Environ. Sci. Technol. 1982, 16, 536-539
Acknowledgments
We thank the Technical Operations Group, National Water Research Institute for aid in collecting the samples, M. Whittle of the Great Lakes Biolimnology Laboratory, GLBL, for the fish samples, and R. Kwiatkowski of the Water Quality Branch, K. Lum of the Environmental Contaminants Division, and H. Shear of GLBL for their cooperation in sample collection. We also thank R. Bourbonniere of the Aquatic Ecology Division for the Lake Huron sediment samples. Literature Cited Young, D. R.; Heesen, T. C.; Gossett, R. W. Water Chlorination: Environ. Impact Health Eff., Proc. Conf. 1979 1980,3,471. Fischer, A.; Slemrova, J. Vom Wasser 1978,51,33. Schwarzenbach, R. P.; Molnar-Kubica, E.; Giger, W.; Wakeham, S. G. Enuiron. Sci. Technol. 1979,13,1367. Niimi, A. J. Bull Environ. Contam. Toxicol. 1979,23,20. Jan, J.;Malnersic, S. Bull. Environ. Contam. Toxicol. 1980, 24,824. “Assessment of Testing Needs: Chlorinated Benzenes”; Report EPA-560/11-80-014, United States Environmental Protection Agency, 1980.
(7) “Water-Related Environmental Fate of 129 Priority Pollutants”; Report EPA-440/4-79-029b, United States Environmental Protection Agency, 1979. (8) Oliver, B. G.; Bothen, K. D. Anal. Chem. 1980,52,2066. (9) Oliver, B. G.; Bothen, K. D., J.Environ. Anal. Chem. 1982, in press. (10) Matter-Miiller, C.; Gujer, W.; Giger, W.; Stumm, W. B o g . Water Technol. 1980,12,299. (11) “Surface Water Data Ontario”; Inland Waters Directorate, Environment Canada, 1979. (12) Kemp, A. L. W.; Harper, N. S. J. Great Lakes Res. 1976, 2,324. (13) Kaiser, K. L. E. Environ. Sci. Technol. 1978,12,520. (14) “Hazardous Waste Disposal Sites in New York State”,New York State/Department of Environmental Conservation, 1980; Vol. 3. (15) “Inventory of Major Municipal and Industrial Point Source Dischargers in the Great Lakes Basin”; International Joint Commission, Great Lakes Water Quality Board, 1979. (16) Farmer, J. G. Can. J. Earth Sci. 1978,15,431. (17) “Kirk Othmer Encylopedia of Chemical Technology”;Wiley: New York, 1979; Vol. 5, p 797. (18) United States Tariff Commission, Washington, D.C. “Synthetic Organic Chemicals 1940-1972”. (19) Konemann, H.; Van Leeuwen, K. Chemosphere 1980,9,3. Received for review June 16,1981.Accepted December 3,1981.
