RICE SEED TOXICITY TESTS FOR ORGANIC AND INORGANIC SUBSTANCES WUNCHENG WANG* Illinois State Water Survey, P.O. Box 697, Peoria, IL 61652, U.S.A. (Received: February 1992)
Abstract. Plant seed toxicity tests can be used to evaluate hazardous waste sites and to assess toxicity of complex effluents and industrial chemicals. Conventional plant seed toxicity tests are performed using culture dishes containing filter paper. Some reports indicate that filter papers might interfere with the toxicity of inorganic substances. In this study, a plastic seed tray was used. Rice was used as the test species. A comparison of results in the literature and this study revealed that variation of test species, methods, exposure duration, and other factors may affect the test results. The results of this study showed that the order of decreasing toxicity of metal ions was Cu>Ag>Ni>Cd>Cr(VI)>Pb>Zn>Mn>NaF for rice. The test results were similar to those reported in the literature for lettuce Ag>Ni>Cd,Cu>Cr (VI)>Zn>Mn, millet Cu,Ni>Cd>Cr(VI)>Zn>Mn, and ryegrass C u > N i > M n > > P b > C d > Z n > AI>Hg>Cr>Fe. The order of decreasing toxicity of organic herbicides was paraquat, 2,4-D> >glyphosate>bromacil.
1. Introduction Plant seed toxicity tests have recently become an important part of ecotoxicology to evaluate hazardous waste sites, as well as to test the toxicity of complex effluents and industrial chemicals (Wong and Bradshaw, 1982; Thomas et al., 1986; Adema and Henzen, 1989; Wang, 1987; Wang and Williams, 1990; Walsh et al., 1991). Plant test guidelines have also been adopted by various regulatory agencies to assess the environmental impacts of pesticides, industrial chemicals, food additives, cosmetics, and other substances (Hoist and Ellwanger, 1982; U.S. Food and Drug Administration, 1987; Organization for Economic Cooperation and Development, 1984). Recent overview and review papers on the tests are available (Fletcher et aL, 1985; Fletcher et al., 1990; Wang, 1991a). Based on a review of chemical registration requirements under the Toxic Substances Control Act, plant tests play a relatively small role in comparison to the emphasis on animal tests for regulation of potentially toxic substances (Benenati, 1990). Plant seed toxicity tests are typically performed using culture dishes containing filter papers. Ratsch and Johndro (1986) and Gorsuch et al. (1990) reported that filter papers may interfere with chemical toxicity. Upon further study, this author found that filter paper had three measurable effects: adsorption of cationic metal ions, stimulation of test specimens in control samples, and root attachment on papers, especially in the presence of toxicants to make it difficult to handle specimens (Wang, 1993). To avoid errors, a seed tray device was designed to be * Present address: U.S. Geological Survey, 720 Gracern Rd, Suite 129, Columbia, SC 29210, U.S.A. Environmental Monitoring and Assessment 29: 101-107, 1994. Q 1994 KluwerAeademie Publishers. Printed in the Netherlands.
