Environ. Sci. Technol. 1992, 26, 609-613
Uptake and Translocation of Selected Organic Pesticides by the Rooted Aquatic Plant HydMa vertici//ata Royle Mark L. Hlnmant and S. J. Klaine*vf
Department of Biology, Memphis State University, Memphis, Tennessee 38 152 Rooted aquatic vascular plants are exposed to both overlying water and sediment and are able to absorb nutrients and chemicals from both of these environments. Translocation of sediment-retained contaminants by plants presents the possibility of contaminant redistribution into the water column or food chain. For [14C]atrazineexposure in solution, uptake and release approached equilibrium within 1 and 2 h for shoot and root tissue, respectively. [14C]Lindaneand [14C]chlordaneconcentrationsin the root and shoot reached equilibrium within 24 and 144 h, respectively. Bioconcentration factors (BCF) for hydrilla were 9.62, 38.15, and 1060.95 for atrazine, lindane, and chlordane, respectively. BCF estimation methods using log octanol-water partition coefficient and log water solubility based on data from fish or duckweeds were not predictive of values for hydrilla. All three chemicals were translocated acropetally from the roots in a spiked sediment. When the 2-3-cm segment of shoot beyond the roots was considered, atrazine was in equilibrium after 6 h of root exposure. All shoot segments had similar concentrations of atrazine within 48 h after root exposure, although this concentration was slightly less than for the whole-plant exposure to the chemical in solution. Lindane concentration was in equilibrium after 192 h of root exposure at approximately 20X less than the shoot concentration for the whole-plant exposure. Chlordane concentration did not change significantly after 96 h of root exposure and was 780X less than the shoot concentration for the whole-plant exposure.
Introduction The uptake of contaminants by aquatic biota has long been of concern. Contaminants released into the aquatic environment will establish an equilibrium between the various compartments in the system. Generally, an inverse relationship exists between the water solubility of the organic compounds and the partitioning into the sediment and biota. In aquatic systems that have extensive growths of submersed vegetation, there is a possibility of these plants being a sink for organic contaminants. Most bioconcentration studies utilize fish or invertebrates; thus few results for aquatic plants have been reported. Since fauna have much greater lipid concentrations than plants, they may not serve as good models for plant uptake. A reduced vascular system and lack of a transpirational stream in submersed aquatic angiosperms have prompted controversy as to whether acropetal translocation of solutes occurs. The ability of these plants to translocate various nutrients acropetally has been established (1-5). If rooted submersed species can mobilize sediment-retained organic compounds and translocate them into the shoot of the plant, the hazard assessment of contaminated sediments may need to include this redistribution pathway. Present address: Exxon Biomedical Sciences, Inc., Mettlers Road, CN 2350, East Millstone, N J 08875-2350. t Present address: Department of Environmental Toxicology, Clemson University, One TIWET Dr., P.O. Box 709, Pendleton, SC 29670. 0013-936X/92/0926-0609$03.00/0
The objective of this study was to examine the role of submersed aquatic angiosperms in bioconcentrating and translocating chemicals between environmental compartments. Specifically, the bioconcentration of selected dissolved organic pollutants by the submersed vascular plant Hydrilla verticillata Royle was determined. Further, the acropetal movement of these compounds during root exposure was evaluated.
