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Oil shale process water affects activity of vesicular-arbuscular fungi and rhizobium 4 years after application to soil
Biochem. Vol. 18, No. 4, pp. 451455, Printed in Great Britain
Soil Bid.
1986
0038-0717/86 $3.00 + 0.00 Pergamon Journals Ltd
OIL SHALE PROCESS WATER AFFECTS ACTIVITY OF VESICULAR-ARBUSCULAR FUNGI AND RHIZOBIUM 4 YEARS AFTER APPLICATION TO SOIL PETER
D.
STAHL
and S. E.
WILLIAMS
Division of Plant Science, University of Wyoming, Laramie, WY 82071, U.S.A. (Accepted 25 January 1986) Summary-The effects of different concentrations of water from oil shale processing on the vesicular-arbuscular (VA) fungal and Rhizobium activity of an arid land soil were investigated 4 yr after contamination. Effects were assessed in field plots and by a greenhouse bioassay. Addition of process water had marked effects on the chemistry of the soil and increased concentrations of Ca, Mg, Na, NO, and NH, as well as raising the electrical conductivity. VA infection analysis of roots from field plots and the bioassay combined with counts of VA fungal spores indicate reduced mycorrhizal activity in treated soils. The individual species of VA fungi were found to be affected differently by the process water. Roots of the legume yellow sweetclover developed fewer nodules in soils treated with retort water. An acetylene reduction assay indicated that nitrogenase activity was reduced in nodules from soils treated with undiluted process water.
lNTRODLJCTION
Water from oil shale processing, a by-product of in-situ retorting, is generated in substantial amounts during oil extraction and is considered to be biologically hazardous (Scott, 1922; Rao et al., 1979). This
effluent originates from three sources during the retorting process: combustion, dehydration of minerals and intrusion of groundwater (Farrier et al., 1978). These waters typically contain organic compounds in concentrations up to 2% and inorganics to 5% (w/w). The process water used in this study, from in-situ retort site Omega-9, has toxic effects on plants (Skinner et al., 1979), fish (Lebsack et al., 1979) and other aquatic biota (Bergman et al., 1978). Hersman and Klein (1979) have shown that retorted Paraho oil shale has varied and significant effects on several important microbially-mediated soil processes and on the major groups of soil micro-organisms. The importance of VA mycorrhizal fungi and Rhizobium to plant-soil systems is well documented and it is known that the distribution and activity of these micro-organisms are influenced by soil conditions. Klein et al. (1979) found that N, fixation, both by free-living and legume-Rhizobium associations, is sensitive to oil shale process water. Schwab and Reeves (1979) reported that retorted oil shale added to soil reduced the formation of mycorrhizae on plants growing in that soil. Because it is probable that oil shale retort water will contact soil through unintentional exposure or waste disposal, it is important to obtain more information on the effects of this waste water on symbiotic soil micro-organisms. Our objective was to investigate the effects, after 4 yr, of application of oil shale process water to soil on the VA fungi and Rhizobium in an otherwise undisturbed soil. This was accomplished by assessing the VA fungal activity in field treatment plots and by using treated soils in a bioassay to determine VA infection and Rhizobium-nodulation potentials 4 yr after process water application.
MATERIALS
AND METHODS
Small plots in a native sagebrush-grassland were treated with different concentrations of Omega-9 retort water. Four years after treatment, soil and roots from the plots were collected and analyzed for VA fungal spore populations. A bioassay determined VA fungal and Rhizobium infectivities, and soil physical and chemical characteristics were ascertained. Roots were examined for frequency of VA mycorrhizal infection. Omega-9 oil shale process water has high pH (9.4) total dissolved organic C (1000 mg l-‘), bicarbonateC (15,940 mg l-l), thiosulfate (2740 mg I-‘), sulfate (1990 mg I-‘), NH:-N (3470 mg l-t), Kjeldahl N (3420 mg l-r), soluble salts (20,400 mhos cm-’ specific conductance), sodium (4333 mg 1-t) and low total phosphorus (3.2 mg 1-l) (Fox et al., 1978). The field site, located in an oil shale region, is approximately half way between Rock Springs and Green River, Wyoming, at the United States Department of Energy’s retort site (Omega-9). This semi-arid area (245cm precipitation yr-‘) was classified by Kuchler (1964) as a sagebrush steppe. The dominant plant species on the study site is Artemisia tridentata; subdominant species include Atriplex confertifolia, Oryzopsis hymenoides, Sitanion hystrix, Ceratoides lanata and Pascopyron smithii.