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WUNCHENGWANG
used specifically for plant seed toxicity tests of organic and inorganic substances (Wang, 1993). Rice is one o f the species recommended for phytotoxicity (Organizaiton for Economic Cooperation and Development, 1984). In addition to being the most important economic crop in the world, rice can be considered a wetland species and the test results are relevant to aquatic environment. Several reports have indicated that rice is more sensitive to toxicity of complex effluents than lettuce, another recommended species (Organization for Economic Cooperation and Development, 1984; Hoist and Ellwanger, 1982; Wang, 1990. 1991a; Wang and Keturi, 1990). The objective of this study was to perform rice seed toxicity tests for nine inorganic substances and four organic herbicides. Rice (Oryza sativa) was used as the test species because previous studies had indicated that it was a promising test species (Wang and Keturi, 1990; Wang, 1990). The results of the current study were compared with the results from the literature. 2. Methods
The chemical substances used for this study were AgNO3, CdC12, K2CrO4, CuCI2, MnC12 • 4H20, NaF, NiCI2, Pb(C2H302)2 • 3H20, and ZnC12, all reagent grade and tested individually. For Ag, Cd, Cr, and NaF, each chemical was tested twice with six concentrations and four replications. The remaining chemicals (Cu, Mn, Ni, Pb, and Zn) were tested once with seven concentrations and five replications. Herbicide 2,4-D was purchased from Aldrich Chemical Co. (Milwaukee, WI). Technical or analytical standards bromacil (95%), glyphosate (96.6%), and paraquat (99.4%) were obtained from Du Pont, Monsanto, and ICI, respectively. Deionized water was used as dilution water and water control. Rice seeds were purchased in bulk and kept at -10°C. The test conditions are summarized in Table I. The test specimens were pretreated with hypochlorite solution (10% Clorox ® solution) for 20 minutes and rinsed ten times with deionized water. Each dish contained a seed tray, 31 mL of test solution, and 12 rice seeds. Seed trays were recycled; they were first soaked in a diluted acid bath, rinsed, soaked in a detergent bath, and then rinsed again. After incubation in the dark at 25-25.2°C for 144 h, all radical roots in each dish were severed, air dried for 24 h, and weighed to the nearest 0.1 mg. The ICs0 values (the concentrations that caused 50% inhibition relative to the control sample) and 95% confidence limits were calculated using the moving average method (Peltier and Weber, 1985). The t test was used to compute the significant differences between mean values. 3. Results and Discussion
Toxicant concentrations were selected to give the toxic effects for 10-100% inhibition of root development. Results in Figures 1 and 2 are plotted in semi-log
103
RICE SEED TOXICITY TESTS
TABLE I Summary of rice seed toxicity tests. Test type
Static
Seed pretreatment Temperature Light quality Test vessel
10% Clorox® solution for 20 min 25-25.2 ° C Dark 100 x 15 mm plastic culture dish plus a seed tray 31mL 12 6_7 (a) 0.5
Test solution Seeds/vessel Concentrations Dilution factor Water control and dilution water Replicates/test Test/toxicant Test duration Test endpoint
Deionized water 4_5 (b) 1_2 (c) 144 h dry root biomass
(a) Six concentrations for Ag, Cd, Cr, and NaF and seven for Cu, Mn, Ni, Pb, Zn, and organic herbicides. (b) Four replicates for Ag, Cd, Cr, NaF and five for Cu, Mn, Ni, Pb, Zn, and organic herbicides. (c) Two tests for Ag, Cd, Cr, and NaF and one for Cu, Mn, Ni, Pb, Zn, and organic herbicides.
• oz
60
Cu NaF
0.1
I
10
100
CONCENTRATIONf Mg/L
Fig. 1. Rice seed toxicity tests of inorganic substances.
1000
104
WUNCHENG WANG lOO
z
80
p..
'-" 60
=
zuJ (z:
40
~_ 20 Ill[
.01
I
I
I'11111
I
.1
I
I
[ i I I1|[
I
I
|
1.0 CONCENTRATIONt
I I Ill I
10
I
I
I
IIIII
I
I
I
100
mg/L
Fig. 2. Rice seed toxicity tests of herbicides.
