age of the
183
fish (Norstrom, Mckinnon & Freitas, 1976; Jewett et al., 2003). Our comparison
184
between THg and MeHg concentrations in muscle tissue of chub from different
185
locations was therefore conducted after their values were age-adjusted (Figs 2 and
186
3). The diagram in Fig. 2 shows a significant difference in THg concentrations
187
(p
S0308-8146(13)01475-1 http://dx.doi.org/10.1016/j.foodchem.2013.10.041 FOCH 14827
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
21 March 2013 22 July 2013 8 October 2013
Please cite this article as: Sedlá čková, L., Kružíková, K., Svobodová, Z., Mercury speciation in fish muscles from major Czech rivers and assessment of health risks, Food Chemistry (2013), doi: http://dx.doi.org/10.1016/ j.foodchem.2013.10.041
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mercury speciation in fish muscles from major Czech rivers
1
and assessment of health risks
2 3 1
4
Lenka Sedláčková*, Kamila Kružíková, Zdeňka Svobodová
5 6
Department of Veterinary Public Health and Toxicology, Faculty of Veterinary Hygiene and
7
Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Palackého 1/3, Brno, 612
8
42, Czech Republic. [email protected], [email protected], [email protected]
9 10
1
Corresponding author: E-mail address: [email protected] (L. Sedláčková)
11 12
ABSTRACT
13
The aim of this work was to determine the mercury and methyl mercury content in
14
muscle tissue of chub (Leuciscus cephalus L.), to assess the health risks of eating the fish
15
and to determine the number of fish meat servings that are suitable for weekly
16
consumption. Total mercury concentrations were determined using a single-purpose
17
atomic absorption spectrophotometer AMA 254. Methylmercury concentrations were
18
determined by gas chromatography. The location where the highest total mercury
19
concentrations in fish muscle tissues were found was the Vltava – Vraňany (0.236 ±
20
0.1001 mg/kg-1), and the highest methylmercury concentration was found at the Labe –
21
Obříství (0.231 ± 0.1056 mg/kg-1). The conclusion based on the data ascertained is that
22
the locations from which the lowest number of fish meat servings can be eaten are the
23
Vltava – Vraňany and the Labe – Obříství. The results of this study helped evaluate
24
contamination levels of rivers that flow out of the Czech Republic.
25 26
1
27
Keywords
28
Total mercury, methylmercury, Leuciscus cephalus L., hazard index
29 30
1. Introduction
31
Mercury is one of the major pollutants of the aquatic environment. Mercury
32
bioaccumulates in aquatic organisms, persists in bottom sediments for long
33
periods of time and is released into the environment from both natural and
34
anthropogenic
35
Svobodová, 2008). An important indicator of mercury pollution in the aquatic
36
environment is fish, where the highest mercury concentrations are found mainly in
37
muscle tissue (Čelechovská, Svobodová, Žlábek & Macharáčková, 2007). The
38
most common entry route for mercury into the human body is the consumption
39
of fish from mercury-polluted areas, with the greatest health hazard posed by its
40
organic form, i.e. methylmercury (Clarkson, 1990; Shimshack, Ward & Beatty,
41
2007; WHO, 1990). For that reason, attention has been focused on fish as the
42
main source of human exposure to methylmercury. The effects of methylmercury
43
in the body adversely impact the nervous, cardiovascular and immune systems
44
(Miero, Pacheco, Pereira & Duarte, 2009). As fish is an important part of the
45
human diet, it is necessary to monitor the levels of total mercury and its organic
46
form in fish muscle tissues.
47
In 2011, attention was given to outlet section of several major rivers in the Czech
48
Republic where elevated levels of mercury contamination of fish were observed,
49
and also to pollution in the rivers before they flow out of the Czech Republic.
50
The aim of the study was to determine the total mercury and methylmercury
51
levels
sources
(Havelková,
Dušek,
Némethová,
Poleszczuk
&
2
52
in muscle tissues of the indicator species, i.e. the chub (Leuciscus cephalus L.) at
53
12 locations on major rivers in the Czech Republic, and to assess health risks
54
associated with eating fish from the locations studied.