Within the 25 x 50 m study site, 25 1 x 1 m treatment plots were randomly established and soil samples from each were obtained prior to treatment. Five plots were treated on 28 June 1978 with a 5 cm depth of retort water (RW), five plots with a 5 cm depth of a 2:l mixture of retort water to deionized water (2RW: lW), five plots with a 5 cm depth of I :2 mixture of retort water to deionized water (lRW:2W), five plots with deionized water (W) and five plots were not treated (Control). Soil samples were collected again on 14 September 1978 for physical and chemical analysis. On 11 October 1982, 15 kg of the upper 20 cm of
452
PETER
D. STAHL and S. E. WILLIAMS
soil was removed from each plot and roots of Oryzopsis hymenoides were collected from as many of the plots as possible. Samples were refrigerated at 4°C until used and the roots were stained in lactophenolcotton blue (Davidson and Christensen, 1977). Most of the soil collected was used in the bioassay with the remainder being analyzed for VA spore populations and soil physical and chemical characteristics. Analyses of VA spore populations of treated and control soils were as described by Stahl and Christensen (1982). Spores were extracted from four 100 g soil subsamples by the sucrose flotation method (Allen et al., 1979). Four subsamples were extracted per field plot. Species present and their spore densities were determined. Absolute density is defined as the mean number of spores per 100 g dry soil and relative density as the number of spores of a particular species as a percentage of the total number of spores. Roots of Oryzopsis hymenoides were assayed for VA mycorrhizal infection using the technique of Allen and Allen (1980). A bioassay was designed to evaluate the VA mycorrhizal and Rhizobium infectivity potentials of the soils treated in this study. Melilotus ojicinalis (yellow blossom sweetclover) was grown in soil from the field site in the greenhouse. Soil collected from each treatment plot was sieved (~4.8 mm) and used to fill five 20 cm (2068 cm’) pots which were planted with 10 sweetclover seeds. Two weeks after planting, seedlings were thinned to five per pot. After 100 days of growth, entire plants were harvested and roots and shoots were separated. Roots were immediately assayed for nitrogenase by the acetylene reduction method (Hardy et al., 1973). Soon afterwards, root systems were weighed and examined for Rhizobiuminduced nodules which were then removed; also, a sample of fine roots was removed, weighed and inspected for VA infection. The roots, shoots and nodules were then dried in a forced air oven at 60°C for 24 h and weighed. Soil (100 g) from each pot was collected at the time of harvest for analysis of its VA spore population. Soil physical and chemical analyses were made by the Soils Testing Laboratory at the University of Wyoming. RESULTS
Soil characteristics
Results of physical-chemical analyses of soil from field treatment plots both before and after process water application are given in Table 1. All soil from the field study site had a fine clay texture. Addition of the process water to the test plots had marked effects on the chemistry of the soil solution as evidenced by the results of the September 1978 soil analyses. Soils to which process water was applied showed substantial increases in concentrations of Ca, Mg, Na, N03, NH, and electrical conductivity. Other analyses which showed no statistical differences either within a sampling period or within treatment levels include organic matter percentage, cation exchange capacity and extractable K. By October 1982, the examined chemical characteristics of treated soils appear to have returned to a condition similar to those of the pretreatment and control soils (Table 1).