relationship. Root elongation was selected because it is known to be more sensitive to toxicity than seed germination (Palazzo and Leggett, 1986). For this study, the seed storage was less than one year, and the germination rate was better than 85% in all control samples. A previous study had shown that the rice seed germination rate did not change over a three-year storage time, and that among ten plant species, rice was the most sensitive to a metal engraving effluent (Wang and Keturi, 1990). In the series of experiments, the mean coefficient of variation of root biomass of all control tests (20) was 12%, attesting the rather consistent test results. Many toxicants showed toxicity above a threshold concentration, yet a stimulation effect at concentrations below the threshold. For example, 50 mg/L Mn inhibited root development significantly, 19% (p < 0.05), and 25 mg/L Mn stimulated root development significantly, 38% (p < 0.05). Similar inhibition and stimulation effects were reported with other toxicants (Bagby and Sherrard, 1981). The decreasing order of metal phytotoxicity for rice in this study was C u > A g > Ni>Cd>Cr(VI)>Pb>Zn>Mn>NaF. This order is similar to that reported in the literature using ryegrass C u > N i > M n > P b > C d > Z n > A I > H g > C r > F e (Wong and Bradshaw, 1982), lettuce Ni>Cd,Cu>Cr(VI)>Zn>Mn, and millet Cu,Ni>Cd> Cr(VI)>Zn>Mn (Wang, 1987; Ratsch and Johndro, 1986). Variation between these trends can be attributed to different test species (rice, ryegrass, lettuce, and millet), test methods (static and renewal), water controls (deionized water, 0.5 g/L Ca(NO3)2, and hard, standard water), test indicators (root elongation and biomass), and other unknown factors. IC50 values are typically used to express the intrinsic toxicity of a test substance. Table II gives the ICs0 values and 95% confidence limits of all metal results for the current rice study and the IC50 values from five published studies. Silver was found to be the most toxic with an IC50 value of 0.007 mg/L for lettuce (Ratsch and Johndro~ 1986). This value was two orders of magnitude lower than the value from the current study on rice: 0.55 mg/L. Ratsch and Johndro conducted the tests using the aeration-immersion method and incubation under light. The method allows
RICE SEED TOXICITY TESTS
105
TABLE II Comparison of plant seed toxicity test results of six studies (A-F), all expressed in ICsos, 50% inhibitory effect concentrations (95% confidence limits), mg/L.
Species
A Ryegrass
B Lettuce
Ag Cd Cr(VI) Cu Mn NaF Ni Pb Zn
1.85 2.00 0.02 0.45 0.18 1.7 1.6
0.007 0.14 660 . -
C Lettuce 0.84 0.16 . . -
D Lettuce
E Lettuce
58 22 -
2.4 3.7 2.4 35 0.83
31
11
.
F Rice 0.55 (0.50-0.61) 1.4 (1.3-1.5) 4.8 (3.9-5.7) 0.22 (0.2-0.25) 100 (92-109) 320 (280-360) 0.85 (0.76--0.94) 9.7 (8.8-10.7) 26 (23-29)
A: Wong and Bradshaw (1982), ryegrass root length, renewal, 14 days. B: Ratsch and Johndro (1986), lettuce root length, static, 5 days. C: Adema and Henzen (1989), lettuce shoot wet biomass, static, 14 days. D: Thomas et al. (1986), lettuce seed germination, static, 5 days. E: Wang (1987), lettuce root length, static, 5 days. F: Current study, rice root dry biomass, static, 6 days.
total submersion and constant whirling of the specimens in the test solution, as opposed to the static, partial submersion used in the current study. In addition, Ag ions are photosensitive and may remain in solution or precipitate dependent on light conditions during the experiment (Wang, 1990). Walsh e t aL (1991) reported that toxicity of complex effluents such as sewage, metal plating, and coking plant differed under light and dark conditions. The toxicity of a coking plant effluent was greater under light than in the dark because the toxicant(s) inhibited photosynthesis and/or the toxicant(s) was photosensitized. On the other hand, the toxicity o f a sewage treatment plant effluent was lost under light probably because toxicant(s) was photodegraded. The relative sensitivity o f lettuce and rice to metal toxicity was mixed. For instance, the Cd IC50 values for lettuce and rice were 0.14 and 1.4 mg/L, respectively, but the values reversed for NaF (Table II). There are reports indicating that rice is invariably more sensitive to effluent toxicity than lettuce, based on the results o f o v e r 30 effluent samples from municipal, dairy, metal processing, photoprocessing, and wood-treating industries (Wang, 1990, 1991b; Wang and Keture, 1990). Taxonomic differences between higher plants have been reported to have a much greater influence on plant response to chemical toxicity than did the testing methods (Fletcher e t al., 1990).