55 56
2. Materials and methods
57
2.1. Sampling sites
58
Samples were collected at 12 locations on 11 rivers in the Czech Republic.
59
To monitor the total mercury and methylmercury content, localities on rivers that
60
are leaving the Czech Republic were chosen: Labe – Děčín (506.7 m3/s-1),
61
Odra – Bohumín (38.9 m3/s-1), Morava – Lanžhot (120.0 m3/s-1). More localities
62
situated at tributaries to the main rivers, upstream of the first migration barrier;
63
tributaries of Labe: Vltava – Vraňany (161.2 m3/s-1) a Ohře – Louny (64.0 m3/s-1);
64
tributaries of Vltava: Berounka – Srbsko (36.0 m3/s-1), Otava – Topělec (26.0
65
m3/s-1), Lužnice – Bechyně (24.3 m3/s-1), Sázava – Nespeky (25.2 m3/s-1);
66
tributaries of Morava: Dyje – Pohansko (43.9 m3/s-1). Localities heavily populated
67
with chemical industries: Labe – Obříství (235.0 m3/s-1), where chlorine
68
production in which mercury is used, and Vltava – Vraňany (161.2 m3/s-1) and
69
Kaučuk Kralupy chemical industries. Localities situated downstream from big
70
urban agglomeration: Brno (384,277 inhibitants) Svratka – Židlochovice (27.2
71
m3/s-1), Praha (1,272,690 inhibitants) Vltava – Vraňany and Ostrava (302,456
72
inhibitants) Odra – Bohumín.
73 74
2.2. Sampling of sediment
3
75
Sampling of sediments (8 samples from each localities) were carried out
76
according to a methodology accepted and used in the Czech hydrometeorological
77
institute (CHMI) within state monitoring of water quality in compliance with EU
78
directives. Samples of so called ‘fresh sediments‘, that are altering in the course of
79
the year, were taken. Samples were taken from a surface layer of sediment to a
80
depth of 3 - 10 cm (depending on the thickness of deposited sediment) from at
81
least 3 sampling sites within one locality. In the case of deeper streams, a sampler
82
of raker type was used (constructed to collect a thin layer of fresh sediment). For
83
shallow streams with problematic quantity of sediment, a stainless steel flat spoon
84
was used. After sampling from several sites, sediments were mixed, homogenized
85
and stored in plastic, glass or stainless steel sample containers at up to 4 oC, and
86
transported to a workplace where they were immediately frozen. Once frozen
87
samples were transferred to laboratories.
88 89
2.3. Sample collection
90
A total of 130 indicator fish (Leuciscus cephalus L.) were caught. The fish were
91
caught during May and June in 2011 at 12 locations on eleven rivers in the Czech
92
Republic (Fig. 1). At each location, 9 to 12 chub were captured, except at the
93
Ohře – Louny where only 6 chub were captured. The fish were captured using
94
electrofishing gear. The fish were then weighed, and a scale sample collected for
95
age verification and muscle tissue sampled for mercury content. The fish muscle
96
samples were collected from the cranial area dorsal to the lateral line. Muscle
97
samples were placed in polyethylene bags, labelled and stored at – 18 °C until
98
analysis.
4
99
2.4. Determination of total mercury and methylmercury concentrations
100
Total mercury (THg) concentrations in fish muscle (wet weight) and sediment
101
(dry weight) samples were determined using a single-purpose atomic absorption
102
spectrophotometer AMA 254 (Advanced Mercury Analyzer), where mercury can
103
be determined without sample pretreatment (detection limit: 1 μg/kg-1 recovery:
104
82 ± 6%) (Kružíková, Svobodová, Valentová, Randák & Velíšek, 2008a).
