Organochlorine pesticide residues and heavy metals in two common dolphins from Ghanaian coastal waters
E Nyarko1, BO Botwe1, OO Ogunnowo1, NC Oburu1, MA Addo2 & PK Ofori-Danson1
1Department of Oceanography & Fisheries, University of Ghana, P. O. Box LG 99, Legon-Accra, Ghana 2Department of Nuclear Engineering and Material Science, National Nuclear Research Institute, Ghana Atomic Energy Commission,
P. O. Box LG 80, Legon-Accra, Ghana
Abstract: Organochlorine (OC) pesticide residues (heptachlor, p,p′-DDT, p,p′-DDE, γ-HCH, δ-HCH, α-endosulfan, β endosulfan and methoxychlor) and heavy metals (Cu, Mn, Cd and Hg) were analyzed in the blubber, liver and muscle tissues of two species of dolphins (Stenella clymene and Grampus griseus) from the coastal waters of Ghana. Concentrations of OC pesticide residues were measured using gas chromatograph-electron capture detector (GC-ECD) while concentrations of heavy metals were measured using instrumental neutron activation analysis (INAA). Concentrations of OC pesticide residues ranged from < 0.01 to 6.54 ngg-1 wet weight. Concentrations of OC pesticide residues increased with lipid content and were significantly higher (p < 0.05) in the blubber than the liver and muscle, indicating the lipophilic nature of the OC pesticides. The γ-HCH residue was most frequently detected at relatively higher concentrations than the other OC pesticide residues detected. Concentrations of heavy metals ranged from < 0.01 to 0.77 µgg-1 dry weight and were highest in the liver. Although Cu was detected in relatively higher concentrations than the other metals, the concentrations of heavy metals were generally higher than the levels of OC pesticides detected in the tissues of the dolphins. Human health risk analysis revealed that average daily exposures to these contaminants from consumption of dolphin meat did not pose appreciable risks of cancer and non-cancer health effects. Although the concentrations of OC pesticide residues and heavy metals were below levels which are toxic to dolphins, there is the potential for chronic toxicity due to the continued release of these contaminants into the environment. There is therefore the need for continuous monitoring of OC pesticides and heavy metals in marine organisms, especially in dolphins, which are considered, threatened according to the International Union of Conservation of Nature (IUCN).
Keywords: Organochlorine pesticide residues, heavy metals, dolphins, human health risks, Ghanaian coastal waters Introduction The world’s oceans are repositories for pollutants from both natural and anthropogenic sources. There are no known natural sources of organochlorine (OC) pesticides in the environment. Although there are natural sources of heavy metals in the marine environment, such as weathering of rocks and volcanic eruptions under the sea (Clark, 2003), heavy metal pollution in the marine environment is mainly from anthropogenic sources (Botkin and Keller, 1995). Concerns about OC pesticides and heavy metals in the environment have arisen because they are toxic and persistent pollutants that can bioaccumulate and biomagnify along food chains. Marine wildlife that occupy higher trophic levels are particularly at increased risk of OC pesticide and heavy metal contamination (Walter et al., 2002). Small cetaceans such as dolphins lack the ability to metabolize persistent OCs. They have high metabolic rates and due to their elevated trophic position within food webs, they have a high potential of accumulating persistent OC pesticides and heavy metals (Hayteas and Duffield, 2000). OCs and heavy metals have been found in the tissues of cetaceans (Kannan et al., 1993; Law et al., 2001; Law et al., 2003; Stockin et al., 2007). OC pesticides and heavy metals in marine mammals can cause numerous health problems in the marine mammals as well as humans who consume them (Reijnders, 1986; De Guise et al., 1994; Kuiken et al., 1994; Schwacke et al., 2002; Jepson et al., 2005;