106
WUNCHENGWANG
The Cu ICs0 value for this study was 0.22 mg/L (rice), and 2.4 mg/L (lettuce) in an earlier study (Wang, 1987). The major difference between these two studies was the use of deionized water in the current study verses hard, standard water (containing 160-180 mg/L hardness, as CaCO3) (American Public Health Association et al., 1989). Manganese was reported to be extremely toxic, with an IC50 value of 0.45 mg/L for ryegrass (Wong and Bradshaw, 1982), compared to 35 mg/L for lettuce (Wang, 1987) and 100 mg/L for rice in the current study. The reason(s) for the discrepacy is (are) unknown. It should be pointed out, however, that Wong and Bradshaw conducted phytotoxicity tests using the renewal method for 14 days of exposure, while the other studies employed the static method for 5-6 days of exposure. There was also a substantial difference of Pb toxicity between the Wong and Bradshaw study and the current study: 1.7 and 9.7 mg/L, respectively. The results of four organic herbicides are given in Figure 2. The ICs0 values shown in the figure vary over three orders of magnitude. The most toxic to rice were paraquat and 2,4-D, while the least toxic was bromacil. For comparison, the toxic value of the most toxic metal, Cu, was intermediate in phytotoxicity between the organic herbicides (Figures 1 and 2). The results of this and other studies indicate that the rice seed toxicity test is useful for toxicity evaluation of inorganic and organic chemical compounds. The test is especially suitable for herbicide compounds, as a group they may be extremely toxic to higher plants. Because herbicides are widespread in the environment, phytotoxicity tests using higher plants are ideal for assessing herbicide contamination in the environment.
Acknowledgements I thank Du Pont, Monsanto, and ICI for donating herbicides used in this study.
References Ademan, D.M.M. and Henzen, L.: 1989, 'A Comparison of Plant Toxicities of some Industrial Chemicals in Soil Culture and Soilless Culture', Ecotoxicol. Environ. Saf 18, 219-229. American Public Health Association, American Water WorksAssociation, and WaterPollution Control Federation: 1989, Standard Methods for Examination of Water and Wastewater, 17th edition, Washington, D.C. Bagby, M.M. and Sherrard, J.H.: 1981, 'CombinedEffectsof Cadmium and Nickel on the Activated Sludge Process', J. Water Poll. Control Fed. 53, 1609-1619. Benenati, E: 1990, 'Plants - Keystoneto Risk Assessment',ASTM STP 1091, American Soc. Testing & Materials, Philadelphia, PA. Fletcher, J.S., Mnhitch, M.S., Vann, D.R., McFaflane, J.C., and Benenati, EE.: 1985, 'PHYTOTOX Database Evaluation of Surrogate Plant Species Recommendedby the U.S. Environmental Protection Agency and the Organization for Economic Cooperation and Development', Environ. Toxicol. Chem. 4, 523-532. Fletcher, J.S., Johnson, EL., and McFarlane, J.C.: 1990, 'Influenceof GreenhouseversusFieldTesting and TaxonomicDifferenceson Plant Sensitivityto ChemicalTreatment', Environ. Toxicol. Chem. 9, 769-776.