105
Methylmercury (MeHg) was determined by gas chromatography with an electron
106
capture detector (GC/ECD). Samples were prepared by acidic digestion and
107
extraction with toluene (Maršálek & Svobodová, 2006). For the determination, a
108
gas chromatograph GC 2010A (Shimadzu GmbH, Czech Republic), a capillary
109
column DB 608 (30 m x 0.53 mm x 0.83 μm; J&W Scientific, Chromservis,
110
Czech Republic) and an electron capture detector (ECD) (Shimadzu GmbH,
111
Czech Republic) were used. Evaluation was made using GC Solution software
112
(Shimadzu GmbH, Czech Republic) and MS Excel software. The limit of
113
detection was 21 μg/kg-1 and the limit of quantification was 62 μg/kg-1 (recovery
114
89 ± 2.5%) (Kružíková et al., 2008a). The accuracy of the results
115
of THg and MeHg determinations were validated using standard reference
116
materials BCR-CRM 463 and 464 (IRMM, Belgium), respectively.
117
To express the portion of the organic form of mercury as a percentage of total
118
mercury (Hg-MeHg in THg), MeHg concentrations were converted to Hg
119
represented in that organic form.
120
In the sediment, dry matter was determined at 105°C until constant weight.
121 122
2.5. Health hazard assessment
5
123
Health hazards were assessed by a method according to Kannan et al. (1998)
124
using a THg reference dose (RfD) of 0.3 µg/kg-1 body weight per day as set forth
125
by US EPA (1989).
126
127 128 129 130 131 132 133 134
D c I W
Estimated dose Concentration of mercury in fish (µg/g wet weight) Fish intake rate: Moderate consumer: 1000 g/year – 2.74 g/day A fisher's family member: 10,000 g/year – 27.4 g/day Average body weight: 70 kg
RfD HI
Reference dose value: 3 x 10-4 mg/kg/day Hazard index
135 136 137 138 139 140
A hazard index (HI) less than 1 indicates that fish consumption poses no risk. The
141
HI was calculated from an average annual consumption of freshwater fish in the
142
Czech Republic, which is 1 kg by a moderate consumer and 10 kg by a fisher's
143
family member.
144
To determine the maximum number of fish meat servings that can be eaten in a
145
week, and which pose no health hazard to humans, the provisional tolerable
146
weekly intake (PTWI) limit of 1.6 µg MeHg per kg bodyweight per week was
147
used (WHO, 1990), with the portion size being 170 g.
148 149 150 151 152 153
D EL W
Estimated dose PTWI exposure limit: 1.6 µg/kg Consumer average weight: 70 kg
154
6
155 156 157
NTL c
Fish muscle weight for reaching the toxicological limit Level of pollutant in muscle tissue of fish from a given location
159
p
170 g serving
160
pp
Number of portions
158
161 162
2.6. Statistical evaluation
163
Weight, age of fish and THg and MeHg in fish muscle from each location was
164
tested for normal distribution using the Shapiro-Wilk test. Parameters that were
165
not normally distributed were analyzed with nonparametric statistical tests. The
166
Kruskal-Wallis test followed by multiple comparison was used to determine
167
differences among groups. To allow for comparison between individual locations,
168
THg and MeHg contents in muscle tissue were age-adjusted for correlation
169
analyzes due to the significant age difference among the groups.
170 171
3. Results and discussion
172
The weight of chub captured at individual locations together with their age and
173
the number of fish analyzed are given in Table 1.
174
Statistical evaluation showed significant differences between the age of fish from
175
the locations compared, and also between the weight of fish from those individual
176
locations. The location where the highest mean THg concentration in muscle
177
tissue of chub was found was the Vltava – Vraňany (0.236 ± 0.1001 mg/kg-1), and
178
the lowest mean THg concentration (0.071 ± 0.0249 mg/kg-1) was found at the
179
Berounka – Srbsko. The highest mean MeHg concentration (0.231 ± 0.1056
7
180
mg/kg-1) was at the Labe – Obříství, and the lowest (0.065 ± 0.0321 mg/kg-1) at
181
the Lužnice – Bechyně.
182
THg and MeHg tissue concentrations in fish increase with increasing age of the
183
fish (Norstrom, Mckinnon & Freitas, 1976; Jewett et al., 2003). Our comparison
184
between THg and MeHg concentrations in muscle tissue of chub from different
185
locations was therefore conducted after their values were age-adjusted (Figs 2 and
186
3). The diagram in Fig. 2 shows a significant difference in THg concentrations
187
(p