Field site VAM analysis
In October 1982, soils at the field site to which oil shale process water had been added had VA fungal spore populations which differed in several ways from the Control and W treatments (Table 2). Retort water-treated soils had consistently lower total spore numbers and different absolute and relative densities of the individual species than the soils to which no process water had been added. VA spore populations from the W treatments were very similar to those from the control plots. Densities of Glomusfasciculatum and G. mosseae were similar at all sites except the IRW :2W treatments where there was an increase in G. mosseae. Absolute densities of G. microcarpum and Entrophospora infrequens were significantly less in all retort-treated soils. The most concentrated process water applications had the most significant effects on VA spore populations. VAM infection levels in roots of Oryzopsis hymenoides from retort water-treated plots were lower than in roots from the controls (Table 3). Bioassay
The greenhouse bioassay conducted using soil from the field plots provided data to compare with those from the field site (Tables 2 and 3). VA fungal spore populations in these soils were changed markedly by the bioassay (Table 2). Total spore density was increased in all soils by the growth of M. oficinalis. Spore populations from the control and W treatments substantially increased in total spore density but the relative densities of the component species remained similar with the exception of an increase in the relative density of G. mosseae. In contrast, the spore populations of the retort water-treated soils not only increased in total density but there were also important changes in the absolute and relative densities of the individual VA species. Most noticeably affected were G. microcarpum and G. mosseae. Their densities were greatly increased by the conditions of the bioassay. These two species, along with E. infrequens, were also most affected by retort water application in the field. Spores of E. infrequens were not recovered from retort water-treated plots in the field but were recovered from the soils after the bioassay. Biomass data of the M. ojicinalis is given in Table 4. Plants grew significantly less in the soils treated with undiluted and 2RW: 1W oil shale retort water. Root biomass appeared to be more affected by retort water treatment than did shoot biomass. M. ojkinalis roots were nodulated in all soil treatments tested in the bioassay (Table 4). Sweet clover plants grown in the retort water-treated soils had fewer nodules than plants grown in control and W treated soils. The acetylene reduction assay indicates that nitrogenase activity of nodules was similar in all treatments except RW which had a significantly lower mean value for C2H4 produced mg-’ dry nodule h-‘. DISCUSSION
The variation in response (spore production) by the different endomycorrhizal fungi to the retort water treatments suggests that the individual species are affected disparately by the retort water. G. micro -
O.lOaA O.llaA O.lOaA 0.09a A 0.12aA
0.14aA 0.16aB 0.30b B 0.44c B 0.47~ B
0.16a bA 0.12aA 0.08a A 0.12a bA 0.20b C
8.2a7A* 8.5~1A 8.3a A 8.4a A 8.3a A
7.9a B 8.0a B 7.9a A 1.9a B 7.8a B
8.Oa AB 8.2a AB 8.IaAB 8.2aA 8.3a A
EC? (S m-‘)
0.9a BA 0.9a A I.OaA I.OaA
0.9a A
1.2aAB 1.2aAB 0.9a A I.laA 5.0b B
2.laB 2.2a A 3.8bB 4.9b B 5.5b B
6.5a B
6.5a B 9.6abB 9.9b B 11SbB
I.laA 1.3aA 1.4aA I.laA 0.8a A
Mg’ (m-equiv I-‘)
1.4aA 2.IaA 1.9a A I.4aA 1.2aA
Ca3 (m-equiv I-‘)
Enrrophospora infiequens Glomns fascicularum G. microcarpwn G. mosseae Total number of spores
Species of VA fungi
I7 -+ 5 128 &32 118+28 21&4 284+61
Field
Control
20+8 208-131 142234 54+ 18 424 + 86
Bioassay 8+4 154+23 130521 16&8 308 + 51
__---____ FieId
W
22.Oa B 22.0a B 34.Oa B 29.Oa C 22Sa C
16+8 168 + 35 106+23 132+31 422 & 102
0 108k46 850 28+8 144+45
0 75f8 1016 22 + 9 107k42 0 98227 20+ II 74+ 19 192 +42
5k3 151 f32 65+21 85 + 30 306_+86 15+6 2CQ+45 160+_21 87k21 462 _t 15
Field
eO.05.
0.03a A O.OOaA 0.03a A 0.03a B 0.03a A
38.8 39.5 38.8 37.8 30.6
1O.OaB 9.9a B I3.2a B 9.9a B
7.9a B 8.laB 8.0a B 8.IaB 8.ia B
41.4 42.3 46.7 44.8 44.5
12.0aB
0.02aA
0.03a 0.02a 0.02a 0.04a
B A A A
43.7 50.6 48.2 46.4 48.2
A A A A A
Saturation (%)
3.9a 3.8a 3.0a 3.8a 3.5a
PO,@ tPRg-‘)
O.OIaA O.Ola A 0.02a A O.OlaA 0.02a A
Total’ N (%)
RW
II +7 I63 +27 46rt16 98 + 19 318 +77
-I___ Bioassay
and NH,+ determined using autoanalyzer.