Wells et al., 2005). These findings have generated interest in monitoring the levels of OC pesticides and heavy metals in small cetaceans such as dolphins, the world over. Dolphins are migratory species and therefore have a broad and global distribution (Evans, 1994; Rice, 1998). They also feed from a wide range of food sources and can therefore serve as biomonitors of geographical, historical and global patterns of pollutants such as OC pesticide residues and heavy metals. About 18 species of dolphins have been identified in the coastal waters of West Africa (Jefferson et al., 1997) of which 14 have been identified in Ghanaian coastal waters (Ofori-Danson et al., 2001). Stenella clymene (S. clymene) is the most abundant species of dolphins in the coastal waters of Ghana, constituting about 34.5 % of the dolphin species (Ofori-Danson et al., 2001). Dixcove is the largest landing site of dolphins along the coast of Ghana (Ofori-Danson et al., 2001) where dolphins are usually by-caught with drift gillnets predominantly used by fishermen. Currently, dolphins are being exploited for meat in Ghana to supplement fish protein due to the decline in fish landings and rapid population growth and therefore high levels of contaminants in by-caught dolphins from Ghanaian coastal waters can have serious health implications for consumers. As there is no information on contaminant levels in by-caught dolphins from the coastal waters of Ghana, this study aimed to investigate the levels of OC pesticide residues and heavy metals in two common dolphins namely S. clymene and Grampus griseus (G. griseus) from the coastal waters of Ghana and to assess the potential human health risks associated with the consumption of dolphin meat. Data collection and analyses Tissue samples were taken from S. clymene and G. griseus, commercially by-caught common dolphins from Ghanaian coastal waters at Dixcove. The length range of the dolphins was 99-150 cm. Blubber, liver and muscle samples for OC analysis were taken using standard protocols as described by Kuiken et al. (1994) and Jepson et al. (2005). Briefly, samples of blubber adjacent to the dorsal fin, muscle and liver were excised from each species using a stainless steel knife. Samples were wrapped with aluminium foil previously cleaned with hexane and placed in polyethylene bags for analysis of OC pesticide residues. Similarly, blubber, liver and muscle tissues were cut and wrapped in plastic bags for trace elements analysis using the method described by Zhou et al. (2001). The samples were placed on dry ice in an ice chest in the field and later kept in a freezer in the laboratory until they were ready for analysis. For analyses of OC pesticide residues, the frozen dolphin tissue samples (blubber, liver and muscle) were allowed to thaw and OC pesticide residues extracted by the method described by Stockin et al., (2007). Briefly, approximately 10 g portions of the tissue samples were removed and chopped into small cubes (approximately 1 cm3). The samples were accurately weighed and placed into a blender. About 30 g powdered sodium sulphate was then added to each sample and blended until a free-flowing mixture was obtained. Each sample was subsequently packed into a Soxhlet extraction thimble and placed in a Soxhlet extractor. Before extraction, internal standards (100 ng each of PCB 30 and PCB 204) were added to each sample. The samples were extracted with 200 ml dichloromethane/hexane (1:1 v/v) mixture for at least 16 h. 5 ml portions of the extracts were transferred into tared flasks and evaporated to constant weight in an oven at 40 oC. The lipid content was measured by weight difference. The remaining extracts were rotary evaporated at 40 oC to near-dryness and the residual extracts cleaned-up as described by Bergen et al. (1993) and Pastor et al. (1993). Briefly, the residual extracts were re-dissolved in 2 ml n-hexane and quantitatively transferred into test-tubes. Lipid was removed by the addition of 1 ml concentrated sulphuric acid (previously washed with n-hexane) to the extracts. After equilibration, the clear organic portions were transferred to silica gel columns overlain with anhydrous sodium sulfate as described by Perugini et al. (2004). The eluates were concentrated to 2 ml, using a gentle stream of nitrogen gas, and then transferred into glass vials for chromatographic analysis. Recovery analysis was carried out on samples fortified with pesticide standards at 1 ngg-1. Blanks were also prepared and analyzed with the samples to check contamination from solvents, reagents and vials. Analyses of OC pesticide residues in blubber, liver