Environ. Sci. Technol. 1985, 19, 828-831
Morris, E. D.; Niki, H. J. Phys. Chem. 1974,78,1337-1338. Atkinson, R.; Carter, W. P. L.; Darnall, K. R.; Winer, A. M.; Pith, J. N., Jr. Int. J . Chem. Kinet. 1980,12,179-837. Killus, J. P.; Whitten, G. 2.Atmos. Environ. 1982, 16, 1973-1988.
(23) Leone, J. A.; Seinfeld, J. H. Int. J. Chem. Kinet. 1984,16, 159-193.
Received for review August 10,1984. Revised manuscript received January 4, 1985. Accepted March 19, 1985.
Variability of Aluminum Concentrations in Organs and Whole Bodies of Smallmouth Bass (Mieropferus do/omleui) William G. Brumbaugh“ and Donald A. Kane Columbia National Fisheries Research Laboratory, U.S. Fish and Wildlife Service, Columbia, Missouri 65201
w Variability of aluminum concentrations in smallmouth bass (Micropterus dolomieui) was evaluated by analysis with graphite furnace atomic absorption spectrophotometry. Fish analyzed as whole bodies were compared to fish which had selected organs analyzed individually and separately from the carcass. Gastrointestinal tract contents contained highly variable amounts of aluminum and caused bias and increased variability when included in whole-body samples. Since aluminum concentrations in tissues of stomach and intestine were similar to those in the whole body (less gastrointestinal tract contents), the entire gastrointestinal tract and contents could be removed to reduce bias and variability without measurably altering the “true” whole-body aluminum concentrations. Of the organs analyzed, gill filaments had the highest and most variable aluminum concentrations and may have contributed to within-fish whole-body variability because of incomplete homogenization.
Introduction High soluble concentrations of metals in lakes and streams are often associated with low pH levels as a result of the mobilization or dissolution from sediments or terrestrial soils. As for most metals, solubility of aluminum exhibits a strong inverse relation to pH (1,2). The presence of elevated soluble aluminum concentrations at low pH can adversely affect aquatic organisms. The most thoroughly investigated biological consequence of elevated aluminum concentrations has been the toxicity to fish and sublethal effects on fish (3, 4). Exposure to aluminum and low pH may result in fish mortality because of respiratory stress caused by gill damage and mucous clogging of the gill membrane and to the loss of sodium and chloride ions (5, 6). Sublethal concentrations of aluminum have been shown to cause histopathologicalchanges in the liver, kidney, skin, muscle, and gills and interfere with reproductive physiology in trout (6, 7). These sublethal effects and the influence of aluminum bioaccumulation are gaining interest among researchers. Bioaccumulation of contaminants is often used as an indicator of the ”health” of fish populations (8) and is usually measured in whole-body fish. Accurate assessment of a particular contaminant may require special considerations and can be affected by collection methods, sample preparation procedures, and analysis technique. Analysis of whole fish for aluminum presents many problems. A representative, uncontaminated sample is difficult to obtain, and measurement must be made in a relatively nonuniform and complex matrix. Results from a round robin survey for metals in water samples showed aluminum measurements to be in greatest error (9),which exemplifies the difficulty of measuring low levels of this 828
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element. Since aluminum is the third most abundant element in the earth’s crust, caution must be exercised to avoid dust contamination and excessive sample handling. Aluminum also is a major component of most laboratory glassware and can be leached out by certain reagents (10). Fusion of aluminum from glassware during dry ashing of samples can cause positive errors for which blanks may not give proper correction (11). Aluminum may exhibit anomalous behavior when determined by graphite furnace atomic absorption, although the use of improved graphite materials has greatly reduced this problem (12). The primary objective in the present study was to ascertain potential field and analytical sources of variability and error when whole-body fish samples are analyzed for aluminum.