Materials and Methods Experimental Approach and Design. Plant uptake of the test chemicals was evaluated by timed exposure of the plant to radiolabeled chemical dissolved in sterile nutrient medium. After exposure, the plants were divided into shoots and roots. The tissue was then combusted at 900 "C, and the 14C02was trapped and quantified. To evaluate the acropetal movement of the chemicals, the exposure chamber was divided into separate compartments of sediment and overlying water. The radiolabeled chemical was retained in the sediment compartment by an agar-Teflon diffusion barrier. Thus, presence of the labeled compound in the shoot above the barrier after root exposure indicated movement of the chemical through the plant. Plant Culture and Maintenance. Monoecious Hydrilla uerticillata Royle tubers had the outer tissue layers removed and were soaked in 1% NaClO for 10 min to produce algal- and fungal-free plant cultures. The plants were grown in autoclaved 10% Hoagland's nutrient medium augmented with 200 mg of NaHC03/L (10% Hoagland's medium) (6). The plants were propagated vegetatively under aseptic conditions and maintained under constant fluorescent illumination (40-50 beinsteins/ m2.s) at 25 f 1 "C. Rooted apical shoots of 8-14 nodes were used in all experiments. Stock colonies were transferred to new medium at 3-week intervals. Chemical Exposure to Plants. To evaluate uptake, plants were exposed to the test chemicals in 1-qt widemouth Mason jars with inverted 100 X 15 mm Petri plate bottoms as lids. The chambers were acid-washed with Nochromix and autoclaved prior to use. Whole plants were exposed to individual test chemicals in 10% Hoagland's medium with no sediment present. The chemical concentration of the solution was monitored, and the plants were transferred to new solution if the concentration decreased by 10% of the original value. Three exposure chambers were set up for each treatment, and each served as a single replicate for statistical purposes. To evaluate translocation from root exposure to a chemical in sediment, a divided exposure chamber was used (Figure 1). The model sediment consisted of autoclaved, nitric acid washed, fine sand (0.7-1.0 mm) in sterile glass culture tubes (100 X 25 mm) with push-on caps (Bellco Co.). The test chemical was dissolved in 50% Hoagland's medium (without Fe-EDTA) and added to 10 g (dry weight) of sand at a ratio of 3.8:l (w/v; sand-medium). A 0.3-cm-thick layer of 1.5% agar was placed on the sediment and covered with a precut, autoclaved Teflon barrier (0.5-mm thickness) and two more layers of 1.5%
0 1992 American Chemical Society
Environ. Sci. Technol., Vol. 26, No. 3, 1992 609
10 1
1
Push-on cap
Glass culture
0.75 c m p l a n t i n g hole
tube (100 x 25 rnm)
0.3 cm t h i c k 1.5% a g a r
.01
0.5 m m T e f l o n b a r r i e r
--s--
shoot
.I
T
1
10
100
Time (hours)
Figure 2. Mean f SD of ['4C]atrazine concentration in hydrilla shoot and root tissue after timed whole-plant exposure to the chemical in solution.
0.3 c m t h t c k 1.5X a g a r
1 0 g acid-washed f i n e sand w i t h 50% Hoaoland's Medium
Figure 1. Test-tube exposure chamber for measuring root uptake and translocation of sediment-incorporated chemicals.
Table I. Mean Chemical Concentration (fSD) at Equilibrium for Plant Tissues Exposed to the Chemical in Solution
chemical
solution concn, mg/L
concn, p g / g of tissue dry wt root shoot
agar to give a total diffusion barrier thickness of approx7.56 f 1.07 atrazine 0.057 3.25 i 0.65 imately 1 cm. Approximately 25 mL of 10% Hoagland's 0.12 25.54 f 5.76 lindane 74.80 f 7.15 medium was used in each chamber. One rooted hydrilla chlordane 0.005 56.44 i 12.56 143.58 f 25.32 plant was inserted through a 0.75-cm-diameter hole in the Teflon in each culture tube. Each chamber was considered used to evaluate burn and trapping efficiency (94.5%). a single replicate for statistical purposes. Data Management. Data summary and calculations Each plant was exposed to the spiked sediment for a were performed by microcomputer spreadsheet programdefined period of time. The plant was then rinsed in 10% ming. Bioconcentration factors (BCF) were calculated by Hoagland's medium to remove any chemical