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A comparison of trace element concentrations in cultured and wild carp (Cyprinus carpio) of Lake Kasumigaura, Japan
Ecotoxicology and Environmental Safety 53 (2002) 348–354
A comparison of trace element concentrations in cultured and wild carp (Cyprinus carpio) of Lake Kasumigaura, Japan M.G.M. Alam,a A. Tanaka,b G. Allinson,c,* L.J.B. Laurenson,c F. Stagnitti,c and E.T. Snowa a
b
School of Biological and Chemical Sciences, Deakin University, 221 Burwood Highway, Burwood, Victoria 3125, Australia Environmental Chemistry Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-0053, Japan c School of Ecology and Environment, Deakin University, P.O. Box 423, Warrnambool 3280, Victoria, Australia Received 19 September 2000; received in revised form 4 February 2002; accepted 15 April 2002
Abstract The concentrations of 13 elements were determined in the muscle, liver, intestine, kidney, and gonads of cultured and wild carp caught at two sites in Lake Kasumigaura, Japan, between September 1994 and September 1995. Despite having a reputation for being heavily polluted, the carp were not heavily burdened with metals. Our results suggest that despite their dietary differences, the wild and cultured fish were accumulating and distributing metals in the same manner and that aquaculture practices are not increasing metal concentrations in these fish. Metal concentrations were lowest in muscle, and did not exceed established quality standards for fish. The differences in metal concentrations between cultivated and wild carp are negligible and should pose no health problems for consumers of either type of fish. r 2002 Elsevier Science (USA). All rights reserved. Keywords: Lake Kasumigaura; Carp; Heavy metals; Cage culture; Aquatic pollution
1. Introduction Metallic elements are environmentally ubiquitous, readily dissolved in and transported by water, and readily taken up by aquatic organisms. Metals enter the aquatic environment by atmospheric deposition, by erosion of the geological matrix, or from anthropogenic sources, such as industrial effluents, and mining wastes. Bioaccumulation of trace metals by fish, together with the underlying mechanisms, has been extensively studied in field and laboratory studies (Boudou and Ribeyre, 1989). Essentially, fish assimilate metals by ingestion of particulate material suspended in water, ingestion of food, ion exchange of dissolved metals across lipophilic membranes, e.g., the gills, and adsorption on tissue and membrane surfaces. Excretion of metals occurs via the feces, urine, and respiratory membranes. Metal distribution between the different tissues depends on the mode
*Corresponding author. Fax: +61-3-5563-3462. E-mail address: [email protected] (G. Allinson).
of exposure, i.e., dietary and/or aqueous exposure, and can serve as a pollution indicator. The common carp, Cyprinus carpio L., is one of the most extensively cultivated fish species in the world (Jhingran, 1977). Cultured carp consume artificial diets, generally commercial pellets containing 32% protein and 3.5% lipids. In natural lake environments, on the other hand, the major food items of wild carp are found in the sediment. Omnivorous, sediment-dwelling, fish species such as carp may therefore accumulate heavy metals more readily than pelagic species as a result of exposure to the generally higher metal content of sediment compared with the water column (Phillips, 1980). No detailed study has been undertaken to compare and contrast the concentrations of trace metals in cultivated and wild carp in Lake Kasumigaura, despite the fact that fish are considered an essential part of the diet in the region. Fish were therefore collected from two sites: culture cages, and an area not used for carp culture. Metal concentrations in five tissues (muscle, liver, intestine, kidney, and gonads) were determined by
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inductively coupled plasma-atomic emission spectrophotometry (ICP-AES) and inductively coupled plasmamass spectrometry (ICP-MS). The data are examined to determine if there are significant differences in metal concentrations between the wild and cultured carp. Where relevant, the concentrations measured are compared with the maximum values permitted by the Australian Food Standards Code and the provisional Codex limits, and the human health implications for heavy consumers of this food are discussed.
2. Materials and methods 2.1. Study site: Lake Kasumigaura Lake Kasumigaura (Fig. 1) is the second largest lake in Japan. The lake is located approximately 60 km northeast of the Tokyo metropolitan area (361020 N; 1401350 E) near the Pacific Ocean and just west of the Kashima industrial area. Lake Kasumigaura has a surface area of 168 km2 ; a capacity of 800 106 m3 ; and a drainage area of 2200 km2 in which 850,000 people reside. The lake is shallow with two large bays (Takahamairi and Tsuchiurairi bays), a mean depth of 4:0 m (maximum depth, 7:3 m; near the mouth of Takahamairi Bay), a flat bottom, and an elevation only slightly above sea level. The level of the lake is controlled to within 750 cm by a gate on the lake outlet. The temperature difference between the surface water and deeper water is usually small (o21C; except
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for a few calm days in summer) because the wind is almost always sufficient to drive a daily turnover. Therefore, distinct thermal stratification does not develop, even during summer, and an anoxic hypolimnion is rarely observed. The water temperature at the lake center is at its lowest, 41C; in February and highest, 301C; in August. The mean water renewal rate is 220 days (Otsuki et al., 1993). There have been 104 fish species from 44 families recorded in Lake Kasumigaura (Fukushima et al., 1995). These include both saltwater and freshwater species, since the lake was connected directly to the Pacific Ocean until about 30 years ago. Pond smelt, white bait, eel, and catfish were the major species before 1965, but from 1965 to 1985 yields of pond smelt and bait decreased, while the goby and freshwater prawn increased. Recently, the populations of many naturalised fishes in the lake, such as the largemouth bass, bluegill, and pejurry have increased. Floating-net-cage culture was introduced to Lake Kasumigaura in 1951. By 1982, this method accounted for 42% of carp production in Japan because of its high productivity per unit area and ease of management, and harvesting (Kafuku and Ikenoue, 1983; Suzuki, 1986). The maximum production of 8800 tons yr1 of carp was recorded in 1978. Since 1978, production has declined, although about 5000–7000 tons of carp are still produced annually. Cages range in area from 25 to 100 m2 and are constructed to float to a depth of 1:5 m: An additional meter of netting around the top of the cages helps retain the fish from jumping. Stocked with yearlings of 100–200 g at no more than 75 fish m2 ; a cage is able to produce marketable carp ð1 kgÞ in the 6 months from April to October if fed high-quality, pelleted feeds. The annual production yields average 100–200 kg m2 (Kafuku and Ikenoue, 1983). 2.2. Sample collection
Fig. 1. Location of Lake Kasumigaura within Japan, and the positions of the Tamatsukuri and Tamari sites ðÞ within the lake.