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107
Gorsuch, J.W., Kringle, R.O., and Robicclard, K.A.: 1990, 'Chemical Effects on Germination and Early Growth of Terrestrial Plants', ASTMSTP 1091, Amer. Soc. Testing & Materials, Philadelphia, PA. Hoist, R.W. and Ellwanger, T.C.: 1982, Pesticide Assessment Guidelines. Subdivision J. Hazard Evaluation: Non-Target Plants. Office of Pesticides and Toxic Substances. U.S. Environmental Protection Agency, Washington, D.C. Organization for Economic Cooperation and Development: 1984, Terrestrial Plants: Growth Tests. OECD Guidelines for Testing of Chemicals. No. 208, Paris, France. Palazzo, A.J. and Leggett, D.C.: 1986, 'Effect and Disposition of TNT in a Terrestrial Plant', J. Environ. Qual. 15, 49-52. Peltier, W.H. and Weber, C.I. (Eds.): 1985, Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms. EPA/600/4-85/013. U.S. Environmental Protection Agency, Cincinnati, OH. Ratsch, H.C. and Johndro, D.: 1986, 'Comparative Toxicity of Six Test Chemicals to Lettuce Using Two Root Elongation Test Methods', Environ. Monit. Assess. 6, 267-276. Thomas, J.M., Skalske, J.R., Cline, J.E, McShane, M.C., Simpson, J.C., Miller, W.E., Peterson, S.A., Callahan, C.A., and Greene, J.C.: 1986, 'Characterization of Chemical Waste Site Contamination and Determination of its Extent Using Bioassays', Environ. Toxicol. Chem. 5, 487-501. U.S. Food and Drug Administration: 1987, Environmental Assessment Technical Handbook. Center for Food Safety and Applied Nutrition and Center for Veterinary Medicine, Washington, D.C. Walsh, G.E., Weber, D.E., Simon, T.L., and Brashers, L.K.: 1991, 'Toxicity Tests of Effluents with Marsh Plants in Water and Sediment', Environ. Toxicol. Chem. 10, 517-525. Wang, W.: 1987, 'Root Elongation Method for Toxicity Testing of Organic and Inorganic Pollutants', Environ. ToxicoL Chem. 6, 409-414. Wang, W.: 1990, 'Toxicity Assessment of Pretreated Industrial Effluents Using Higher Plants', Res. J. Water Poll Control Fed. 62, 853-860. Wang, W.: 1991a, 'Literature Review on Higher Plants for Toxicity Testing', Water, Air, Soil Poll. 59, 381-400. Wang, W.: 1991b, 'Higher Plants (Common Duckweed, Lettuce, and Rice) for Effluent Toxicity Assessment', ASTM STP 1115, Amer. Soc. Testing & Materials, Philadelphia, PA. Wang, W.: 1993, 'Comparative Rice Seed Toxicity Tests using Filter Paper, Growth Pouch-TM, and Seed Tray Methods', Environ. Monit. Assess. 24, 257-266. Wang, W. and Keturi, E: 1990, 'Comparative Seed Germination Tests Using Ten Plant Species for Toxicity Assessment of a Metal Engraving Effluent', Water, Air, Soil Poll. 52, 369-376. Wang, W. and Williams, J.: 1990, 'The Use of Phytotoxicity Tests (Common Duckweed, Cabbage, Millet) for Determining Effluent Toxicity', Environ. Monit. Assess. 14, 45-58. Wong, M.H. and Bradshaw, A.D.: 1982, 'A Comparison of the Toxicity of Heavy Metals, Using Root Elongation of Ryegrass, Lolium perenne', New Phytol. 91, 255-261.
Abstract. Plant seed toxicity tests can be used to evaluate hazardous waste sites and to assess toxicity of complex effluents and industrial chemicals. Conventional plant seed toxicity tests are performed using culture dishes containing filter paper. Some reports indicate that filter papers might interfere with the toxicity of inorganic substances. In this study, a plastic seed tray was used. Rice was used as the test species. A comparison of results in the literature and this study revealed that variation of test species, methods, exposure duration, and other factors may affect the test results. The results of this study showed that the order of decreasing toxicity of metal ions was Cu>Ag>Ni>Cd>Cr(VI)>Pb>Zn>Mn>NaF for rice. The test results were similar to those reported in the literature for lettuce Ag>Ni>Cd,Cu>Cr (VI)>Zn>Mn, millet Cu,Ni>Cd>Cr(VI)>Zn>Mn, and ryegrass C u > N i > M n > > P b > C d > Z n > AI>Hg>Cr>Fe. The order of decreasing toxicity of organic herbicides was paraquat, 2,4-D> >glyphosate>bromacil.