or sand on dividing the mean ( n = 6) equilibrium concentration of the surface of the plant and divided into root and 1-cm chemical in the whole plant on a wet weight basis by the shoot segments. Each unit of plant tissue was weighed to aqueous chemical concentration used in the whole-plantthe nearest 0.001 g and stored in aluminum foil packages exposure experiments. Descriptive and comparative stauntil further analysis. Tissue wet weights were converted tistics were performed by Statview 512 ( a = 0.05). Difto dry weights with the following equations ( n = 25): root ferences in the chemical concentration between sampling dry weight = root wet weight X 0.0521 - 0.0002, r2 = 0.95; times and the establishment of equilibrium were detershoot dry weight = shoot wet weight X 0.085 - 0.0001, r2 mined by Fisher's protected least significant difference. = 0.85; apical tip dry weight = apical tip wet weight X 0.08 - 0.000089, r2 = 0.86. Results and Discussion Quantitative Analysis of Chemicals. Chemicals used Uptake. The uptake of atrazine from solution by hywere uniformly ring labeled [l4C]atrazine [2-chloro-4drilla was rapid. The shoot and root tissue reached (ethylamino)-6-(isopropylamino)-s-triazine] (specific acequilibrium with the external medium within 1 h (Figure tivity (sa), 22.0 pCi/mg; 99.0% pure; courtesy of Ciba2). After equilibrium was reached, there was no statisGeigy Corp.), uniformly ring labeled [ 14C]lindane [ytically significant change in atrazine concentrations in the hexachlorocyclohexane] (sa, 31.6 pCi/mg; 99.9% pure; tissues through the duration of the experiment (24 h). The Sigma Chemical Co.), and uniformly ring labeled [I4C]chlordane [ 1,2,4,5,6,7,8,8-octachloro-3a,4,7,7a-tetrahydro- atrazine concentration at equilibrium for hydrilla roots was lower than for shoots (Table I). The roots of aquatic 4,7-methanoindane] (sa, 33.4 pCi/mg; 99.9% pure; courtesy so plants constitute 1-3% of the total plant biomass (3, of Velsicol Chemical Co.). Atrazine at 0.057 and 0.050 root uptake of the compound would only constitute a small mg/L, lindane at 0.12 and 0.12 mg/L, and chlordane at fraction of the total uptake when the entire plant is ex5.5 and 5.5 pg/L were used for uptake and translocation posed to the same concentration of chemical in solution. studies, respectively. Aqueous and solvent solutions were In order to compare with the literature, BCF values were analyzed by adding 1mL of solution to 10 mL of Ecoscint computed using the equilibrium concentrations of chemical A scintillation cocktail (National Diagnostics) and counting per wet weight of plant. The BCF for atrazine with hyon a Beckman Model LS-7000 scintillation counter. drilla was 9.62 for this study. BCF estimation equations Quench was corrected using a quenched 14Cstandard set which use the octanol-water partition coefficient (KO,) and (Nuclear-Chicago). Plant tissue was combusted to 14C02 water solubility (S),and are based on data from fish (81, with an R. J. Harvey Model OX-400 biological oxidizer at are as follows: 900 "C in a stream of oxygen. The I4CO2was trapped in 10 mL of Carbon 14 Cocktail (R. J. Harvey Instrument log BCF = 0.76 log KO, - 0.23 Corp.). The bubbling chamber was rinsed with 3 mL of methanol (HPLC grade), which was added to the scintillog BCF = 2.791 - 0.564 log S lation cocktail before counting. All values for plant tissue BCF values of 64.09 and 86.01 were calculated from the were reported as micrograms of chemical per gram tissue log KO, (2.68) and S (33.0 mg/L) (91, respectively. The dry weight. External standards of [14C]methyl methlipid content of submersed plants is much less than fish; acrylate (sa, 366 dpm/mg; New England Nuclear) were 610
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150
5
125
F
f
11
I shoot
5 e
ma
D
l5
a
F
m .
3 u)
75
.
i= m
Y
11
10
a
._ N
m
3 .01
.1
1
10
100
1000
v.
01 I
lime (hours)
5
5:
[“C]liine concentration in hydrilla shoot and root tissue after timed whole-plant exposure io the chemical in solution. F~~UIO 3. Mean f SD Of
0 1
4
6
12
24
48
72
96
Time (hours) Flgure 5. Mean f SD of [“C]airaAne concentration in hydrilla root and I c m shoot segments after timed exposure of roots to the
chemical in sediment.
.