and muscle tissues of dolphins were carried out using gas chromatograph (Hewlett-Packard series II model 6890) equipped with an electron capture detector. A capillary column (Varian VF-5MS, length 30 m, ID 0.25 mm, film thickness 0.025 µm) was used. For optimal operation of the GC, injector temperature was set at 180oC; column temperature at 60 oC for 2 min and then increased to 180oC at a rate of 25 oC/min, where it was held for 1 min before increasing to 300 oC at 5 oC/min. This column temperature was maintained for 31.8 min while keeping the detector temperature at 310 oC; make-up gas flow rate at 29.0 ml N2/min; and column flow rate at 1.0 ml/min. Injected volumes of extracts were 1.0 µl. Analyte peaks were identified by their retention times compared to the corresponding retention times of the pesticide standards. No independent method of confirmation was applied. Triplicate analyses were performed for all the samples. Recovery values were calculated from calibration curves constructed from the concentration and peak area of the chromatograms obtained with OC pesticide standards. Detection limits of the GC were found by determining the lowest concentrations of the residues in the tissues that could be reproducibly measured at the operating conditions of the GC. All
solvents and reagents used were of pesticide grade and were purchased from Sigma Aldrich Chemicals (USA). Pesticide standards were obtained from Dr. Ehrenstorfer (Ausburg, Germany). Individual OC pesticide stock solutions were prepared in isooctane. Standard mixtures were prepared from stock solutions (50-100 mg in 100 ml acetone) and then diluted to working calibration standards at three concentration levels with acetone/hexane (9:1 v/v). All solutions were stored at 4oC. For heavy metals analyses, samples were thawed, placed in clean dry vials and then freeze-dried for 48 hrs to remove all water from the tissues. The samples were homogenized by grinding to powder using agate mortar and pestle. Homogenized sub-samples of 100 ± 3 mg were weighed onto clean polyethylene films, wrapped airtight and heat-sealed. The sealed samples were packed in clean plastic capsules, heat-sealed and subjected to instrumental neutron activation analysis (INAA) as described by Sefor-Armah et al. (2004), Nyarko et al. (2006) and Addo et al. (2008). For short-lived radionuclides (Cu and Mn), samples were irradiated for 60 s and activities measured after a short decay period while for the medium-lived radionuclides (Cd and Hg), samples were irradiated for 3600 s and activities measured after 48 h decay period. The activities were measured with a high purity germanium (HPGe) n-type coaxial detector (GR 2518, Canberra Industries Inc.). The samples were counted (600 s for short irradiation and 2 h for medium irradiation) and the counts (areas) under the photo peaks of the metals were converted into concentrations using the comparator method (Ehmann and Vance, 1991). Biological standard reference materials (Hay Powder and Oyster Tissue standard reference materials from the National Institute of Standards and Technology) were also analyzed to validate the INAA method employed for this study while blanks were prepared and analyzed in the same manner as the analytical samples to check contamination. All containers used were acid washed with 1% hydrochloric acid solution to remove all binding traces of metals, oven-dried and stored in a desiccator at room temperature until use. The potential human health risks from consumption of dolphin meat contaminated with OC pesticide residues and trace metal among the Ghanaian population were assessed following the method described by Jiang et al., (2005), which is based on the derivation of hazard ratios (HRs). MOFA (2004) estimates the per capita consumption of fish for Ghana to be 25 kg per annum. According to Ofori-Danson et al. (2001), by-caught dolphins constitute about 8% of the total fish landings in Ghana. Thus, the annual per capita consumption of dolphin in Ghana was estimated to be 2 kg (i.e. 8% of 25 kg) and the daily per capita consumption to be 5.48 g. The average daily consumption of dolphin per body weight for the Ghanaian population was estimated to be 0.0913 gkg-1 body weight by dividing the daily per capita consumption (5.48 g) by the average body