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Determination of polychlorinated biphenyls and polyaromatic hydrocarbons in frog liver
~
Pergamon
War. Sci. Tech. Vol. 34, No. 7-8, pp. 153-156, 1996. Copyright © 1996 IAWQ. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved.
PH: S0273-1223(96)OO738-X
0273-1223/96 $15'00 + 0'00
DETERMINATION OF POLYCHLORINATED BIPHENYLS AND POLYAROMATIC HYDROCARBONS IN FROG LIVER Mirjana Vojinovic-Miloradov*, Danica Buzarov*, Jasna Adamov*, Smiljka Simic** and Ester Popovic** * University ofNovi Sad, Institute of Chemistry
** University ofNovi Sad, Institute ofBiology, rrg Dositeja Obradovica 3, 21000 Novi Sad, Yugoslavia
ABSTRACT The aim of this work was to detennine the content of the residues of the most commonly detected organohalogens and polychlorinated biphenyls in biomaterial. It is also the introduction of frogs as a new fine indicator of pollution in aquatic and terrestrial systems, due to their abundance all over Europe and their habitats in still and running waters. The residues of organohalogens and polyaromatic hydrocarbons in frog liver samples were taken from four sites - the river Danube and canal DID in Vojvodina (northern part of Serbia), from the river Dubasnica near the town of Bor and in the Zlot canyon, Serbia,Yugoslavia. They were detennined using gas chromatography with mass detector (GCIMS) and they indicate the high level of contamination of the chosen aquatic and terrestrial ecosystems by HCH, PCB, PAH and herbicides. Copyright © 1996 IAWQ. Published by Elsevier Science Ltd.
KEYWORDS Benzo(a)pyrene; frog liver; GCIMS; PAH; PCBs; water pollution. INTRODUCTION In the detennination of water quality, monitoring approach is frequently used allover the world. There are many organisms which can be chosen as the pollution indicators. Amphibians make a good indicator group because they can inhabit an area in relatively large numbers (they are often the most abundant vertebras), they live in different water systems allowing direct comparison, and most of them need a variety of microhabitats to complete their life cycles, which means they can provide an earlier warning than other indicators. Frogs may not be beautiful, but there is more and more evidence that they are useful to health, and they can also serve as a very fine pollution indicators. Species from genus Rana (R. ridibunda, R. dalmatina, Bombina variegata) belong to the very abundant group of Anura. They inhabit Middle, East and West Europe, and live both in still and running waters 153
M. VOJINOVIC-MILORADOV et at.
154
(ponds. lakes, fishponds, old river basins, moors). They prefer waters with grassy banks or with abundant water vegetation. According to the literature, the residues of organochlorine contaminants and PAHs have not so far been determined in this type of biological materials. The investigated group of organohalogens is a family of lipophilic, resistent compounds with large concentration factors and they can be determined from the frog liver because of their deposition in fat tissue. MATERIALS AND METHODS The frogs were collected in April and May of 1994 from the river Danube at the site called Ribnjak (Novi Sad), from the irrigation canal system Danube-Tisa-Danube (DTD) near Vrbas in Vojvodina, and in two sites in the industrial and mining area in the river Dubasnica (near the town of Bor and in Zlot canyon), Serbia, Yugoslavia. The livers of these frogs were separated and the fat was extracted by a mixture of methanol:chloroform:water (2:2: 1). After filtration the solvent was evaporated at 50°C and the samples were dissolved in hexane and purified by concentrated sulphuric acid. After rinsing with water the solvent from the samples was evaporated. Analytes were enriched by solid-phase extraction using silica SPE cartridges and dissolved in 250111 of dichloromethane. They were analysed by gas chromatography with mass detector (GCIMS).
All chemical used were of p.a. quality. lInstrument configuration Gas chromatograph Detector IGC parameters Injection port Inject. port temp. Column Oven temp. program Column flow rate Injection volume IMS parameters Mode Ions monitored
Dwell time Solvent delay time
HP 5890 Series II HP 5972A MS Det. splitless 300°C 30 m x 250 mm x 0.25 mm HP5-MS 40°C - 1 min, 25°C/min - 140°C 10°C/min - 320°C, 2 min hold 50 kPa helium - constant flow rate (1 ml/min) Imm SIM (selected ion monitoring) ~he PCBs and PAHs were monitored using three groups of molecular Ions. The time-windows for each group were obtained from a previously acquired full scan analysis. 100 mslion 5 min
RESULTS AND DISCUSSION In ~e literature there are very few data on pollution by residues of organochlorine compounds and PCB in the livers of frogs. It is well ~own that the liver is the organ which accumulates the toxicants that enter the living organism in the very high degree.