100
Table 11. Mean Chemical Concentration ( S D ) at Equilibrium far Plant Parts after Root Exposure to the
1
Chemical
chemical
2
.a1
m
I
4. Mean
.I
1
10
Time (hours)
100
1000
* SD of [”C]chbr~!aneamcentnth in hydriib shoot
and root tissue after timed whole-plant exposure to the chemical in solution.
thus a smaller value would be expected. A BCF estimation equation based on the floating Lemnaceae (duckweeds) (10)is as follows: log BCF = 0.491 log K , + 0.0562
This equation produced a BCF of 23.82. Floating-leaved and emergent species have a greater percentage of ether extractable (lipid) component than the submersed species. Presumably, this is due to the waxes on the surface of the leaves exposed to the air (5). The shoot and root concentrations of both lindane and chlordane were in equilibrium with the environment within 24 and 144 h, respectively (Figures 3 and 4). After equilibrium was reached, there was no significant change in chemical concentrations in the tissues through the duration of the experiment (192h). The equilibrium concentration for hydrilla roots was less than for shoots (Table I). The BCF for lindane with hydrilla was 38.15. This was much smaller than the values (BCF = 325-470) reported for fish (8)or calculated from regression equations based on fish data (log K , = 5.2,BCF = 5272;S = 7.0 mg/L, BCF = 1802)(9). The estimation based on duckwed data (BCF = 407) still overestimates the value seen in this study. The BCF for chlordane in this study was 1061. As with lindane, this value was much smaller than reported by Bysshe (8) for fish (BCF = 11 400-37800). Fish-based estimations using log K , (5.58)and S (0.06mg/L) (9)were 10252 and 3141,respectively. The duckweed-based estimation was 625,which more closely approximates the results of this study. On the basis of the discussion above, BCF generated from duckweed data should overestimate BCF for submersed plants. This is not apparent for chlordane. The equation generated hy Lockhart et el. (10)to estimate the
concentration, p g / g of tissue dry w t shwt rwt &l em 1-2 em 2-3 em
atrazine 2.68 2.73 lindane chlordane 0.86
* 0.59 2.33 * 0.22 &
6.96 2.44 1.M)
* 3.86 * 1.55
6.93 6.44 0.67 0.29
* 3.55
* 3.36 * 0.23
6.52 f 4.93 3.96 2.87 0.18 0.08
* *
BCF of materials with duckweed is based on a linear regression analysis of 10 materials of varying log K, (0.296-6.57). It is likely that the resulting equation is less accurate at the extremes of the range of K , values. Since the log Km for chlordane is greater than 5.5,the BCF may not he as accurately predicted as for those chemicals with log K , values between 2 and 4. Translocation. Acropetal translocation of atrazine from the roots occurred (Figure 5). The roots reached equilibrium with the sediment atrazine concentration within 6 h, with no statistically significant change in concentration for the duration of the study (96 h) (Table 11). The atrazine concentrations in the 1-cm shoot segment above the root were highly variable and no statistical difference was present. The chemical concentrations in the 1-2 and 2-3-cm segments were statistically different from the 1-h concentration within 24 h, but were consistent after 48 h, for the duration of the study. Atrazine is translocated in the apoplastic (nonliving) component of the plant structure (17). Neumann et al. (72) suggested that compounds with pK, values less than 7 are able to accumulate in the alkaline medium of the phloem sap and are thus phloem-mobile. In contrast, basic compounds, such as atrazine, are concentrated in the apoplast. Triazines vary in mobility within submersed aquatic plants. In Heteranthera dubia, ametryn [Z-ethylamino4-(isopropylamii0)-6-(methylthio)-~-triazine] and prometryn ~2,4-his~isopropylamino~-6-(methylthio)-s-triazine] move only basipetally (toward the root). In contrast, simazine [2-chloro-4,6-bis(ethylamino)-s-triazine] is translocated both acropetally and basipetally. But in Potamgeton crispus, simazine is translocated only hasipetauy
(17).
Translocation suppression may be an active process on the part of the plant. Sutton and Singham (13)found that Myriophyllurn aquaticurn did not translocate 2,4-D [(2,4-dichlorophenoxy)aceticacid] acropetally from the Environ. Sci. Technoi.. Voi. 26.
No. 3, 1992 611
Table 111. Hypothetical Chemical Mass in Plant Tissue at Different Maximal Biomass Densities for a 23.7-ha Lake
chemical
porewater concn, mg/L
atrazine lindane chlordane
0.05 0.12 0.0055
chemical mass for plant biomasap g 64 g o f 89Ogof pdw/m2 pdw/ma 74.00 43.56 2.32
1376.12 834.40 127.40
'pdw, plant dry weight.