NOTES Cleaning Procedures for Removal of External Deposits from Plant Samples Hlroshi Saiki
\
Graduate School of Environmental Sciences, The University of Tsukuba, Sakura-mura, Ibaraki 305, Japan
Osamu Maeda’ Institute of Biological Sciences, The university of Tsukuba, Sakura-mura, Ibaraki 305, Japan
Foliar samples of Japanese cedar (Cryptomeria japonica), Japanese white oak (Quercus myrsinaefolia),and Japanese red pine (Pinus densiflora) were washed with running tap water, a 2% detergent solution, or 0.2 M HC1. The concentrations of 16 elements were determined by inductively coupled argon plasma spectrometry to compare the effects of the three cleaning procedures on removal of external deposits. Among the three washing procedures, the most effective was the acid washing, which removed significant proportions of the total Al, Cr, Fe, Pb, Ti, and other elements from crude foliar samples without causing any measurable loss of K. It was ascertained by scanning electron microscopy that this treatment effectively removed particulate fallout on leaf surfaces without doing any visual damage to the plant material. Outdoor plant materials are subjected to surface contamination with atmospheric dust, soil, or many other environmental substances. So that the effect of airbone dust on vegetation can be investigated, levels of an element in a plant and its burdens will have to be evaluated separately. For this purpose a suitable cleaning procedure 536
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must be used to remove external deposits on plant material before analysis. Several kinds of procedures such as washing with tap water (1) or treatment with a dilute detergent solution (2)or dilute hydrochloric acid (3) have been employed for cleaning contaminated foliar samples. There is, however, conflicting evidence in the literature as to the effectiveness of such procedures ( 4 ) . For the study of outdoor samples, the cleaning procedure must be able to treat a large number of samples within a short time and must be effective for as many elements as possible. The aim of our work was to compare the effect of three brief cleaning procedures on the external deposits of foliar samples. We have analyzed washed and unwashed leaves of three evergreen trees for 16 elements including exo- and endogenous constituents. Visual observations were also made on the treated and untreated material. Sample Preparation and Cleaning. Needles of Japanese cedar (Cryptomeria japonica) fronting the electric railroad, leaves of roadside trees of Japanese white oak (Quercus myrsinaefolia), and 2-year old needles of Japanese red pine (Pinus densiflora) in a roadside stand were collected from the campus of the University of Tsukuba
0013-936X/82/0916-0536$01.25/0
0 1982 American Chemical Soclety
Table I. Mean Concentration (pg/g Dry Weight) of 16 Elements in Unwashed Foliar Material (15 Samples for Each Species)a cedar oak pine 798 (39) 735 (62) 269 (10) 32.3 (7.8) 5.24 (0.85) 10.6 (1.3) 25700 (2600) 3840 (170) 1430(30) 2.15 (0.25) cr 3.84 (0.21) 10.6 (1.1) 10.6 (3.8) 26.2 (3.7) cu 77.7 (4.9) Fe 787 (24) 832 (75) 553 (33) 1021 (40) K 4220 (70) 10800 (1000) 3280 (260) 1070 (40) Mg 1 6 3 0 ( 2 8 ) 552 (42) 535 (40) Mn 397 ( 9 ) 2.02 (0.18) b Mo 0.623 (0.127) 2.26 (0.39) b Ni 3.90 (0.53) 1270 (70) 860 (50) P 836(17) 8.84 (1.32) 7.40 (0.97) Pb 6.40 (0.91) Ti 15.7 (0.4) 62.0 (5.3) 25.1 (3.6) 4.98 (0.57) 2.25 (0.14) V 2.07 (0.48) 52.5 (6.7) 43.2 (4.9) Zn 23.2 (3.5) Numbers in parentheses represent standard deviations. Below detection limit.
A1 Ba Ca
and ita neighborhood in December 1981. Harvested material (500 g fresh weight) was well mixed and divided into four portions, one of which was left unwashed for control. The other three portions were washed respectively with a large volume of slowly running tap water, about 1L of a 2% domestic detergent (Cherrina, Kao Inc., mainly