Samples were obtained from a local aquaculturist between July 1994 and September 1995. Each month, two cultured carp were removed from the carp cultivation nets at Tamatsukuri in Lake Kasumigaura (Fig. 1, Site 1), and two fish were caught in the lake at a point far from the carp cultivation, near Tamari village (Fig. 1, Site 2). Both cultured and wild carp were rinsed with double-distilled water and then sealed in polyethylene bags for transportation to the laboratory. The pelleted fish food used by the aquaculturist was also provided for analysis. The cultured fish were stocked into five different cages at different times between March 1993 and May 1994 (Cage A, March 1993; Cage B, July 1993; Cage C, November 1993; Cage D, January 1994; Cage E, May 1994). Carp were also sampled from the cages at different times: Cage A, July, August, and September,
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1994; Cage B, October, November, and December 1994; Cage C, January, February, and March 1995, Cage D, April, May, and June 1995; Cage E, July, August, and September 1995. Thus, in all cases, the fish were sampled between 13 and 17 months after stocking. 2.3. Age determination Carp ages were determined by visual examination of magnified scales. Scales were collected from each individual from the left side under the posterior half of the pectoral fin. After being removed from the fish, scales were rinsed with distilled water, and immersed in ethanol for 3–4 h: The scales were then dried on paper towels. The clean scales were placed between two glass microscope slides, and labeled. Only scales exhibiting symmetry, distinct circuli, and smooth unbroken margins were aged with a binocular microscope (Nikon, Tokyo) at 100 magnification. 2.4. Sample preparation The total length and weight of each sample fish were recorded prior to dissection of muscle, liver, kidney, gonad, and intestine of fish using a Teflon knife on a clean, glass working surface free from metals. Muscle samples were separated using the method of UNEP (1984) below the dorsal fin without skin. The dissection was carried out only on one side of the carp, taking care not to cut too deep to avoid cutting into viscera and thus contaminating the fillet. Special care was taken to pull the skin away from the muscle with a pair of tweezers, so that the outer skin did not contaminate the flesh. All samples were stored at 201C prior to the metal analyses. To prevent trace metal contamination of the samples by laboratory equipment, all laboratoryware was soaked in 2 M HNO3 for 48 h; and rinsed five times with distilled water, and then five times with deionized water prior to use. Deionized water, of resistivity greater than 18 O cm1 ; was prepared by passing singly distilled water through a Milli-Q Water Purification System. The tissue samples were digested with concentrated nitric acid. A portion of the dissected sample visually approximating 1 g was precisely weighed, and then the material transferred to a 100 mL Teflon beaker. Thereafter, 10 mL of ultrapure concentrated HNO3 (63% w/v, Kanto Chemical Co, Tokyo, Japan) was added slowly to the sample. The Teflon beaker was covered with a Teflon watch glass, and the sample heated at 2001C on a hot plate for 3 h: Thereafter, the solution was evaporated slowly to near dryness. Two millilitres of 1 N HNO3 was added to the residue and the solution evaporated again on the hot plate. By repeating the additional digestion twice, all organic material in every sample was completely digested. After cooling, a further
10 mL of 1 N HNO3 was added. The solution was quantitatively transferred to 100 mL polyethylene bottles and diluted 10-fold with Milli-Q water. Before analysis, the samples were filtered through a 0:45-mm nitrocellulose membrane filter. Sample blanks were prepared in the laboratory in a similar manner to the field samples. 2.5. Sample analysis All samples were analyzed three times for Ag, Al, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Se, and Zn, by ICPAES (ICAP-750 Nippon Jarrel Ash, Tokyo, Japan) and ICP-MS (Model HP 4500 Yokogawa Analytical Systems Inc, Tokyo, Japan). The final reported values are the means of replicate determinations. Standard solutions were prepared from SPEX CertiPrep stock solutions. Sample blanks were analyzed after every 7–10 samples. Two standard reference materials (DORM-2 and DOLT-2, National Research Council Canada, Ottawa, Ontario, Canada) were analyzed for each trace element, one per set. For the measurement of metal and metalloids, choice of internal standard elements for matrix correction, and the development of a refined metal correction, In (indium) and TI (thallium) were chosen to produce accurate ICP-MS results. Detection limits were set at three times the standard deviation of the concentrations recorded for the blank. The concentrations were calculated on a wet weight basis. 2.6. Data analysis For the purpose of data analysis, where the trace elements were detectable only at concentrations below the limit of determination (LOD), the results were arbitrarily set equal to 0.5 LOD. Principal component analysis (PCA) was conducted using the discriminate routine in Statistica 4.1 for Macintosh grouping by site and using the concentrations of metals and tissues as independent variables. The relationships between site and fish age and site and fish length were determined by analysis of variance (ANOVA). Differences in concentrations in the five tissue types with site were also determined by ANOVA. Post hoc tests (SNK, LSD, and Fisher’s tests) were used to determine statistically significant differences following ANOVA.
3. Results Analysis of the dogfish muscle and liver standard reference materials DORM-2 and DOLT-2 (National Research Council, Canada) found all trace elements to be within 5–15% of the expected values (85–94% recovery) (Table 1). To check our analytical results, two internal standards were used. Errors were
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Table 1 Summary of DORM-2 and DOLT-2 certified metal concentrations and concentrations determined in this study Element
Reference material DORM-2 Certified value
Observed value
DOLT-2 Certified value
Observed value
Ag Al As Cd Co Cr Cu Fe Mn Ni Pb Se Zn
0.04170.013a 10.971.7 18.071.1 0.04370.008 0.18270.031 34.775.5 2.3470.16 142710 3.6670.34 19.473.1 0.06570.007 1.4070.09 25.672.3
0.05270.04 11.0470.03 17.470.1 0.04870.010 0.17870.03 35.672.3 2.3670.21 145713 4.0470.97 17.970.1 0.06270.020 1.3770.16 27.571.0
0.60870.032 25.272.4 16.671.1 20.870.5 0.2470.05 0.3770.08 25.871.1 1103747 6.8870.56 0.2070.02 0.2270.02 6.0670.49 85.872.5
0.71370.090 26.7173.4 15.572.3 21.7270.66 0.227 0.98 0.3971.04 23.6972.8 1189738 7.0671.1 0.2370.92 0.2470.88 5.6770.64 87.071.2
a
All values are mg kg1 dry wt.