weight of an adult, which was set at 60 kg (Jiang et al., 2005; Wagman, 2000). The average daily exposure to each contaminant (µgkg-1 body wt) for the Ghanaian population was then obtained from the product of the dolphin consumption and the contaminant concentration (µgg-1) according to (Jiang et al., 2005). HRs were then derived for each contaminant by dividing average daily exposure by the benchmark concentration (for cancer risk) or by the oral reference dose (for non-cancer risk). The benchmark concentrations used were obtained from Dougherty et al. (2000) and represent exposure concentrations at which lifetime cancer risk is one in one million (1/1,000,000) of the population while oral reference doses (RfDs) used were those of USEPA (1990). Benchmark concentrations and oral RfDs of the OC and trace metal contaminants are shown in Table 1. Results OC pesticide residues The average concentrations of OC pesticide residues in blubber, muscle and liver tissues of S. clymene and G. griseus are shown in Table 2. The OC pesticide residues detected were p,p′-DDT, p,p′-DDE, γ-HCH, δ-HCH, α-endosulfan, β-endosulfan, heptachlor, and methoxychlor. The highest concentration (6.54 ngg-1 wet wt.) was recorded for p,p′-DDE. Average concentrations of p,p′-DDT ranged from < 0.01 to 0.42 ngg-1 wet wt. while average concentrations of its metabolite, p,p′-DDE ranged from < 0.01 to 6.54 ngg-1 wet wt. In G. griseus, concentrations of p,p′-DDE residues were significantly higher (p < 0.5) than the concentrations of p,p′-DDT. The DDE/ΣDDT ratios (ratio of the concentration of p,p′-DDE to the sum of the concentrations of p,p′-DDT and p,p′-DDE) ranged from 0.40 to 0.94. Only γ-HCH and δ-HCH isomers of HCH were detected, the γ-HCH isomer being most frequently detected. The concentrations of γ-HCH residues were significantly higher (p < 0.05) than the concentrations of the other OC pesticide residues in all tissues analyzed except in the blubber of G. griseus. The highest concentration of γ-HCH (3.35 ngg-1 wet wt.) was detected in the blubber of S. clymene while the lowest concentration (0.08 ngg-1 wet wt.) was found in the liver of S. clymene. Only the blubber and muscle of G. griseus contained measurable levels of α-endosulfan residues. In both S. clymene and G. griseus, β-endosulfan residues were detected in only the blubber at average concentrations of 0.02 ngg-1 while heptachlor was detected in only the blubber and muscle at average concentrations ranging from 0.01 to 0.05 ngg-1 wet wt. Methoxychlor was detected in only the blubber (0.62 ngg-1 wet wt.) of S. clymene but it was detected in blubber, muscle and liver tissues of G. griseus at average concentrations ranging from 0.05 to 1.27 ngg-1 wet wt. Lipid content and OC pesticide residue concentrations were significantly higher (p < 0.05) in G. griseus
than in S. clymene. Generally, OC pesticide residues concentrations and lipid content of the tissues were in the order blubber > muscle > liver. Table 1: Benchmark concentrations (Dougherty et al., 2000) and oral RfD values (USEPA, 1990) of OC pesticides and
heavy metals
Contaminant
Cancer benchmark concentration
(µg/kg x day)
Oral RfD
(µg/kg x day)
DDTa
0.003
0.5
HCHb
0.00077
0.3
Endosulfanc
-
6
Heptachlor
0.00022
0.5
Methoxychlor
-
5
Cadmium
-
1
Manganese
-
140
Mercury
-
0.3
a = listed as p,p′-DDT
b = listed as γ-HCH
c = listed as total-endosulfan (α-endosulfan + β-endosulfan + endosulfan sulfate)
Table 2: Concentrations of organochlorine pesticide residues (ngg
-1 wet wt.) in by-caught dolphins from the coastal waters
of Ghana
Dolphin
species
Tissue
Lipid
(%)
p,p′-
DDT
p,p′-
DDE
γ-
HCH
δ-
HCH
α-
Endosulfan
β-
Endosulfan
Heptachlor
Methoxychlor
S.
clymene
Blubber
72.6
0.03
0.02
3.35
0.10
ND
0.02
0.01
0.62
Muscle
3.7
ND
ND
0.11
ND
ND
ND
0.02
ND
Liver
1.0
0.03
ND
0.08
ND
ND
ND
ND
ND
G.
griseus
Blubber
73.7
0.42
6.54
1.46
0.09
0.08
0.02
0.05
1.27
Muscle
6.96
0.04
0.14
0.83
0.02
0.02
ND
0.01
0.34
Liver
1.7
0.03
0.05
0.75
ND
ND
ND
ND
0.05
ND = not detected (i.e. < 0.01 ngg-1
wet wt.)
Heavy metals The results of the analysis of the standard reference materials are shown in Table 3, and were generally in good agreement with the certified values. The average concentrations of heavy metals in blubber, muscle and liver tissues of S. clymene and G. griseus are shown in Table 4.