Experimental Section Sample Collection. Smallmouth bass (Micropterus dolomieui) were collected from Chatuge Reservoir, which is located on the border of Georgia and North Carolina. The reservoir receives runoff from forested watersheds that are poorly buffered (13),and there are no known sources of significant contamination from agriculture within the watershed. Mean alkalinity in the reservoir is about 140 pequiv/L, and average pH is 6.3 for 52 measurements taken over a 6-year period. Aluminum concentrations are widely variable, ranging from 0.06 to 0.94 mg/L (14). After collection by gill netting, electrofishing, and rotenone application, fish were placed in large plastic bags, frozen, and shipped to the Columbia National Fisheries Research Laboratory. Sample Preparation. All dissection tools and homogenization equipment were rinsed with dilute HC1 and then ultrapure water (15-18 Ma-cm specific resistivity) before each sample was processed. Frozen fish used for wholebody analysis were sectioned with a band saw and passed twice through a small meat grinder. Fish which were dissected before analysis were placed on a clean polyethylene sheet, and the liver, kidney, gill filaments, and gastrointestinal tract were removed with stainless steel scalpels and surgical scissors. The gastrointestinal tract was sliced open, and the contents were flushed into preleached beakers with ultrapure water. Large, intact food items were removed and not included in the analysis of the “gut” contents. Analysis of gut tissue included the stomach, intestine, and pyloric caeca. The remaining “carcass”was homogenized by chopping it into sections on a Teflon cutting board and then passing it 3 times through a small meat grinder. Sample Digestion. Digestion glassware was washed sequentially with 16 M HNO,, 12 M HC1, and ultrapure H20. Polyethylene sample bottles were leached for 48 h with a 6 M HN03-2 M HC1 solution, rinsed, and filled with US. Copyright. Published 1985 by the American Chemical Society
Table I. Comparison of Digestion Methods for the Determination of Aluminum in Tissue (Mean Aluminum Concentrations f SD; n = 5) sample type digestion blank
NBS bovine liver CNFRL" striped bass a
HN03/pressure
digestion method HN03/HC104/HzS04
dry ash
8.1 f 3.6 pg/L 0.81 f 0.36 pg/gb 2.2 f 0.7 pglg 18.8 f 3.3 pg/g
41.0 f 14 pg/L 4.1 f 1.4 pg/gb 1.8 f 1.5 pg/g 31.3 f 1.6 pg/g
7.9 f 3.1 pg/L 0.79 0.31 pg/gb 2.2 f 0.6 pg/g 21.7 f 2.3 pg/g
Columbia National Fisheries Research Laboratory.
Converted to tissue equivalent concentration.
ultrapure water until needed. An HN03 pressure digestion technique (8) routinely used at our laboratory for determining metals was evaluated for aluminum. Since no standard biological reference materials are certified for aluminum, results were evaluated by comparison with two more rigorous digestion methods: wet ashing with the tertiary mixture "OB, HC104, and H2S04(15)and dry ashing (21) in quartz beakers. The digestion evaluation was done with two powdered tissue samples: National Bureau of Standards bovine liver and a lyophilized, cryogenically ground whole fish (striped bass, Morone soxatillis) prepared at our laboratory. These two materials are fairly homogeneous and contain relatively low and relatively high concentrations of aluminum. A four-point method of standard additions was used for all determinations in the digestion comparison. Results were in good agreement for the bovine liver, which is a very uniform tissue, but were less satisfactory for the wholebody striped bass (Table I). It is likely that the tertiary acid mixture was more effective in attacking acid-insoluble residue which was observed in the striped bass digestates (HC104 is commonly used as an aid in solubilizing acidresistant silicates). However, since the blanks were unacceptably high for the tertiary mixture and the dry ash procedure required more operator attention, the "OB/ pressure digestion was chosen for the analysis. For smallmouth bass samples, 2.0-2.5-g wet weight aliquants were digested except for kidneys which were limited to 0.4-0.8 g. Gut contents were not weighed; only the total mass of acid-extractable aluminum contained within each gut was determined. Whole-body and carcass samples were digested in duplicate, but single aliquants were digested for each of the other tissue types. Since gut content samples could not be conveniently transferred to digestion tubes, they were digested on a hot plate by refluxing with 16 M HN03 in beakers covered with watch glasses. Samples were first heated at 90 "C to evaporate rinse water, 5 mL of HN03was then added, and the samples were covered and left at 100 "C overnight. The temperature was then increased to 150 "C, and samples were taken to dryness. After 3 mL of HN03 was added, samples were heated for 1 h at 150 "C, quantitatively transferred to polyethylene bottles, and diluted to 50 mL with 1% HC1 (v/v). Sample Analysis. Aluminum was determined with a Perkin-Elmer Model 5000 atomic absorption spectrophotometer equipped with an HGA-500 graphite furnace and an AS-1 autosampler. Peak areas were measured at 396.2 nm with continuum source background correction. A L'vov platform was used to improve precision and reduce matrix effects (16). Samples of gill, liver, kidney, gut, and gut content were analyzed by calibration using acid-matched standards. Single spike additions were performed on two samples of each tissue type to check for possible matrix effects (Table 11). Due to a matrix enhancement of aluminum signal (20-40%), whole-body and carcass samples were analyzed
Table 11. Spike Addition Recoveries for Tissues Analyzed by Direct Calibrationarb tissue type liver liver kidney kidney gill gill gut gut gut content gut content
aluminum concentration, pg/L sample sample + spike % recovery 39 30 0 33 4Ogc 301 133 119 173 37
247 227 183 244 576 516 338 307 379 257
104 99 94 105 85 107 102 94 103 110
'All digestates spiked a t an effective A1 concentration of 200 pg/L. bMean recovery fSD = 100.3 f 7.5%. CConcentrationafter additional 10-fold dilution.