1
24
48
96
144
192
696
Tims(hours)
6. Mean f SD of ["C]liMane concentration in hydrilla root and l-cm shoot segments after timed exposure of roots to ihe chemical in sedimnt.
*re
-
2.0
?
0.5
I I 1 1
24
48
96 144 T i m (hours)
192
696
npur 7. Mean f SD of ["Clchbrdane concentrarmn h hy&ii!a root and l-cm shoot segments after timed exposure of mot8 to Ihe
chemical In sediment.
roots. When the plant was treated with the metabolic inhibitor dinitrophenol, translocation did occur. Acropetal translocation of lindane from hydrilla roots occurred, but was not extensive for the duration of this study (Table II, Figure 6). Lindane was quickly taken up by the roots and then the root concentration of chemical steadily decreased. Concentrations in the roots were not statistically different after 24 h and to the end of the study at 696 h. Lindane concentrations in the 0-1-em shoot segment were statistically different within 96 h. The greatest concentration occurred a t 96 h, with a steady decrease thereafter. Lindane concentration in the 1-2-cm segments was statistically different from the 1 h value within 24 h and then remained similar from 24 to 696 h. The lindane concentrations in the 2-3-cm segments were different within 192 h and consistent thereafter. Chlordane was translocated acropetally from the roots, but not extensively (Figure 7). Instead, it waa steadily bioconcentrated by the roots (Table 11). The chemical concentrations were statistidy different within 48 hand consistent after 192 h. The chlordane concentrations a t 696 h averaged 0.86 pg/g of plant dry weight (pdw), which is significantly less than the equilibrium uptake value of 56.44%/g of pdw for exposure to the chemical in solution. The chlordane in the (tl-cm segment increased throughout the study, with a statistical difference within 96 h The concentrations were similar after 96 h and for the remainder of the study. The chemical concentrations in the 1-2-cm segments were significantly different within 192 h, when a maximal concentration occurred. The 2-3-cm segments had chlordane concentrations that were statis612
Emhon. Sci. Techmi.. Vol. 26, No. 3, 1992
t i d y different within 96 h. The concentrations were similar for the remainder of the study. Chemical Redistribution within Aquatic Systems. This study has shown the potential for chemical redistribution in aquatic ewystems by rooted aquatic vascular plants. Fate and transport studies of chemicals from terrestrial systems frequently consider assimilation and transport by plants. This is not the case for studies of aquatic systems. Although bioconcentration into a biotic component may be determined, plant-mediated translocation of chemicals from the sediment into the water column is not considered. Is the quantity of chemical redistributed by these plants significant in the hazard assessment process? Consider a hypothetical situation in which two lakes of 23.7 ha are infested with hydriia at different biomass densities at the seasonal maximum, 64 and 890 g of pdw/m2 (14). h u m e (simplistically) that the concentration of chemical in the plant tissue is approximately that of the equilibrium concentration in the 2-3-cm segment of the shoots (Table 11) in this study. As shown in Table III, the hypothetical redistribution concentrations can be significant. Hazard assessment for a water body may require consideration of this pathway, depending on the designated use of the system. The primary concern of this repositioning of chemicals withiin the ecosystem is the potential for movement of the chemical into uncontaminated compartments. Fragmentation and senescence allow the plant biomass to move laterally within aquatic systems. Fragmentation is the primary form of reproduction and range expansion for many submersed plants (4). Phytophagous fauna can introduce hydrophobic compounds into the food chain. Grass carp (Ctempharyngodon idello Val.) are commonly used as a biological control in aquatic weed management programs. The adult fish can consume between 0.8 and 2 times their weight in hydrilla daily, with a 20-9470 utilization of the plant biomass (15,16).The plant biomass that is not assimilated is then available for detritivors. Conclusions Bioconcentration of the chemicals examined was inv e d y related to the water solubity of the chemical, while the translocation was directly related to the water solubility. Atrazine concentration in the plant tissue was at equilibrium with the external concentration within 1and 2 h for shoot and root tissue, respectively. Lindane and chlordane concentrations in the root and shoot reached equilibrium within 24 and 144 h, respectively. Bioconcentration factors for hydrilla were 9.62,38.15, and 1060.95 for atrazine, lindane, and chlordane, respectively. BCF estimation methods using log K , and water solubility based on data from fish or duckweeds were not predictive of values for hydrilla. The smaller lipid content of the
submersed plant species may account for the failure of the models. All three chemicals were translocated acropetally from roots exposed to a spiked sediment. Atrazine in the 2-3-cm segment of the shoot was in equilibrium after 6 h of root exposure. All shoot segments measured had similar concentrations of the compound within 48 h, although this concentration was slightly less than for the whole-plant exposure to the chemical in solution. Lindane concentrations in the 2-3-cm segment were in equilibrium after 192 h of root exposure and were approximately 20 times less than the shoot concentration for the whole-plant exposure. Chlordane in the 2-3-cm segment did not change significantlyafter 96 h of root exposure and was 780 times less than the shoot concentration for the whole-plant exposure. Registry No. Atrazine, 1912-24-9;lindane, 58-89-9; chlordane, 12789-03-6.