The results of gas-chromatographic analysis of the frog liver samples are given in Tables 1,2. and 3.
Detennination of polychlorinated biphenyls
155
Table 1. The content of contaminants in liver samples from species R. ridibunda taken from the Danube at the site Ribnjak [10- 3 ~g/g of liver]
Contaminant a-HCH ~-HCH
y-HCH LHCH P/P-DDE o/P-DDT PIP-DDT L DDT PCB 28 PCB 52 Atrazine Fluoranthene Benzofluoranthene Benzo(a)pyrene
Content 1.29 397.2 22.44 420.93 10.52 10.52 12.44 7.19 93.38 0.88 4.16 20.24
Table 2. The content of contaminants in liver samples from species R. Ridibunda taken from the canal DTD, Vrbas [10- 3 ~g/g of liver]
Contaminant a-HCH ~-HCH
y-HCH LHCH P/P-DDE o/P-DDT p,P-DDT L DDT PCB 28 PCB 52 Atrazine Fluoranthene Benzofl uoran thene Benzo(a)pyrene
Content 58.9 0.04 1.84 60.78 1.13 1.13 1.12 0.65 6.64 10.81 52.5 1.62
According to the results from the given Tables, it is important to emphasize that in all samples of frog liver the isomers of HCH, the metabolites of DDT and lower PCBs were detected by mass detector. The other compounds, like herbicide atrazine, and polyaromatic hydrocarbons (fluoranthene, benzofluoranthene, benzopyrene) were detected, although the experimental procedure of our laboratories was designed from monitoring the organochlorine compounds and PCBs only. The highest level of pollution was discovered in the river Dubasnica in the vicinity of Bor, which is easily explained by facts that it is the highly industrial area, with thermoelectric plant, mining and coloured metal industry and chemical fertilizer factory. The pollution in the same river is importantly lower at Zlot canyon, which is at the greater distance from the industrial area.
M. VOJINOVIC-MILORADOV et al.
156
The pollution of DTD canal at Vrbas is not so high, although it lies in the highly developed agricultur~ are~. Actually it has the lowest level of pollution of all investigated samples. It is comparable to the pollutIOn In the river Danube ( which is slightly higher) which flows through the same area. Table 3. The content of contaminants in liver samples from species R. Dalmatina and Bombina variegata taken from the River Dubasnica near Bor and from species R. Ridibunda taken in Zlot Canyon [I 0- 3 ~g/g of liver]
a-HCH
Content In the vicinity of Bor 24.11
Content At Zlot canyon 0.53
p-HCH
2587.31
158.73
y-HCH
24.60
6.42
2636.02
165.68
13.06 7.42 10.34 30.82
6.79
Contaminant
LHCH p,P-DOE o/P-DDT p,P-DDT LOOT PCB 28 PCB 52 PCB 101 PCB 118 PCB 180 Atrazine Fluoranthene Benzofluoranthene Benzo(a)pyrene
16.92 9.52 5.50 6.91 5.97 93.72 2.42 8.37 46.05
-
6.79 4.47 2.51 2.75
-
26.06 0.30 1.47 8.27
CONCLUSION Since the residues of organochlorine compounds, PCBs and other toxicants were detected in all samples of frog liver, this method is a very suitable way of monitoring the state of the environment. It is important to emphasize that frogs, which are very abundant and easy accesible and which eat both water organisms and insects, can be used as very fine indicators of the level of pollution in aquatic and terrestrial ecosystems. REFERENCES Vojinovic, M., Simic, S., Pavkov, S. and Popovic, E. (1993). The residues of organochlorine compounds in frog liver as indicator of environmental contamination, Szegedi Okologiai Napok, 24. Tiszakutato Anket, Vojinovic-Miloradov, M., Marjanovic, P., Buzarov, D., Pavkov, S., Dimitrijevic, Lj. and Miloradov, M. (1992). Bioaccumulation of polychlorinated biphenyls and organochlorine pesticides in selected fish species as an indicator of the pollution of aquatic resources in Vojvodina, Yugoslavia, Wat. Sci. Tech. (9-11),2361-2364 Vojinovic-Miloradov, M., Simic, S., Pavkov, S. and Popovic, E. (1994). Froshe als belastungs-indikatoren bestimmung der organohalogenen residuen in der leber der froshe der gattung Rana I. (Amphibia: Anura), 30. Arbeotstagung der lAD, ZUOZ, Schweitz - Wisenschaftliche Kurzreferate, 220-224
Pergamon
War. Sci. Tech. Vol. 34, No. 7-8, pp. 153-156, 1996. Copyright © 1996 IAWQ. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved.