containing LAS and sodium pyrophosphate) solution, or 0.2 M hydrochloric acid solution for 4 min and further washed in running tapwater for 1 min. Each washed portion was rinsed four times for 2 min periods in a large volume of distilled water. After such cleaning procedures, each of the four portions was oven-dried for 24 h at 105 OC, subdivided into 15 parts, and powdered by an electric coffee mill. Chemicals Analysis. A 500-mg sample of dried material was digested with acid mixture (20 mL of concentrated "OB, 1mL of 70% HClOJ. The digested material was adjusted to a standard volume of 20 mL with 4 M "OB and centrifuged at 2000 rpm for 5 min. The supernatant was analyzed for the concentration of 16 elements by inductively coupled argon plasma spectrometry by using Jarrel-Ash Plasma Atomcompo Model-975 under the following conditions: incident power, 1.1kW; reflected power, 5 W; argon flow rates (coolant gas) 17 L/min, (sample gas) 0.5 mL/min, (plasma gas) 1L/min; sample aspiration rate, 1.2 mL/min; viewing height, 12 mm above the coil. Analyzed elements and analytical wavelengths (nm) were A1 (309.2), Ba (455.4), Ca (317.91, Cr (267.7), Cu (324.7), Fe (259.9), K (766.5), Mg (279.5), Mn (256.7), Mo (202.2), Ni (231.6), P (314.9), Pb (220.3), Ti (334.9), V (292.4), and Zn (213.8). Elementary analysis was made for each of 15 powdered subsamples for each of the four treatments. Visual Observation. Washed and unwashed pine
Table 11. Relative Contenta of Elements in Materials Washed with Detergent (D), Hydrochloric Acid (H), or Water (W) pine cedar oak H D W A1 H D W D H W 70b 74b 77b33b 37b 40b 63b 67b 70b H D W H W D Ba H W D 7 5b 81 86 60b 7 56 71b 98 104 106 W H D H W D Ca D H W 100 101 104 57b 68b 81b 98 101 102 D H W D Cr H W H D W 87 92 76b 56b 74b 76b 2gb 47b 53b H D W W cu H D H W D 74b 106 26b 49b 51b 57b 56b . 68b 79 Fe H H W D D W W D H 59b 60b 62b 56b 59b 62b 30 32b 35 K D H W D W H D H W 96 103 105 98 99 104 100 104 105 H D W H W D H W D Mg 96 101 102 58b 75b 76b 98 102 103 Mn D W H H W H W D D 88b 91b 93b 70b 82b 101 102 87b 104 Mo H W D W D H 61b 396 79b 91 63b 7 5b Ni H W D W H D 52b 60b 6fjb 956 70 100 P W D H D H W W H D 99 99 100 85b gob 93b 103 105 109 Pb H W D H H D W D W 80 97 30b 36b 69 70 58 93 95 Ti H D W D H W D H W 60b 65b 65b 26b 27b 28b 54b 57b 62b V H W D H D W H W D 76b 48b 72b 24b 40b 44b 56b 59b 62b Zn H W D W H D H D W 67b 75b 86 5Bb 61b 62b 7 5b 81b 110 a Calculated for mean value and with the content in unwashed material control) taken as 100. Any two values underscored by the same line are not highly significantly different (P> 0.01). Highly significantly less than the control (P< 0.01). ~
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Flgurs 1. Surface views (X250 magnification) of washed and unwashed pine needles: U, unwashed: W. water washed: D. detergent washed; H. HCI washed. Scale bars at the left corner indicate 50 pm.
needles were cut into lengths of 1cm, were fixed by osmic tetroxide vapor for 24 h, and were dried in a desiccator for 24 h. Dried material was coated with gold and observed by a scanning electron microscope (Hitachi Model-430).
Results and Discussion Table I shows the mean and standard deviation of 15 measurements of 16 elementary concentrations in unwashed (control) samples of three evergreen foliar maseea. Analytical error due to background noise and optical interference (5) was estimated to be less than 5% for all elements except for Mo in pine needles and Ni in oak leaves. In washed samples, mean concentrations of most elements were lower than those in the control Crable IO. The analysis of variance was made on total 46 mxa (3materiala X 16 elements - 2 undetectable cases), each of which consisted of four treatments with 15 replications. Results indicated that there were highly significant differences among the four means of 35 cases except for the cases of 588 E m . Sci. Technd.. Vd. 16, No. 8, 1982
Ba, Ca, K, Mg, and P in cedar needles, K in oak leaves, and Ca, K, Mg, Mn, and P in pine needles. Then the Student-Neuman-Keul’s test (6)was employed on the 35 cases to judge the significance of differences among means. The test revealed that there were 34 cases in which the mean in HC1-washed samples was highly significantly less than the mean in the control (Table 11). The mean in detergent- and water-washed samples was highly significantly less than that in the control in 28 and 25 cases, respectively. The test also permitted decisions as to which differences among the means for treated samples were significant (Table 11). Judging from results, we could summarize the effects of three washing treatments on removal of elements from foliar masses as follows: HCI washing was the most effective in 15 cases; detergent washing was the most effective in 3 cases;there was no case in which water washing was the most effective; HC1 and detergent washings were similar and more effective than water washing in 4 cases; detergent and water washings were similar and more ef-