Table 2 Summary of metal concentrations in fish food Element
Concentration ðmg g1 dry wtÞ
Al As Cd Co Cr Cu Fe Mn Ni Pb Se Zn
16.370.4 13.370.7 0.10070.001 BDLa 1.2370.19 6.3970.35 26072 72.471.8 1.0870.002 BDL 5.4070.59 70.071.4
a
Below detection limit.
consistently less than 10%. The concentrations of these elements measured in fish feed were reported in a previous study on the impact on carp culture of metal concentrations in the water and sediment of Lake Kasumigaura (Alam et al., 2001), and here are summarized in Table 2. The concentrations of the 13 elements Ag, Al, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Se, and Zn,) measured in the wild and cultured carp are summarized in Table 3. Values quoted are on a wet weight basis, and have not been corrected for analyte recoveries from certified reference materials. Values quoted, therefore, represent a conservative evaluation of metal concentrations. Fish ages, lengths, and weights are listed in Table 4. The predominant pathways for heavy metal uptake, target organs, and organism sensitivity are highly variable, and are dependent on factors such as metal concentration, age, size, physiological status, habitat
preferences, feeding behavior, and growth rates of fish (Chapman et al., 1996). Principal component analysis, using the concentrations of metals as independent variables, were unable to demonstrate differences between the sites in terms of the suite of metals accumulated by the fish or their distribution in the fish. This result suggests that, despite their habitat and dietary differences, both the wild and cultured carp were accumulating dietary metals in essentially the same manner. There was no statistically significant difference in age between the wild and cultured specimens ðP > 0:05Þ; although wild fish were statistically significantly longer than cultured carp ðPo0:05Þ: Fish length was used therefore as a surrogate for age in our data analysis. No statistically significant correlations ðPo0:05Þ between carp length and metal concentration in muscle tissue were found. Freshwater fin fish are known to maintain constant internal metal concentrations, with the result that concentrations of essential elements do not increase with age (Chapman et al., 1996); so it is surprising that statistically significant correlations ðPo0:05Þ between carp length and metal concentration were found in the intestine (Zn) and kidney (Cd, Mn). Post hoc analysis (SNK and LSD) between cultured and wild fish determined that there were statistically significantly higher concentrations of As (liver, muscle), Cd (intestine, kidney), and Pb (intestine) and statistically significantly lower concentrations of Ag (intestine and kidney), Al (intestine), Fe (gonads), Mn (intestine), Se (intestine, kidney, liver, gonads), and Zn (intestine, kidney) in cultured fish ðPo0:05Þ: There were no statistically significant differences in the concentrations of Co, Cr, Cu, and Ni in any organ in wild and cultured fish, nor of As, Cd, Pb, Ag, Al, Fe, Mn, and Se in organs not otherwise specified ðP > 0:05Þ:
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Table 3 Summary of metal concentrations in the intestine, kidney, liver, muscle and reproductive organs of wild (a) and cultured (b) Lake Kasumigaura carp Element
Organ metal concentrations ðmg kg1 Þ Intestine
Kidney
Liver
Muscle
Gonads
(a) Wild carp Ag Al As Cd Co Cr Cu Fe Mn Ni Pb Se Zn
0.004 (191)a 2.678 (145) 0.076 (67) 0.033 (131) 0.029 (109) 0.070 (24) 1.646 (99) 65.90 (138) 4.646 (231) 0.060 (105) 0.030 (58) 0.760 (41) 447 (57)
0.005 (112) 0.948 (77) 0.102 (48) 0.273 (78) 0.357 (51) 0.070 (23) 2.014 (27) 88.14 (71) 1.569 (61) 0.092 (97) 0.042 (93) 1.195 (28) 330 (55)
0.003 (70) 0.877 (110) 0.087 (128) 0.010 (87) 0.026 (170) 0.079 (63) 0.741 (62) 203 (68) 0.390 (59) 0.055 (178) 0.087 (163) 0.898 (44) 201 (82)
0.003 0.778 0.095 0.009 0.005 0.067 0.249 2.729 0.307 0.041 0.031 0.300 5.433
0.002 (152) 0.906 (176) 0.063 (157) 0.009 (66) 0.020 (449) 0.068 (5) 1.263 (162) 185 (195) 0.566 (64) 0.042 (88) 0.037 (109) 0.641 (74) 108 (79)
(b) Cultured carp Ag Al As Cd Co Cr Cu Fe Mn Ni Pb Se Zn
0.0011 (63) 0.7753 (73) 0.0991 (59) 0.1082 (130) 0.0195 (67) 0.0727 (29) 1.3763 (33) 20.96 (44) 1.5256 (90) 0.1020 (306) 0.0459 (95) 0.6196 (37) 270 (121)
0.0013 (57) 0.5293 (57) 0.1052 (52) 0.3851 (64) 0.3459 (57) 0.0715 (22) 2.0105 (36) 73.50 (50) 1.5892 (48) 0.1332 (309) 0.0428 (77) 0.9884 (34) 175 (62)
0.0018 (150) 0.1568 (97) 0.1568 (122) 0.0094 (61) 0.0236 (72) 0.0949 (88) 0.5949 (45) 179 (67) 0.3599 (53) 0.1335 (300) 0.0904 (208) 0.7491 (59) 106 (95)
0.0019 0.5833 0.1789 0.0074 0.0053 0.0757 0.3322 4.1246 0.1766 0.0397 0.0319 0.2256 5.4490
a
(45) (51) (41) (59) (33) (0) (47) (41) (71) (98) (116) (38) (35)
(46) (63) (48) (11) (35) (35) (63) (47) (33) (77) (66) (40) (40)
0.0010 (72) 0.8119 (115) 0.0624 (46) 0.0086 (54) 0.0347 (133) 0.0716 (24) 0.6073 (52) 67.02 (132) 0.3225 (52) 0.3485 (337) 0.0290 (113) 0.5248 (59) 74.42 (116)
Values in parentheses indicate the coefficient of variance (%).