PH: S0273-1223(96)OO738-X
0273-1223/96 $15'00 + 0'00
DETERMINATION OF POLYCHLORINATED BIPHENYLS AND POLYAROMATIC HYDROCARBONS IN FROG LIVER Mirjana Vojinovic-Miloradov*, Danica Buzarov*, Jasna Adamov*, Smiljka Simic** and Ester Popovic** * University ofNovi Sad, Institute of Chemistry
** University ofNovi Sad, Institute ofBiology, rrg Dositeja Obradovica 3, 21000 Novi Sad, Yugoslavia
ABSTRACT The aim of this work was to detennine the content of the residues of the most commonly detected organohalogens and polychlorinated biphenyls in biomaterial. It is also the introduction of frogs as a new fine indicator of pollution in aquatic and terrestrial systems, due to their abundance all over Europe and their habitats in still and running waters. The residues of organohalogens and polyaromatic hydrocarbons in frog liver samples were taken from four sites - the river Danube and canal DID in Vojvodina (northern part of Serbia), from the river Dubasnica near the town of Bor and in the Zlot canyon, Serbia,Yugoslavia. They were detennined using gas chromatography with mass detector (GCIMS) and they indicate the high level of contamination of the chosen aquatic and terrestrial ecosystems by HCH, PCB, PAH and herbicides. Copyright © 1996 IAWQ. Published by Elsevier Science Ltd.
KEYWORDS Benzo(a)pyrene; frog liver; GCIMS; PAH; PCBs; water pollution. INTRODUCTION In the detennination of water quality, monitoring approach is frequently used allover the world. There are many organisms which can be chosen as the pollution indicators. Amphibians make a good indicator group because they can inhabit an area in relatively large numbers (they are often the most abundant vertebras), they live in different water systems allowing direct comparison, and most of them need a variety of microhabitats to complete their life cycles, which means they can provide an earlier warning than other indicators. Frogs may not be beautiful, but there is more and more evidence that they are useful to health, and they can also serve as a very fine pollution indicators. Species from genus Rana (R. ridibunda, R. dalmatina, Bombina variegata) belong to the very abundant group of Anura. They inhabit Middle, East and West Europe, and live both in still and running waters 153
M. VOJINOVIC-MILORADOV et at.
154
(ponds. lakes, fishponds, old river basins, moors). They prefer waters with grassy banks or with abundant water vegetation. According to the literature, the residues of organochlorine contaminants and PAHs have not so far been determined in this type of biological materials. The investigated group of organohalogens is a family of lipophilic, resistent compounds with large concentration factors and they can be determined from the frog liver because of their deposition in fat tissue. MATERIALS AND METHODS The frogs were collected in April and May of 1994 from the river Danube at the site called Ribnjak (Novi Sad), from the irrigation canal system Danube-Tisa-Danube (DTD) near Vrbas in Vojvodina, and in two sites in the industrial and mining area in the river Dubasnica (near the town of Bor and in Zlot canyon), Serbia, Yugoslavia. The livers of these frogs were separated and the fat was extracted by a mixture of methanol:chloroform:water (2:2: 1). After filtration the solvent was evaporated at 50°C and the samples were dissolved in hexane and purified by concentrated sulphuric acid. After rinsing with water the solvent from the samples was evaporated. Analytes were enriched by solid-phase extraction using silica SPE cartridges and dissolved in 250111 of dichloromethane. They were analysed by gas chromatography with mass detector (GCIMS).
All chemical used were of p.a. quality. lInstrument configuration Gas chromatograph Detector IGC parameters Injection port Inject. port temp. Column Oven temp. program Column flow rate Injection volume IMS parameters Mode Ions monitored
Dwell time Solvent delay time
HP 5890 Series II HP 5972A MS Det. splitless 300°C 30 m x 250 mm x 0.25 mm HP5-MS 40°C - 1 min, 25°C/min - 140°C 10°C/min - 320°C, 2 min hold 50 kPa helium - constant flow rate (1 ml/min) Imm SIM (selected ion monitoring) ~he PCBs and PAHs were monitored using three groups of molecular Ions. The time-windows for each group were obtained from a previously acquired full scan analysis. 100 mslion 5 min
RESULTS AND DISCUSSION In ~e literature there are very few data on pollution by residues of organochlorine compounds and PCB in the livers of frogs. It is well ~own that the liver is the organ which accumulates the toxicants that enter the living organism in the very high degree.