1. Introduction Plant seed toxicity tests have recently become an important part of ecotoxicology to evaluate hazardous waste sites, as well as to test the toxicity of complex effluents and industrial chemicals (Wong and Bradshaw, 1982; Thomas et al., 1986; Adema and Henzen, 1989; Wang, 1987; Wang and Williams, 1990; Walsh et al., 1991). Plant test guidelines have also been adopted by various regulatory agencies to assess the environmental impacts of pesticides, industrial chemicals, food additives, cosmetics, and other substances (Hoist and Ellwanger, 1982; U.S. Food and Drug Administration, 1987; Organization for Economic Cooperation and Development, 1984). Recent overview and review papers on the tests are available (Fletcher et aL, 1985; Fletcher et al., 1990; Wang, 1991a). Based on a review of chemical registration requirements under the Toxic Substances Control Act, plant tests play a relatively small role in comparison to the emphasis on animal tests for regulation of potentially toxic substances (Benenati, 1990). Plant seed toxicity tests are typically performed using culture dishes containing filter papers. Ratsch and Johndro (1986) and Gorsuch et al. (1990) reported that filter papers may interfere with chemical toxicity. Upon further study, this author found that filter paper had three measurable effects: adsorption of cationic metal ions, stimulation of test specimens in control samples, and root attachment on papers, especially in the presence of toxicants to make it difficult to handle specimens (Wang, 1993). To avoid errors, a seed tray device was designed to be * Present address: U.S. Geological Survey, 720 Gracern Rd, Suite 129, Columbia, SC 29210, U.S.A. Environmental Monitoring and Assessment 29: 101-107, 1994. Q 1994 KluwerAeademie Publishers. Printed in the Netherlands.
102
WUNCHENGWANG
used specifically for plant seed toxicity tests of organic and inorganic substances (Wang, 1993). Rice is one o f the species recommended for phytotoxicity (Organizaiton for Economic Cooperation and Development, 1984). In addition to being the most important economic crop in the world, rice can be considered a wetland species and the test results are relevant to aquatic environment. Several reports have indicated that rice is more sensitive to toxicity of complex effluents than lettuce, another recommended species (Organization for Economic Cooperation and Development, 1984; Hoist and Ellwanger, 1982; Wang, 1990. 1991a; Wang and Keturi, 1990). The objective of this study was to perform rice seed toxicity tests for nine inorganic substances and four organic herbicides. Rice (Oryza sativa) was used as the test species because previous studies had indicated that it was a promising test species (Wang and Keturi, 1990; Wang, 1990). The results of the current study were compared with the results from the literature. 2. Methods
The chemical substances used for this study were AgNO3, CdC12, K2CrO4, CuCI2, MnC12 • 4H20, NaF, NiCI2, Pb(C2H302)2 • 3H20, and ZnC12, all reagent grade and tested individually. For Ag, Cd, Cr, and NaF, each chemical was tested twice with six concentrations and four replications. The remaining chemicals (Cu, Mn, Ni, Pb, and Zn) were tested once with seven concentrations and five replications. Herbicide 2,4-D was purchased from Aldrich Chemical Co. (Milwaukee, WI). Technical or analytical standards bromacil (95%), glyphosate (96.6%), and paraquat (99.4%) were obtained from Du Pont, Monsanto, and ICI, respectively. Deionized water was used as dilution water and water control. Rice seeds were purchased in bulk and kept at -10°C. The test conditions are summarized in Table I. The test specimens were pretreated with hypochlorite solution (10% Clorox ® solution) for 20 minutes and rinsed ten times with deionized water. Each dish contained a seed tray, 31 mL of test solution, and 12 rice seeds. Seed trays were recycled; they were first soaked in a diluted acid bath, rinsed, soaked in a detergent bath, and then rinsed again. After incubation in the dark at 25-25.2°C for 144 h, all radical roots in each dish were severed, air dried for 24 h, and weighed to the nearest 0.1 mg. The ICs0 values (the concentrations that caused 50% inhibition relative to the control sample) and 95% confidence limits were calculated using the moving average method (Peltier and Weber, 1985). The t test was used to compute the significant differences between mean values. 3. Results and Discussion
Toxicant concentrations were selected to give the toxic effects for 10-100% inhibition of root development. Results in Figures 1 and 2 are plotted in semi-log
103
RICE SEED TOXICITY TESTS
TABLE I Summary of rice seed toxicity tests. Test type
Static
Seed pretreatment Temperature Light quality Test vessel
10% Clorox® solution for 20 min 25-25.2 ° C Dark 100 x 15 mm plastic culture dish plus a seed tray 31mL 12 6_7 (a) 0.5
Test solution Seeds/vessel Concentrations Dilution factor Water control and dilution water Replicates/test Test/toxicant Test duration Test endpoint
Deionized water 4_5 (b) 1_2 (c) 144 h dry root biomass
(a) Six concentrations for Ag, Cd, Cr, and NaF and seven for Cu, Mn, Ni, Pb, Zn, and organic herbicides. (b) Four replicates for Ag, Cd, Cr, NaF and five for Cu, Mn, Ni, Pb, Zn, and organic herbicides. (c) Two tests for Ag, Cd, Cr, and NaF and one for Cu, Mn, Ni, Pb, Zn, and organic herbicides.