Table 4 Summary of the ages, lengths, and weights of carp Carp
Wild Cultured
Age (yr)
Length (cm)
Weight (kg)
Mean
C.V. (%)
Mean
C.V. (%)
Mean
C.V. (%)
2.10 2.17
24 21
50.02 45.64
10 9
1.52 1.30
36 40
4. Discussion The Japanese consume an average of 90 g of fish and shellfish each day (Ministry of Health and Welfare, 1998), often raw as sashimi and sushi (sashimi being slices of raw fish eaten directly, whereas with sushi the raw fish is served on rice cakes). Contamination of fish is therefore of particular concern to both fisheries and the general public. Chemical factors, such as acidity, buffer capacity, and the presence of calcium and organic compounds, influence the bioavailability and accumulation of metals in fish (Chapman et al., 1996). In our previous study, we determined that there were statisti-
cally significant differences in metal concentrations in the water column and sediments between the aquaculture and wild sites, with metal concentrations higher at the culture site (Alam et al., 2001). These higher concentrations may be due to release of metals from uneaten fish food (metal concentrations provided in Table 2) and fish excreta. Our results suggest that such localized increases in metal concentrations do not appear to be increasing muscle concentrations of any metal, apart from As. The muscle analyzed in this study was found to comply with the Australian Food Standards Code (AFSC) maximum concentrations for arsenic, copper,
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Table 5 Summary of dietary intakes for measured elements when consuming 90 g of carp muscle Reservoir
Intake per 90 g muscle (wet wt.) (mg) As
Cd
Cu
Ni
Se
Zn
Wild Cultured
8.5 16.1
0.79 0.66
22.4 29.9
3.72 3.57
27.0 20.3
489 490
Reference limit
1050a
490a
35 000b
1400c
70d
70 000b
a
UN FAO PWTDI (Provisional Tolerable Weekly Intake). UN FAO PMTDI, (Provisional Maximum Tolerable Daily Intake. c US EPA oral reference dose of 20 mg kg1 day1 : d US Food and Nutrition Board (1989) maximum recommended daily intake of 70 mg: All calculations assume an average male adult body weight of 70 kg: b
lead, and zinc of less than 1.0, 10.0, 1.0, and 150 mg kg1 wet wt, respectively (Australia New Zealand Food Authority, 1998) (Note: The only UN FAO Codex levels for metals in fish are 0.5 and 1:0 mg kg1 wet wt methylmercury in nonpredatory and predatory fish, respectively (R. Ellis, Acting Secretariat to Joint FAO/ WHO Expert Committee on Food Additives, pers. comm.). We therefore use the AFSC maximum concentrations as a benchmark.) Consumption of 90 g of carp muscle, from wild or cultured sources, would provide approximately 10% of the Codex provisional tolerable weekly intake (PTWI) of As, 1% PWTI for Cd, 0.1% of the Codex provisional maximum tolerable daily intake (PMTDI) for Cu, 1% of the U.S. EPA’s oral reference dose for Ni, and 1% of the PMTDI for Zn (Table 5) (R. Ellis, pers. comm.; EPA, 1985). Dietary intake from consumption of 90 g of muscle tissue would also provide approximately 10% of the required daily intake of Fe and 5% of the typical daily intake of Al, but negligible amounts of dietary Ag, Co, Cr, and Pb (Cox, 1995). The intestine, kidney, liver, and gonads of both wild and cultured carp on most occasions failed the ANZFA maximum concentration for zinc. However, since the Japanese do not routinely consume the viscera, this should not lead to deleterious effects to consumers of these fish.
5. Conclusion Lake Kasumigaura is considered to be a highly polluted water body, a view reinforced by the carcasses of large fish often observed on the shoreline. However, our results suggest that carp reared in Lake Kasumigaura are not heavily burdened with metals. In addition, despite their dietary differences, the wild and cultured fish were accumulating and distributing metals in much the same manner. Also, aquaculture practices are not leading to enhanced metal content in the fish. Metal concentrations were lowest in muscle, and did not exceed established quality standards for fish. The
differences in metal concentrations between cultivated and wild carp are negligible, and should pose no health problems for consumers of these fish.
Acknowledgments The authors thank Dr. A. Hamada, former Director of the Ibaraki Prefectural Freshwater Experimental Station, for assistance during the experimental field work, and Professor Humitake Seki of the Institute of Biological Sciences, University of Tsukuba, for his advice and suggestions throughout this research. The research was partly conducted while the authors G.A. and F.S. were guests of the National Institute for Environmental Studies, Tsukuba, Japan, funded by the Japanese Government JGRAFS and STA schemes. The research by E.T.S. and M.G.M.A. at Deakin University was supported in part by the U.S. Environmental Protection Agency’s Science to Achieve Results (STAR) program, and by the Electric Power Research Institute Contract No. EP-p4898/C2396.
References Alam, M.G.M., Tanaka, A., Stagnitti, F., Allinson, G., Maekawa, T., 2001. Observations on the effects of caged carp culture on water and sediment metal concentrations in Lake Kasumigaura, Japan. Ecotoxicol. Environ. Safety 48, 107–115. Australia New Zealand Food Authority, 1998. Food Standards Code. Standard A12, Issue 37. Boudou, A., Ribeyre, F., 1989. Fish as ‘‘biological model’’ for experimental studies in ecotoxicology. In: Boudou, A., Ribeeyre, F. (Eds.), Aquatic Ecotoxicology: Fundamental Concepts and Methodologies, Vol II. CRC Press, Boca Raton, FL, USA, pp. 127–162. Chapman, P.M., Allen, H.E., Godtfredsen, K., Z’Graggen, M.N., 1996. Evaluation of bioaccumulation factors in regulating metals. Environ. Sci. Technol. 30, 448A–452A. Cox, P.A., 1995. The Elements on Earth: Inorganic Chemistry in the Environment. Oxford University Press, Oxford.