Organochlorine pesticide and heavy metals in dolphins from Ghanaian coastal waters 54
Table 3: Comparison of Concentrations in Standard Reference Materials with Local Laboratory Values
Elements Reference Material No. of
Measurements
Certified value
(mgkg-1
)
Measured value
(mgkg-1
)
Ratio
Cu Oyster Tissue 5 71.6 ± 1.6 70.78 ± 8.56 0.99
Cd Oyster Tissue 5 2.48 ± 0.08 2.39 ± 0.20 0.96
Hg Hay Powder 5 0.013 0.012 ± 0.002 0.92
Table 4. Concentrations of heavy metals (µgg
-1 dry wt.) in by-caught dolphins from the coastal waters of Ghana
Dolphin species
Tissue
Cu
Mn
Cd
Hg
S. clymene
Blubber
0.13
0.03
0.02
0.02
Muscle
0.60
0.15
0.16
0.05
Liver
0.67
0.77
0.77
0.16
G. griseus
Blubber
0.05
0.02
0.03
0.01
Muscle
0.38
0.16
0.32
0.04
Liver
0.55
0.30
0.54
0.05
Average concentrations of heavy metals (copper, manganese, cadmium and mercury) ranged from 0.01 to 0.77 µgg-1 dry wt. Cadmium and manganese recorded the highest average metal concentrations (0.77 µgg-1 dry wt.), all in the liver of S. clymene. Compared to the other metals, copper concentrations were highest in all the tissue analyzed except in the liver of S. clymene. Generally, for each tissue, concentrations of mercury were relatively lower than the concentrations of copper, manganese and cadmium. The highest average concentration of mercury (0.16 µgg-1 dry wt.) was recorded in the liver of S. clymene while the lowest average concentration (0.01 µgg-1 dry wt.) was recorded in the blubber of G. griseus. In general, accumulation of heavy metals was highest in the liver and lowest in the blubber. Heavy metal concentrations increased in the order copper > cadmium > manganese > mercury and S. clymene accumulated higher levels of heavy metals than G. griseus. Health risk assessment Fig. 1a shows the hazard ratios for OC pesticide residues. Hazard ratios for heavy metals are shown in Fig. 1b. The hazard ratios for both OC pesticide residues and heavy metals were all less than 1.
Fig. 1. Hazard ratios (HR) for (a) γ- HCH, p,p′-DDT, endosulfan, methoxychlor and heptachlor; (b) manganese, cadmium
and mercury. If HR is greater than 1, then the average exposure exceeds the benchmark concentration. Graphs are defined
as follows: = Cancer HR; = Non-cancer HR
Discussion The occurrence of OC pesticide residues and heavy metals in dolphin tissues can be attributed to bioaccumulation of these contaminants along the marine food chain (USPHS, 1997a, 1997b; Blus et al., 1996; Hayteas and Duffield, 2000). The generally low concentrations of OC pesticide residues and heavy metals in dolphins (S. clymene and G. griseus) suggest low levels of these contaminants in the Ghanaian coastal waters and the deep-sea regions (Chevreuil et al., 1996;
FAO/IOC/IAEA, 1993). However, OC pesticides and heavy metals are persistent (Clark, 1997) and toxic even at low levels and therefore their occurrence in dolphins is of concern. Sources of OC pesticides and heavy metals may include runoff and riverine inflows from land, atmospheric transport from remote areas (Hoff et al., 1992) and discharge of domestic and industrial wastewater into coastal waters (Biney et al., 1987). The observed variations in the concentrations of OC pesticides in the two dolphin species may be due to differences in their dietary sources and in the physicochemical properties of the contaminants such as lipophilicity and biodegradability (O’Hara et al., 1999). Higher levels of OC pesticide residues in the blubber reflect the lipophilic nature of the OC pesticides and their tendency to accumulate in tissues with high lipid content (Larsson et al., 1990; Blus et al., 1996). The relatively lower concentrations of p,p′-DDT compared to its degradation product, p,p′-DDE, as well as the low p,p′-DDE/ΣDDT ratios (0.40-0.94) indicate contamination as a result of previous usage of DDT (Aguilar, 1984) rather than current use as DDT degrades to DDE over time. The presence of the persistent γ-HCH in the dolphins, without the occurrence of the other isomers such as α-HCH, β-HCH and δ-HCH, also indicate previous exposure of the dolphins rather than current exposure to HCHs (Edwards, 1977). Endosulfan is a current-used pesticide and is being widely used in vegetable cultivation and cotton plantations in many countries including Ghana (Ntow et al., 2006). The very low levels of α-endosulfan and β-endosulfan (isomers of endosulfan) detected may be due to the high affinity of these pesticides to adsorb to soil particles, which makes them relatively immobile in soils (EXTOXNET, 