The results of gas-chromatographic analysis of the frog liver samples are given in Tables 1,2. and 3.
Detennination of polychlorinated biphenyls
155
Table 1. The content of contaminants in liver samples from species R. ridibunda taken from the Danube at the site Ribnjak [10- 3 ~g/g of liver]
Contaminant a-HCH ~-HCH
y-HCH LHCH P/P-DDE o/P-DDT PIP-DDT L DDT PCB 28 PCB 52 Atrazine Fluoranthene Benzofluoranthene Benzo(a)pyrene
Content 1.29 397.2 22.44 420.93 10.52 10.52 12.44 7.19 93.38 0.88 4.16 20.24
Table 2. The content of contaminants in liver samples from species R. Ridibunda taken from the canal DTD, Vrbas [10- 3 ~g/g of liver]
Contaminant a-HCH ~-HCH
y-HCH LHCH P/P-DDE o/P-DDT p,P-DDT L DDT PCB 28 PCB 52 Atrazine Fluoranthene Benzofl uoran thene Benzo(a)pyrene
Content 58.9 0.04 1.84 60.78 1.13 1.13 1.12 0.65 6.64 10.81 52.5 1.62
According to the results from the given Tables, it is important to emphasize that in all samples of frog liver the isomers of HCH, the metabolites of DDT and lower PCBs were detected by mass detector. The other compounds, like herbicide atrazine, and polyaromatic hydrocarbons (fluoranthene, benzofluoranthene, benzopyrene) were detected, although the experimental procedure of our laboratories was designed from monitoring the organochlorine compounds and PCBs only. The highest level of pollution was discovered in the river Dubasnica in the vicinity of Bor, which is easily explained by facts that it is the highly industrial area, with thermoelectric plant, mining and coloured metal industry and chemical fertilizer factory. The pollution in the same river is importantly lower at Zlot canyon, which is at the greater distance from the industrial area.
M. VOJINOVIC-MILORADOV et al.
156
The pollution of DTD canal at Vrbas is not so high, although it lies in the highly developed agricultur~ are~. Actually it has the lowest level of pollution of all investigated samples. It is comparable to the pollutIOn In the river Danube ( which is slightly higher) which flows through the same area. Table 3. The content of contaminants in liver samples from species R. Dalmatina and Bombina variegata taken from the River Dubasnica near Bor and from species R. Ridibunda taken in Zlot Canyon [I 0- 3 ~g/g of liver]
a-HCH
Content In the vicinity of Bor 24.11
Content At Zlot canyon 0.53
p-HCH
2587.31
158.73
y-HCH
24.60
6.42
2636.02
165.68
13.06 7.42 10.34 30.82
6.79
Contaminant
LHCH p,P-DOE o/P-DDT p,P-DDT LOOT PCB 28 PCB 52 PCB 101 PCB 118 PCB 180 Atrazine Fluoranthene Benzofluoranthene Benzo(a)pyrene
16.92 9.52 5.50 6.91 5.97 93.72 2.42 8.37 46.05
-
6.79 4.47 2.51 2.75
-
26.06 0.30 1.47 8.27
CONCLUSION Since the residues of organochlorine compounds, PCBs and other toxicants were detected in all samples of frog liver, this method is a very suitable way of monitoring the state of the environment. It is important to emphasize that frogs, which are very abundant and easy accesible and which eat both water organisms and insects, can be used as very fine indicators of the level of pollution in aquatic and terrestrial ecosystems. REFERENCES Vojinovic, M., Simic, S., Pavkov, S. and Popovic, E. (1993). The residues of organochlorine compounds in frog liver as indicator of environmental contamination, Szegedi Okologiai Napok, 24. Tiszakutato Anket, Vojinovic-Miloradov, M., Marjanovic, P., Buzarov, D., Pavkov, S., Dimitrijevic, Lj. and Miloradov, M. (1992). Bioaccumulation of polychlorinated biphenyls and organochlorine pesticides in selected fish species as an indicator of the pollution of aquatic resources in Vojvodina, Yugoslavia, Wat. Sci. Tech. (9-11),2361-2364 Vojinovic-Miloradov, M., Simic, S., Pavkov, S. and Popovic, E. (1994). Froshe als belastungs-indikatoren bestimmung der organohalogenen residuen in der leber der froshe der gattung Rana I. (Amphibia: Anura), 30. Arbeotstagung der lAD, ZUOZ, Schweitz - Wisenschaftliche Kurzreferate, 220-224