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EPA (U.S. Environmental Protection Agency), 1985. Drinking Water Criteria Document for Nickel. EPA Environmental Criteria Assessment Office, EPA/600/X-84-193-1, Cincinnati, OH. Fukushima, T., Aizaki, M., Takamura, N., Hanazato, T., Nohara, S., Iwakuma, T., Ebise, S., 1995. In: Aizaki, M. (Ed.), Environmental Map of Lake Kasumigaura. The National Institute for Environmental Studies, Buyodo (Co) Publisher, Tokyo. Jhingran, V.G., 1977. Fish and Fisheries of India. Hindustan Publishing Corporation (India), Jawahar Nagar, Delhi, India, pp. 954. Kafuku, T., Ikenoue, H., 1983. Modern Methods of Aquaculture in Japan. Elsevier, New York, NY, pp. 1–16. Japanese Ministry of Health and Welfare, Japan, 1998. Results of national survey of nutritive conditions in 1996 (in Japanese). Otsuki, A., Goma, R.H., Aizaki, M., Nojiri, Y., 1993. Seasonal and spatial variations of dissolved nitrogenous nutrient concentrations
in hypertrophic shallow lake, with special reference to dissolved organic nitrogen. Verh. Int. Verein. Limnol. 25, 187–192. Phillips, D.J.H., 1980. Quantitative Aquatic Biological Indicators: Their Use to Monitor Trace Metal and Organochlorine Pollution. Applied Science Publishers Ltd., London, UK, 488pp. Suzuki, R., 1986. Intensive carp rearing in Japan. In: Billard, R., Marcel, J. (Eds.), Aquaculture of Cyprinids. INRA, Paris, pp. 327–333. UNEP, 1984. Sampling of selected marine organisms and sample preparation for trace metal analysis. Reference Methods for Marine Pollution Studies, No. 7, Rev. 2. US Food and Nutrition Board, 1989. Recommended Dietary Allowances. 10th Edition. National Academy of Science, Washington, DC.
A comparison of trace element concentrations in cultured and wild carp (Cyprinus carpio) of Lake Kasumigaura, Japan M.G.M. Alam,a A. Tanaka,b G. Allinson,c,* L.J.B. Laurenson,c F. Stagnitti,c and E.T. Snowa a
b
School of Biological and Chemical Sciences, Deakin University, 221 Burwood Highway, Burwood, Victoria 3125, Australia Environmental Chemistry Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-0053, Japan c School of Ecology and Environment, Deakin University, P.O. Box 423, Warrnambool 3280, Victoria, Australia Received 19 September 2000; received in revised form 4 February 2002; accepted 15 April 2002
Abstract The concentrations of 13 elements were determined in the muscle, liver, intestine, kidney, and gonads of cultured and wild carp caught at two sites in Lake Kasumigaura, Japan, between September 1994 and September 1995. Despite having a reputation for being heavily polluted, the carp were not heavily burdened with metals. Our results suggest that despite their dietary differences, the wild and cultured fish were accumulating and distributing metals in the same manner and that aquaculture practices are not increasing metal concentrations in these fish. Metal concentrations were lowest in muscle, and did not exceed established quality standards for fish. The differences in metal concentrations between cultivated and wild carp are negligible and should pose no health problems for consumers of either type of fish. r 2002 Elsevier Science (USA). All rights reserved. Keywords: Lake Kasumigaura; Carp; Heavy metals; Cage culture; Aquatic pollution
1. Introduction Metallic elements are environmentally ubiquitous, readily dissolved in and transported by water, and readily taken up by aquatic organisms. Metals enter the aquatic environment by atmospheric deposition, by erosion of the geological matrix, or from anthropogenic sources, such as industrial effluents, and mining wastes. Bioaccumulation of trace metals by fish, together with the underlying mechanisms, has been extensively studied in field and laboratory studies (Boudou and Ribeyre, 1989). Essentially, fish assimilate metals by ingestion of particulate material suspended in water, ingestion of food, ion exchange of dissolved metals across lipophilic membranes, e.g., the gills, and adsorption on tissue and membrane surfaces. Excretion of metals occurs via the feces, urine, and respiratory membranes. Metal distribution between the different tissues depends on the mode
*Corresponding author. Fax: +61-3-5563-3462. E-mail address: [email protected] (G. Allinson).
of exposure, i.e., dietary and/or aqueous exposure, and can serve as a pollution indicator. The common carp, Cyprinus carpio L., is one of the most extensively cultivated fish species in the world (Jhingran, 1977). Cultured carp consume artificial diets, generally commercial pellets containing 32% protein and 3.5% lipids. In natural lake environments, on the other hand, the major food items of wild carp are found in the sediment. Omnivorous, sediment-dwelling, fish species such as carp may therefore accumulate heavy metals more readily than pelagic species as a result of exposure to the generally higher metal content of sediment compared with the water column (Phillips, 1980). No detailed study has been undertaken to compare and contrast the concentrations of trace metals in cultivated and wild carp in Lake Kasumigaura, despite the fact that fish are considered an essential part of the diet in the region. Fish were therefore collected from two sites: culture cages, and an area not used for carp culture. Metal concentrations in five tissues (muscle, liver, intestine, kidney, and gonads) were determined by
0147-6513/02/$ - see front matter r 2002 Elsevier Science (USA). All rights reserved. PII: S 0 1 4 7 - 6 5 1 3 ( 0 2 ) 0 0 0 1 2 - X
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inductively coupled plasma-atomic emission spectrophotometry (ICP-AES) and inductively coupled plasmamass spectrometry (ICP-MS). The data are examined to determine if there are significant differences in metal concentrations between the wild and cultured carp. Where relevant, the concentrations measured are compared with the maximum values permitted by the Australian Food Standards Code and the provisional Codex limits, and the human health implications for heavy consumers of this food are discussed.