by standard additions. High levels of calcium present from bone and scales of these samples may have been reponsible for this enhancement (12). As a standardization check, Environmental Protection Agency reference water 5 ("true" = 450 pg/L) was analyzed periodically and found to contain 457 pg/L aluminum (SD = 21 pg/L). Although the instrumental detection limit for aluminum was 2 pg/L, variability in the analytical blanks was the limiting factor in the actual limit of detection for the method (17). The standard deviation of aluminum concentrations of four analytical blanks (a = 6.2 pg/L, SD = 6.0) was multipled by 3 to calculate a limit of detection of 18 pg/L (0.5 pg/g wet weight tissue concentration). Limit of detection for kidney samples (1.0 pg/g wet weight) was higher due to the smaller sample mass available. Digestion blanks for the HN03reflux method used for gut content samples were higher (17 f 3.0 pg/L) but were low compared with the levels of aluminum found in these samples.
Results and Discussion Whole-Body Concentrations. Precision of duplicate aluminum determinations on fish analyzed as whole bodies was poor, and mean concentrations of aluminum varied almost 100-fold (Table 111). Differential accumulation of aluminum in selected organs and a lack of a completely homogeneous sample were suspected to be responsible for variability within samples. Evidence of acid-insoluble residue in some sample digestates also suggested the possibility of bias due to ingested sediments (18) which often contain inherently high concentrations of aluminum. Although this sample dissolution method does not solubilize residual forms of sediment, significant acid leaching of aluminum can be expected. Subsequently, a second group of fish ( n = 10) was prepared by removing selected organs and gut contents and homogenizing the rest of the carcass. In addition to evaluating bias due to gut contents, selected tissues were analyzed to estimate variability due to lack of sample homogeneity. Environ. Scl. Technol., Vol. 19, No. 9, 1985
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analyzed with gut contents intact. Similar results were obtained whether estimated whole-body values for dissected fish without gut contents (3.9 f 3.6; Table V) were compared to undissected fish (10.5 f 19.4; Table 111) or to dissected fish with gut contents included (13.8 f 29.5; Table V). Estimates of whole-body aluminum concentrations for dissected fish were measureably higher for 6 of 10 samples if gut contents were included (Table V). Of the organs analyzed, gill filaments had the highest and most variable aluminum concentrations and may have contributed to within-fish variability in whole-body samples (Table V). A similar trend for gill tissue has been reported (19)for both field-sampled and hatchery-raised rainbow trout (Salmo gairdneri). Aluminum concentrations in liver, kidney, and gut tissues from our fish were relatively low and consistent. Although the aluminum concentration for gut tissue from sample 0876 was high, it was probably not completely purged of ingested sediment. For aluminum concentrations between fish for any two tissue types, only the gill filaments and carcasses were significantly rank correlated (p < 0.05, Spearman r = 0.67). Precision of duplicate determinations, which reflect method variance, was considerably better for carcasses (mean percent difference = 18%;Table VI) than for whole bodies (mean percent difference = 46%; Table 111). This suggests that whole-body samples were relatively inhomogeneous, probably due to uneven distribution of gut contents or gill filaments. Low-level random contamination was likely a minor factor in method variability and probably could have been reduced if samples had been prepared in a clean-air laboratory (20). Although the results of this study probably depend upon variables such as watershed soil type, geographic location, fish species, and method and time of collection,researchers should remain aware of potential problems in measuring aluminum concentrations in aquatic organisms and that concentrations may be highly variable among individuals.
Table 111. Aluminum Concentrations and Precision of Duplicate Determinations for Whole-Body Smallmouth Bass sample no. 0447 0450 0451 0452 0454 0455 0460 0461 0464 0465 0466 0468
aluminum concn, pg/g wet weight run 2 meann run 1 4.0 1.6 2.6 3.1 2.4 2.5 59.8 0.6 8.5 11.8 11.5 0.6
3.1 1.8 4.0 1.9 4.4 2.2 81.1 2.4 20.6 11.6 9.0 1.1
% differenceb (A/Z X 100)
3.6 1.7 3.3 2.5 3.4 2.4 70.4 1.5 14.6 11.7 10.2 0.8
25 12 42 48 59 13 30 161 83 2 24 59
Mean concentration f SD = 10.5 f 19.4 pg/g. ference f SD = 46 f 43 % .
Mean % dif-
Table IV. Quantity of Acid-Extractable Aluminum Measured in Gut Contents of Smallmouth Bass sample no. 0876 0877 0878 0879 0880 0881 0882 0883 0884 0885
AI,
contribution to whole-body concn, pg/g“
17800 305 390 948 29 4 8 28 196 197
89.2 1.5 1.9 4.7 0.1