fective than HC1 washing in 1case; the three treatments were identically effective in 8 cases; none of the three was effective in 11 cases. From above findings, the treatment with hydrochloric acid was consideredto be most effective. The acid reduced the largest number of elements most strongly, and it was the only solution used in our experiments that could significantly reduce Pb in tested foliar masses. There was no element in our experiments that was unaffected by HC1 but was affected by any of others. The acid removed more than 50% of Cu, Mo, Pb, and V from crude cedar needles collected near the electric railroad. It also removed considerably larger proportions of the total Al, Cr, Fe, Pb, Ti, and V from crude oak leaves of roadside trees. The lower levels of these elements in treated samples was most likely to be due to removal of exogenous leaf burdens. No significant reduction of K occurred in any treated samples (Table 11). None of the Ca, Mg, and P in cedar and pine showed measurable change with washing treatments. Almost all of these unaffected elements were probably derived from plant tissues, and the leaching loss during the treatment was probably not serious. The low level of leaching in our experiments agrees with work on apple leaves (4), for which the leaching of K was minimal with 0.3 M HC1. Our findings are, however, contrary to heavy leaching of Ca and Si observed for cut rye grass rinsed in water for several minutes (7). As penetration of the washing reagent into inner tissues of plant material was supposed to influence the magnitude of leaching, the effect of HC1 on inner tissues was investigated by measuring the optical density at 665 nm of acetone extracts of plant material. When affected by HC1, chlorophylls in plant tissues will be changed into phaeophytins, thereby effecting a significant reduction of the optical density (8). During a 15-min treatment with 0.2 M HC1, no reduction of the optical density occurred for intact pine needles, whereas considerable reduction occurred in extracts of chopped pine needles and young leaves of white clover. Thus inner tissues of intact pine needles were not likely to be affected by the brief treatment with acid solution. Pine needles are coated with waxy cuticular layers, which may protect the needles from the acid solution. However, longer treatments with strong acid should be avoided as we observed a partial effect on
intact pine needles treated with 1 M HC1 for 6 h. The effect of washing was also ascertained by visual observations with a scanning electron microscope (Figure 1). It was observed that considerable amounts of particulate fallout on leaf surfaces were removed by all of the washing treatments. The HCl washing was the most effective of the three. Its effect reached into the stomatal cavity without causing any visual damage on the surface of the needle. From the above results, we concluded that the most effective procedure for removal of leaf burdens from crude evergreen leaves is washing with 0.2 M HC1 for a few minutes. This procedure may be applicable to other plant materials covered with developed cuticular layers. Care must, however, be taken when the washing treatment is applied to herbaceous or abraded materials, which are subjected to serious leaching. Acknowledgments
We express our thanks to Nobuhiro Shimojo for his helpful suggestions about digestion of plant material. Thanks are also due to the Chemical Analysis Center of the University of Tsukuba for permitting the use of inductively coupled plasma spectrometric facilities. Literature Cited (1) Ward, N. I.; Brooks, R. R.; Roberts, E. Environ. Sci. Technol. 1977, 11, 917. (2) Iserman, K. Enuiron. Pollut. 1977, 12, 199. (3) Impes, R.; MVUZU, Z.; Nangniot, P. Anal. Lett. 1973,6,253. (4) Mitchell, R. L. J . Sci. Food Agric. 1960, 11, 553. (5) Fassel, V. A.; Kazenberger, J. M.; Winge, R. K. Appl. Spectrosc. 1979, 33, 1. (6) Steel, R. G. D.; Torris, J. H. “Principles and Procedures of Statistics”; McGrow-Hill: New York, 1960; Chapter 7, p 110. (7) Arora, D. K.; Upadhyay, R. K. Plant Soil 1978,49, 691. (8) Lorenzen, C. J. Limnol. Oceanogr. 1967,12, 343.
Received for review July 13,1981. Revised manuscript received February 18,1982. Accepted March 30,1982. This work was partly supported by a grant from the Ministry of Education of Japan for Special Research Project on Environmental Science R 12-6.
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