2. Materials and methods 2.1. Study site: Lake Kasumigaura Lake Kasumigaura (Fig. 1) is the second largest lake in Japan. The lake is located approximately 60 km northeast of the Tokyo metropolitan area (361020 N; 1401350 E) near the Pacific Ocean and just west of the Kashima industrial area. Lake Kasumigaura has a surface area of 168 km2 ; a capacity of 800 106 m3 ; and a drainage area of 2200 km2 in which 850,000 people reside. The lake is shallow with two large bays (Takahamairi and Tsuchiurairi bays), a mean depth of 4:0 m (maximum depth, 7:3 m; near the mouth of Takahamairi Bay), a flat bottom, and an elevation only slightly above sea level. The level of the lake is controlled to within 750 cm by a gate on the lake outlet. The temperature difference between the surface water and deeper water is usually small (o21C; except
349
for a few calm days in summer) because the wind is almost always sufficient to drive a daily turnover. Therefore, distinct thermal stratification does not develop, even during summer, and an anoxic hypolimnion is rarely observed. The water temperature at the lake center is at its lowest, 41C; in February and highest, 301C; in August. The mean water renewal rate is 220 days (Otsuki et al., 1993). There have been 104 fish species from 44 families recorded in Lake Kasumigaura (Fukushima et al., 1995). These include both saltwater and freshwater species, since the lake was connected directly to the Pacific Ocean until about 30 years ago. Pond smelt, white bait, eel, and catfish were the major species before 1965, but from 1965 to 1985 yields of pond smelt and bait decreased, while the goby and freshwater prawn increased. Recently, the populations of many naturalised fishes in the lake, such as the largemouth bass, bluegill, and pejurry have increased. Floating-net-cage culture was introduced to Lake Kasumigaura in 1951. By 1982, this method accounted for 42% of carp production in Japan because of its high productivity per unit area and ease of management, and harvesting (Kafuku and Ikenoue, 1983; Suzuki, 1986). The maximum production of 8800 tons yr1 of carp was recorded in 1978. Since 1978, production has declined, although about 5000–7000 tons of carp are still produced annually. Cages range in area from 25 to 100 m2 and are constructed to float to a depth of 1:5 m: An additional meter of netting around the top of the cages helps retain the fish from jumping. Stocked with yearlings of 100–200 g at no more than 75 fish m2 ; a cage is able to produce marketable carp ð1 kgÞ in the 6 months from April to October if fed high-quality, pelleted feeds. The annual production yields average 100–200 kg m2 (Kafuku and Ikenoue, 1983). 2.2. Sample collection
Fig. 1. Location of Lake Kasumigaura within Japan, and the positions of the Tamatsukuri and Tamari sites ðÞ within the lake.
Samples were obtained from a local aquaculturist between July 1994 and September 1995. Each month, two cultured carp were removed from the carp cultivation nets at Tamatsukuri in Lake Kasumigaura (Fig. 1, Site 1), and two fish were caught in the lake at a point far from the carp cultivation, near Tamari village (Fig. 1, Site 2). Both cultured and wild carp were rinsed with double-distilled water and then sealed in polyethylene bags for transportation to the laboratory. The pelleted fish food used by the aquaculturist was also provided for analysis. The cultured fish were stocked into five different cages at different times between March 1993 and May 1994 (Cage A, March 1993; Cage B, July 1993; Cage C, November 1993; Cage D, January 1994; Cage E, May 1994). Carp were also sampled from the cages at different times: Cage A, July, August, and September,
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1994; Cage B, October, November, and December 1994; Cage C, January, February, and March 1995, Cage D, April, May, and June 1995; Cage E, July, August, and September 1995. Thus, in all cases, the fish were sampled between 13 and 17 months after stocking. 2.3. Age determination Carp ages were determined by visual examination of magnified scales. Scales were collected from each individual from the left side under the posterior half of the pectoral fin. After being removed from the fish, scales were rinsed with distilled water, and immersed in ethanol for 3–4 h: The scales were then dried on paper towels. The clean scales were placed between two glass microscope slides, and labeled. Only scales exhibiting symmetry, distinct circuli, and smooth unbroken margins were aged with a binocular microscope (Nikon, Tokyo) at 100 magnification. 2.4. Sample preparation The total length and weight of each sample fish were recorded prior to dissection of muscle, liver, kidney, gonad, and intestine of fish using a Teflon knife on a clean, glass working surface free from metals. Muscle samples were separated using the method of UNEP (1984) below the dorsal fin without skin. The dissection was carried out only on one side of the carp, taking care not to cut too deep to avoid cutting into viscera and thus contaminating the fillet. Special care was taken to pull the skin away from the muscle with a pair of tweezers, so that the outer skin did not contaminate the flesh. All samples were stored at 201C prior to the metal analyses. To prevent trace metal contamination of the samples by laboratory equipment, all laboratoryware was soaked in 2 M HNO3 for 48 h; and rinsed five times with distilled water, and then five times with deionized water prior to use. Deionized water, of resistivity greater than 18 O cm1 ; was prepared by passing singly distilled water through a Milli-Q Water Purification System. The tissue samples were digested with concentrated nitric acid. A portion of the dissected sample visually approximating 1 g was precisely weighed, and then the material transferred to a 100 mL Teflon beaker. Thereafter, 10 mL of ultrapure concentrated HNO3 (63% w/v, Kanto Chemical Co, Tokyo, Japan) was added slowly to the sample. The Teflon beaker was covered with a Teflon watch glass, and the sample heated at 2001C on a hot plate for 3 h: Thereafter, the solution was evaporated slowly to near dryness. Two millilitres of 1 N HNO3 was added to the residue and the solution evaporated again on the hot plate. By repeating the additional digestion twice, all organic material in every sample was completely digested. After cooling, a further
10 mL of 1 N HNO3 was added. The solution was quantitatively transferred to 100 mL polyethylene bottles and diluted 10-fold with Milli-Q water. Before analysis, the samples were filtered through a 0:45-mm nitrocellulose membrane filter. Sample blanks were prepared in the laboratory in a similar manner to the field samples. 2.5. Sample analysis All samples were analyzed three times for Ag, Al, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Se, and Zn, by ICPAES (ICAP-750 Nippon Jarrel Ash, Tokyo, Japan) and ICP-MS (Model HP 4500 Yokogawa Analytical Systems Inc, Tokyo, Japan). The final reported values are the means of replicate determinations. Standard solutions were prepared from SPEX CertiPrep stock solutions. Sample blanks were analyzed after every 7–10 samples. Two standard reference materials (DORM-2 and DOLT-2, National Research Council Canada, Ottawa, Ontario, Canada) were analyzed for each trace element, one per set. For the measurement of metal and metalloids, choice of internal standard elements for matrix correction, and the development of a refined metal correction, In (indium) and TI (thallium) were chosen to produce accurate ICP-MS results. Detection limits were set at three times the standard deviation of the concentrations recorded for the blank. The concentrations were calculated on a wet weight basis. 2.6. Data analysis For the purpose of data analysis, where the trace elements were detectable only at concentrations below the limit of determination (LOD), the results were arbitrarily set equal to 0.5 LOD. Principal component analysis (PCA) was conducted using the discriminate routine in Statistica 4.1 for Macintosh grouping by site and using the concentrations of metals and tissues as independent variables. The relationships between site and fish age and site and fish length were determined by analysis of variance (ANOVA). Differences in concentrations in the five tissue types with site were also determined by ANOVA. Post hoc tests (SNK, LSD, and Fisher’s tests) were used to determine statistically significant differences following ANOVA.