• oz
60
Cu NaF
0.1
I
10
100
CONCENTRATIONf Mg/L
Fig. 1. Rice seed toxicity tests of inorganic substances.
1000
104
WUNCHENG WANG lOO
z
80
p..
'-" 60
=
zuJ (z:
40
~_ 20 Ill[
.01
I
I
I'11111
I
.1
I
I
[ i I I1|[
I
I
|
1.0 CONCENTRATIONt
I I Ill I
10
I
I
I
IIIII
I
I
I
100
mg/L
Fig. 2. Rice seed toxicity tests of herbicides.
relationship. Root elongation was selected because it is known to be more sensitive to toxicity than seed germination (Palazzo and Leggett, 1986). For this study, the seed storage was less than one year, and the germination rate was better than 85% in all control samples. A previous study had shown that the rice seed germination rate did not change over a three-year storage time, and that among ten plant species, rice was the most sensitive to a metal engraving effluent (Wang and Keturi, 1990). In the series of experiments, the mean coefficient of variation of root biomass of all control tests (20) was 12%, attesting the rather consistent test results. Many toxicants showed toxicity above a threshold concentration, yet a stimulation effect at concentrations below the threshold. For example, 50 mg/L Mn inhibited root development significantly, 19% (p < 0.05), and 25 mg/L Mn stimulated root development significantly, 38% (p < 0.05). Similar inhibition and stimulation effects were reported with other toxicants (Bagby and Sherrard, 1981). The decreasing order of metal phytotoxicity for rice in this study was C u > A g > Ni>Cd>Cr(VI)>Pb>Zn>Mn>NaF. This order is similar to that reported in the literature using ryegrass C u > N i > M n > P b > C d > Z n > A I > H g > C r > F e (Wong and Bradshaw, 1982), lettuce Ni>Cd,Cu>Cr(VI)>Zn>Mn, and millet Cu,Ni>Cd> Cr(VI)>Zn>Mn (Wang, 1987; Ratsch and Johndro, 1986). Variation between these trends can be attributed to different test species (rice, ryegrass, lettuce, and millet), test methods (static and renewal), water controls (deionized water, 0.5 g/L Ca(NO3)2, and hard, standard water), test indicators (root elongation and biomass), and other unknown factors. IC50 values are typically used to express the intrinsic toxicity of a test substance. Table II gives the ICs0 values and 95% confidence limits of all metal results for the current rice study and the IC50 values from five published studies. Silver was found to be the most toxic with an IC50 value of 0.007 mg/L for lettuce (Ratsch and Johndro~ 1986). This value was two orders of magnitude lower than the value from the current study on rice: 0.55 mg/L. Ratsch and Johndro conducted the tests using the aeration-immersion method and incubation under light. The method allows
RICE SEED TOXICITY TESTS
105
TABLE II Comparison of plant seed toxicity test results of six studies (A-F), all expressed in ICsos, 50% inhibitory effect concentrations (95% confidence limits), mg/L.