Literature Cited (1) Mantai, K. E.; Newton, M. E. Root growth in Myriophyllum: A specific plant response to nutrient availability? Aquat. Bot. 1982, 13, 45-55. (2) Bristow, J. M.; Whitcombe, M. The role of roots in the nutrition of aquatic vascular plants. Am. J. Bot. 1971,58, 8-13. (3) Raven, J. A. Energetics and transport i n aquatic plants; Alan R. Liss, Inc.: New York, 1984. (4) Sculthorpe, C. D.T h e biology of aquatic vascular plants; Koeltz Scientific Books: Konigstein, Germany, 1967. (5) Hutchinson, G. E. A treatise on limnology, Vol. Z Z I Limnological botany; John Wiley & Sons: New York, 1975. (6) Klaine, S. J.; Ward, C. H. Environmental and chemical control of vegetative dormant bud production in Hydrilla verticillata. Ann. Bot. 1984, 53, 503-514.
( 7 ) Waisel, Y.; Shapira, Z. Functions performed by roots of some submerged hydrophytes. Isr. J . Bot. 1971,20,69-77. ( 8 ) Bysshe, S. E. Bioconcentration factor in aquatic organisms. In Handbook of chemical property estimation methods: Environmental behavior of organic compounds; Lyman, W. J., Reehl, W. F., Rosenblatt, D. H., Eds.; McGraw-Hill Book Co.: New York, 1982; pp 5.1-5.30. (9) Laskowsiki, D. A.; Goring, C. A. I.; McCall, P. J.; Swann, R. L. Terrestrial Environment. In Environmental Risk Analysis for Chemicals; Conway, R. A., Ed.; Van Nostrand Reinhold Co.: New York, 1982; pp 198-256. (10) Lockhart, W. L.; Billeck, B. N.; de March, B. G. E.; Muir, D.C. G. Uptake and toxicity of organic compounds: Studies with an aquatic macrophyte (Lemna minor). In Aquatic toxicology and hazard assessment: S i x t h symposium; Bishop, W. E., Cardwell, R. D., Heidolph, B. B., Eds.; A S T M Spec. Tech. Publ. 1983, No. 802, 460-468. (11) Ashton, F. M.; Crafts, A. S. Mode of action of herbicides; John Wiley & Sons: New York, 1981. (12) Neumann, S.; Grimm, E.; Jacob, F. Transport of xenobiotics in higher plants: I. Structural prerequisites for translocation in the phloem. Biochem. Physiol. Pflanz. 1985,180, 257-268. (13) Sutton, D.L.; Bingham, S. W. Uptake and translocation of 2,4-D-1-14Cin parrotfeather. Weed Sei. 1970,18,193-196. (14) Harlan, S. M.; Davis, G. J.; Pesacreta, G. J. Hydrilla in three North Carolina lakes. J. Aquat. Plant Manage. 1985,23, 68-71. (15) Smith, C. R.; Shireman, J. V. Grass carp bibliography; Contract No. DAWC 39-80-0035; submitted to US.Army Corps of Engineers, Waterways Experimenta Station, Vicksburg, MS, 1981. (16) Sutton, D.L. Grass carp (Ctenopharyngodon idella Val.) in North America. Aquat. Bot. 1977, 3, 157-164.
Received for review October 29, 1990. Revised manuscript received J u l y 8, 1991. Accepted August 19, 1991.
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