3. Results Analysis of the dogfish muscle and liver standard reference materials DORM-2 and DOLT-2 (National Research Council, Canada) found all trace elements to be within 5–15% of the expected values (85–94% recovery) (Table 1). To check our analytical results, two internal standards were used. Errors were
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Table 1 Summary of DORM-2 and DOLT-2 certified metal concentrations and concentrations determined in this study Element
Reference material DORM-2 Certified value
Observed value
DOLT-2 Certified value
Observed value
Ag Al As Cd Co Cr Cu Fe Mn Ni Pb Se Zn
0.04170.013a 10.971.7 18.071.1 0.04370.008 0.18270.031 34.775.5 2.3470.16 142710 3.6670.34 19.473.1 0.06570.007 1.4070.09 25.672.3
0.05270.04 11.0470.03 17.470.1 0.04870.010 0.17870.03 35.672.3 2.3670.21 145713 4.0470.97 17.970.1 0.06270.020 1.3770.16 27.571.0
0.60870.032 25.272.4 16.671.1 20.870.5 0.2470.05 0.3770.08 25.871.1 1103747 6.8870.56 0.2070.02 0.2270.02 6.0670.49 85.872.5
0.71370.090 26.7173.4 15.572.3 21.7270.66 0.227 0.98 0.3971.04 23.6972.8 1189738 7.0671.1 0.2370.92 0.2470.88 5.6770.64 87.071.2
a
All values are mg kg1 dry wt.
Table 2 Summary of metal concentrations in fish food Element
Concentration ðmg g1 dry wtÞ
Al As Cd Co Cr Cu Fe Mn Ni Pb Se Zn
16.370.4 13.370.7 0.10070.001 BDLa 1.2370.19 6.3970.35 26072 72.471.8 1.0870.002 BDL 5.4070.59 70.071.4
a
Below detection limit.
consistently less than 10%. The concentrations of these elements measured in fish feed were reported in a previous study on the impact on carp culture of metal concentrations in the water and sediment of Lake Kasumigaura (Alam et al., 2001), and here are summarized in Table 2. The concentrations of the 13 elements Ag, Al, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Se, and Zn,) measured in the wild and cultured carp are summarized in Table 3. Values quoted are on a wet weight basis, and have not been corrected for analyte recoveries from certified reference materials. Values quoted, therefore, represent a conservative evaluation of metal concentrations. Fish ages, lengths, and weights are listed in Table 4. The predominant pathways for heavy metal uptake, target organs, and organism sensitivity are highly variable, and are dependent on factors such as metal concentration, age, size, physiological status, habitat
preferences, feeding behavior, and growth rates of fish (Chapman et al., 1996). Principal component analysis, using the concentrations of metals as independent variables, were unable to demonstrate differences between the sites in terms of the suite of metals accumulated by the fish or their distribution in the fish. This result suggests that, despite their habitat and dietary differences, both the wild and cultured carp were accumulating dietary metals in essentially the same manner. There was no statistically significant difference in age between the wild and cultured specimens ðP > 0:05Þ; although wild fish were statistically significantly longer than cultured carp ðPo0:05Þ: Fish length was used therefore as a surrogate for age in our data analysis. No statistically significant correlations ðPo0:05Þ between carp length and metal concentration in muscle tissue were found. Freshwater fin fish are known to maintain constant internal metal concentrations, with the result that concentrations of essential elements do not increase with age (Chapman et al., 1996); so it is surprising that statistically significant correlations ðPo0:05Þ between carp length and metal concentration were found in the intestine (Zn) and kidney (Cd, Mn). Post hoc analysis (SNK and LSD) between cultured and wild fish determined that there were statistically significantly higher concentrations of As (liver, muscle), Cd (intestine, kidney), and Pb (intestine) and statistically significantly lower concentrations of Ag (intestine and kidney), Al (intestine), Fe (gonads), Mn (intestine), Se (intestine, kidney, liver, gonads), and Zn (intestine, kidney) in cultured fish ðPo0:05Þ: There were no statistically significant differences in the concentrations of Co, Cr, Cu, and Ni in any organ in wild and cultured fish, nor of As, Cd, Pb, Ag, Al, Fe, Mn, and Se in organs not otherwise specified ðP > 0:05Þ:
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Table 3 Summary of metal concentrations in the intestine, kidney, liver, muscle and reproductive organs of wild (a) and cultured (b) Lake Kasumigaura carp Element
Organ metal concentrations ðmg kg1 Þ Intestine
Kidney
Liver
Muscle
Gonads
(a) Wild carp Ag Al As Cd Co Cr Cu Fe Mn Ni Pb Se Zn
0.004 (191)a 2.678 (145) 0.076 (67) 0.033 (131) 0.029 (109) 0.070 (24) 1.646 (99) 65.90 (138) 4.646 (231) 0.060 (105) 0.030 (58) 0.760 (41) 447 (57)
0.005 (112) 0.948 (77) 0.102 (48) 0.273 (78) 0.357 (51) 0.070 (23) 2.014 (27) 88.14 (71) 1.569 (61) 0.092 (97) 0.042 (93) 1.195 (28) 330 (55)
0.003 (70) 0.877 (110) 0.087 (128) 0.010 (87) 0.026 (170) 0.079 (63) 0.741 (62) 203 (68) 0.390 (59) 0.055 (178) 0.087 (163) 0.898 (44) 201 (82)
0.003 0.778 0.095 0.009 0.005 0.067 0.249 2.729 0.307 0.041 0.031 0.300 5.433
0.002 (152) 0.906 (176) 0.063 (157) 0.009 (66) 0.020 (449) 0.068 (5) 1.263 (162) 185 (195) 0.566 (64) 0.042 (88) 0.037 (109) 0.641 (74) 108 (79)
(b) Cultured carp Ag Al As Cd Co Cr Cu Fe Mn Ni Pb Se Zn
0.0011 (63) 0.7753 (73) 0.0991 (59) 0.1082 (130) 0.0195 (67) 0.0727 (29) 1.3763 (33) 20.96 (44) 1.5256 (90) 0.1020 (306) 0.0459 (95) 0.6196 (37) 270 (121)
0.0013 (57) 0.5293 (57) 0.1052 (52) 0.3851 (64) 0.3459 (57) 0.0715 (22) 2.0105 (36) 73.50 (50) 1.5892 (48) 0.1332 (309) 0.0428 (77) 0.9884 (34) 175 (62)
0.0018 (150) 0.1568 (97) 0.1568 (122) 0.0094 (61) 0.0236 (72) 0.0949 (88) 0.5949 (45) 179 (67) 0.3599 (53) 0.1335 (300) 0.0904 (208) 0.7491 (59) 106 (95)
0.0019 0.5833 0.1789 0.0074 0.0053 0.0757 0.3322 4.1246 0.1766 0.0397 0.0319 0.2256 5.4490
a
(45) (51) (41) (59) (33) (0) (47) (41) (71) (98) (116) (38) (35)
(46) (63) (48) (11) (35) (35) (63) (47) (33) (77) (66) (40) (40)
0.0010 (72) 0.8119 (115) 0.0624 (46) 0.0086 (54) 0.0347 (133) 0.0716 (24) 0.6073 (52) 67.02 (132) 0.3225 (52) 0.3485 (337) 0.0290 (113) 0.5248 (59) 74.42 (116)
Values in parentheses indicate the coefficient of variance (%).