Species
A Ryegrass
B Lettuce
Ag Cd Cr(VI) Cu Mn NaF Ni Pb Zn
1.85 2.00 0.02 0.45 0.18 1.7 1.6
0.007 0.14 660 . -
C Lettuce 0.84 0.16 . . -
D Lettuce
E Lettuce
58 22 -
2.4 3.7 2.4 35 0.83
31
11
.
F Rice 0.55 (0.50-0.61) 1.4 (1.3-1.5) 4.8 (3.9-5.7) 0.22 (0.2-0.25) 100 (92-109) 320 (280-360) 0.85 (0.76--0.94) 9.7 (8.8-10.7) 26 (23-29)
A: Wong and Bradshaw (1982), ryegrass root length, renewal, 14 days. B: Ratsch and Johndro (1986), lettuce root length, static, 5 days. C: Adema and Henzen (1989), lettuce shoot wet biomass, static, 14 days. D: Thomas et al. (1986), lettuce seed germination, static, 5 days. E: Wang (1987), lettuce root length, static, 5 days. F: Current study, rice root dry biomass, static, 6 days.
total submersion and constant whirling of the specimens in the test solution, as opposed to the static, partial submersion used in the current study. In addition, Ag ions are photosensitive and may remain in solution or precipitate dependent on light conditions during the experiment (Wang, 1990). Walsh e t aL (1991) reported that toxicity of complex effluents such as sewage, metal plating, and coking plant differed under light and dark conditions. The toxicity of a coking plant effluent was greater under light than in the dark because the toxicant(s) inhibited photosynthesis and/or the toxicant(s) was photosensitized. On the other hand, the toxicity o f a sewage treatment plant effluent was lost under light probably because toxicant(s) was photodegraded. The relative sensitivity o f lettuce and rice to metal toxicity was mixed. For instance, the Cd IC50 values for lettuce and rice were 0.14 and 1.4 mg/L, respectively, but the values reversed for NaF (Table II). There are reports indicating that rice is invariably more sensitive to effluent toxicity than lettuce, based on the results o f o v e r 30 effluent samples from municipal, dairy, metal processing, photoprocessing, and wood-treating industries (Wang, 1990, 1991b; Wang and Keture, 1990). Taxonomic differences between higher plants have been reported to have a much greater influence on plant response to chemical toxicity than did the testing methods (Fletcher e t al., 1990).
106
WUNCHENGWANG
The Cu ICs0 value for this study was 0.22 mg/L (rice), and 2.4 mg/L (lettuce) in an earlier study (Wang, 1987). The major difference between these two studies was the use of deionized water in the current study verses hard, standard water (containing 160-180 mg/L hardness, as CaCO3) (American Public Health Association et al., 1989). Manganese was reported to be extremely toxic, with an IC50 value of 0.45 mg/L for ryegrass (Wong and Bradshaw, 1982), compared to 35 mg/L for lettuce (Wang, 1987) and 100 mg/L for rice in the current study. The reason(s) for the discrepacy is (are) unknown. It should be pointed out, however, that Wong and Bradshaw conducted phytotoxicity tests using the renewal method for 14 days of exposure, while the other studies employed the static method for 5-6 days of exposure. There was also a substantial difference of Pb toxicity between the Wong and Bradshaw study and the current study: 1.7 and 9.7 mg/L, respectively. The results of four organic herbicides are given in Figure 2. The ICs0 values shown in the figure vary over three orders of magnitude. The most toxic to rice were paraquat and 2,4-D, while the least toxic was bromacil. For comparison, the toxic value of the most toxic metal, Cu, was intermediate in phytotoxicity between the organic herbicides (Figures 1 and 2). The results of this and other studies indicate that the rice seed toxicity test is useful for toxicity evaluation of inorganic and organic chemical compounds. The test is especially suitable for herbicide compounds, as a group they may be extremely toxic to higher plants. Because herbicides are widespread in the environment, phytotoxicity tests using higher plants are ideal for assessing herbicide contamination in the environment.
Acknowledgements I thank Du Pont, Monsanto, and ICI for donating herbicides used in this study.
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