2006). Thus, they may not be easily dislodged by runoff to reach the coastal waters. The occurrence of α-endosulfan in relatively higher concentrations as compared to β-endosulfan may be due to its higher lipophilicity than β-endosulfan (Guerin and Kennedy, 1992; Antonious et al., 1998). The generally very low levels of heptachlor and methoxychlor may indicate a possible adherence to the ban on their use. The greater OC pesticide concentrations found in G. griseus may be due to the higher lipid content of their tissues compared to the lipid content of S. clymene tissues. Large proportion of lipids present within cetaceans facilitates bioaccumulation (Blus et al., 1996; Hayteas and Duffield, 2000), and therefore G. griseus are at greater risks of OC toxicity. Generally, apart from γ-HCH, concentrations of p,p′-DDT, p,p′-DDE and heptachlor found in this study were much lower than those found in common dolphins from other areas such as the Ganges River in India (Kannan et al., 1993) and New Zealand waters (Stockin et al., 2007). The higher concentrations of heavy metals in the liver, relative to the blubber and muscle, may be related to the mineral storage function of the liver (Wagman, 2000; Clark, 2003). Although the blubber had relatively higher levels OC pesticide residues, it had relatively low levels of heavy metals suggesting that heavy metals are not lipophilic. Copper and manganese are essential trace metals. However, cadmium has no biochemical or nutritional function, and it is highly toxic to both plants and animals (USPHS 1997a, WHO 1992). Mercury is also a toxic and a non-essential trace metal (WHO, 1989; WHO, 1992; USPHS, 1997b; Clark, 2003). Although copper was generally found in relatively high concentrations, liver concentrations of copper in this study were within the range of concentrations (3 to 30 µgg-1 wet weight) thought to represent homeostatic control (Law, 1996). The tolerable levels of copper and manganese observed suggest that these metals are being regulated in the dolphins and their levels in dolphins may not reflect environmental levels. Cadmium and mercury on the other hand are poorly regulated in biological systems (WHO, 1989) and their low levels in both species of dolphins may reflect low environmental levels of these contaminants. OC pesticide residues and heavy metals can exert a broad range of toxic effects on humans. For instance, OCs can cause hormonal dysfunction (Reijnders and Aguilar, 2002), immune suppression (Kuiken et al., 1994; Jepson et al., 2005), reproductive disorders (Reijnders, 1986; Schwacke et al., 2002; Wells et al., 2005) and the development of tumours (De Guise et al., 1994). High levels of copper can cause cirrhosis of the liver in children while mercury can cause cancers (Goyer, 1996). Certain forms of cadmium are also carcinogenic (IARC, 1998) while high levels of manganese exposure can cause neurological impairment (Wennberg, 1991) as well as mental and emotional disturbances (ATSDR, 1997). However, the hazard ratios (HRs) for OC pesticides (Fig. 1a) and heavy metals (Fig. 1b) were all less than 1, indicating that OC pesticide and heavy metal exposure from the consumption of the dolphin meat will not result in any appreciable health effects associated with these contaminants.
Conclusion Generally, low concentrations of OC pesticides and heavy metals were found in by-caught dolphins from Ghanaian coastal waters. The γ-HCH residue was most ubiquitous OC pesticide in dolphin tissues while heavy metals (copper, manganese, cadmium and mercury) were all commonly found in dolphin tissues. The current levels of OC pesticide residues and heavy metals detected in S. clymene and G. griseus would not pose appreciable health risk to the consumers of these dolphins. Most OC pesticides and heavy metals are however persistent and toxic at low concentrations and therefore there is the need
for continuous monitoring of these contaminants in dolphins from the costal waters of Ghana to protect these species and human health.
Acknowledgement This work was financially supported by the International Atomic Energy agency (IAEA) through the RAF7/008 project in Ghana. The authors thank Mr. Nicholas Opata of the Ghana Atomic Energy Commission (GAEC) for providing technical assistance in the use of the nuclear reactor facility. Mr. Paul Osei-Fosu of the Ghana Standards Board is also acknowledged for his technical assistance in the use of the gas chromatograph.
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