Table 4 Summary of the ages, lengths, and weights of carp Carp
Wild Cultured
Age (yr)
Length (cm)
Weight (kg)
Mean
C.V. (%)
Mean
C.V. (%)
Mean
C.V. (%)
2.10 2.17
24 21
50.02 45.64
10 9
1.52 1.30
36 40
4. Discussion The Japanese consume an average of 90 g of fish and shellfish each day (Ministry of Health and Welfare, 1998), often raw as sashimi and sushi (sashimi being slices of raw fish eaten directly, whereas with sushi the raw fish is served on rice cakes). Contamination of fish is therefore of particular concern to both fisheries and the general public. Chemical factors, such as acidity, buffer capacity, and the presence of calcium and organic compounds, influence the bioavailability and accumulation of metals in fish (Chapman et al., 1996). In our previous study, we determined that there were statisti-
cally significant differences in metal concentrations in the water column and sediments between the aquaculture and wild sites, with metal concentrations higher at the culture site (Alam et al., 2001). These higher concentrations may be due to release of metals from uneaten fish food (metal concentrations provided in Table 2) and fish excreta. Our results suggest that such localized increases in metal concentrations do not appear to be increasing muscle concentrations of any metal, apart from As. The muscle analyzed in this study was found to comply with the Australian Food Standards Code (AFSC) maximum concentrations for arsenic, copper,
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Table 5 Summary of dietary intakes for measured elements when consuming 90 g of carp muscle Reservoir
Intake per 90 g muscle (wet wt.) (mg) As
Cd
Cu
Ni
Se
Zn
Wild Cultured
8.5 16.1
0.79 0.66
22.4 29.9
3.72 3.57
27.0 20.3
489 490
Reference limit
1050a
490a
35 000b
1400c
70d
70 000b
a
UN FAO PWTDI (Provisional Tolerable Weekly Intake). UN FAO PMTDI, (Provisional Maximum Tolerable Daily Intake. c US EPA oral reference dose of 20 mg kg1 day1 : d US Food and Nutrition Board (1989) maximum recommended daily intake of 70 mg: All calculations assume an average male adult body weight of 70 kg: b
lead, and zinc of less than 1.0, 10.0, 1.0, and 150 mg kg1 wet wt, respectively (Australia New Zealand Food Authority, 1998) (Note: The only UN FAO Codex levels for metals in fish are 0.5 and 1:0 mg kg1 wet wt methylmercury in nonpredatory and predatory fish, respectively (R. Ellis, Acting Secretariat to Joint FAO/ WHO Expert Committee on Food Additives, pers. comm.). We therefore use the AFSC maximum concentrations as a benchmark.) Consumption of 90 g of carp muscle, from wild or cultured sources, would provide approximately 10% of the Codex provisional tolerable weekly intake (PTWI) of As, 1% PWTI for Cd, 0.1% of the Codex provisional maximum tolerable daily intake (PMTDI) for Cu, 1% of the U.S. EPA’s oral reference dose for Ni, and 1% of the PMTDI for Zn (Table 5) (R. Ellis, pers. comm.; EPA, 1985). Dietary intake from consumption of 90 g of muscle tissue would also provide approximately 10% of the required daily intake of Fe and 5% of the typical daily intake of Al, but negligible amounts of dietary Ag, Co, Cr, and Pb (Cox, 1995). The intestine, kidney, liver, and gonads of both wild and cultured carp on most occasions failed the ANZFA maximum concentration for zinc. However, since the Japanese do not routinely consume the viscera, this should not lead to deleterious effects to consumers of these fish.
5. Conclusion Lake Kasumigaura is considered to be a highly polluted water body, a view reinforced by the carcasses of large fish often observed on the shoreline. However, our results suggest that carp reared in Lake Kasumigaura are not heavily burdened with metals. In addition, despite their dietary differences, the wild and cultured fish were accumulating and distributing metals in much the same manner. Also, aquaculture practices are not leading to enhanced metal content in the fish. Metal concentrations were lowest in muscle, and did not exceed established quality standards for fish. The
differences in metal concentrations between cultivated and wild carp are negligible, and should pose no health problems for consumers of these fish.
Acknowledgments The authors thank Dr. A. Hamada, former Director of the Ibaraki Prefectural Freshwater Experimental Station, for assistance during the experimental field work, and Professor Humitake Seki of the Institute of Biological Sciences, University of Tsukuba, for his advice and suggestions throughout this research. The research was partly conducted while the authors G.A. and F.S. were guests of the National Institute for Environmental Studies, Tsukuba, Japan, funded by the Japanese Government JGRAFS and STA schemes. The research by E.T.S. and M.G.M.A. at Deakin University was supported in part by the U.S. Environmental Protection Agency’s Science to Achieve Results (STAR) program, and by the Electric Power Research Institute Contract No. EP-p4898/C2396.
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