13.9a AB 7.IaA 41.8b B 63.9b B 105.8~ B
8.9a A 9.6a A 9.2a A 9.9a A 9.6a A
NH,-N4 (&?a-‘)
Mean number of spores 100 g soil-’ + standard deviation IRW:2W 2RW:lW _ --___ Bioassay Field Bioassay FieId Bioassay
Table 2. VA spore populations
9.3aB 11.8bA 9.3a A 9.3a B 7.4a B
I5.4a A 17.9a A 36.7b A 43.3b A 44.2b A
16.2a A0 24.la A 18.9aA 27Sa AB 27.3a AB
NO,-N4 (PRg-‘f
HCI added per 20mI solution. NO;
9.0a bA IO&A 8.IbB 16.5~ AB 9.6abAB
7SaA 9.laA i 5.9bcA 20.9~ 8 23.Obc B
9.9a A IO.laA 15.7aAB 12.3a A 8.4a A
Na’ (m-equiv I-‘)
‘Each reported analysis is an average of five subsamples. PH and electrical conductivity ofpaste and saturation extract, respectively (USDA, 1954). ‘Soluble cations according to USDA (1954). Final analysis by inductively coupled plasma. ‘5g soil (wet weight) shaken in 2~ KCI. Flasks sealed and allowed to settle 12 h. One drop Ultrex concentrated ‘Jones (1971). bOIsen and Sommers (1982). ‘Numbers within each sampling period not followed by the same lower case letter are statistically different at P ‘Numbers within each treatment not followed by the same upper case letter are statistically different at P ~0.05.
June 1978’ Control Water (W) IRW:ZW 2RW:IW Retort Water (RW) Post-treatment September 1978 Control W IRW:ZW 2RW: IW RW Post-treatment September 1982 Control W IRW:ZW 2RWIW RW
pH2 (paste)
Table I. Pretreatment and two most-treatment soil analvses from exoerimental olots from near Rock Snrinas. Wvominn. See Materials and Methods for exolanation of labels
PETER D. STAHL and S. E. WILLIAMS
454
offcinalis, although
Table 3. VA infection levels in roots from field plots and bioassay’. See Materials and Methods for explanation of labels
Oryzopsis roots Treatment
in field
Control W IRW:ZW 2RW:lW RW
89 +_8a2 91 +_8a 64+31b 38+26c 36+2Oc
Melilorus roots in bioassay 12 +_22a 67 _t 19a 64 + 22ab 49 + 24b 54 + 19b
‘Values indicate percentage of observed 1 mm root segments inhabited by VA fungi. 2Numerical entries within each column not followed by the same letter are statistically different at P < 0.05.
carpum and E. infreqwens were sensitive to the retort water while G. fasciculatwm appeared to be more tolerant and G. mosseae spores actually increased in density at the treated sites. At the field study site, absolute densities of G. fasciculatum and G. mosseae were much less changed by retort water-treatment than were the densities of G. microcarpum and E. injiiequens, which were lowered significantly. The response of G. mosseae to both retort watertreatment and the bioassay is interesting and unique. This species was recovered in similar absolute densities (17-28 spores per 100 g soil) from all field soil treatments except lRW:2W where it had substantially higher density (74 spores per 100 g soil) and it increased in all bioassayed soils. G. mosseae may have benefited from the lack of competition from the sensitive species in the retort water-treated soils. Daft and Nicolson (1974) investigated VA mycorrhizas of plants colonizing coal waste in Scotland and report an interesting distribution of the three endophytes present. These authors found an endophyte with narrow hyphae to be more prevalent on the most recently colonized spoils but it was gradually replaced by other, more typical, VA mycorrhizal endophytes as plant colonization progressed (and, presumably, soil conditions improved). The substantial changes in spore populations in retort water-treated soils during the bioassay suggest that these soils may lose some of their inhibitive properties as the bioassay progresses. It is possible that the biweekly watering of plants caused leaching of toxic compounds through the soil. This is supported by the doubling and tripling of total spore numbers and increases in the densities of the sensitive species G. microcarpum and E. infrequens in the retort water-treated soils during the bioassay. No leguminous plants were found growing at any of the contaminated field plots 4 yr after treatment. Nevertheless, soil from these sites was shown to support Rhizobium-induced root nodulation of M.
it does appear that nodulation was suppressed in process water treated soils. When nodulation is expressed as dry weight nodules produced g-’ dry weight root (Table 4) it is clear that less nodule tissue was formed on root systems grown in the retort water-treated soils. Nitrogenase activity of the M. oficinalis-Rhizobium nodules, as determined by acetylene reduction, was significantly reduced only in soil treated with full-strength retort water. Hersman and Klein (1979) report that of all the soil microbiological characteristics they investigated nitrogen fixation (acetylene reduction) rate of soil was most affected by the presence of retorted oil shale. Although nodulation was inhibited in retorttreated soils in this study, lesser amounts of nodules were still produced and nitrogenase activity of these nodules was not affected in the IRW :2W and 2RW: IW soils. The symbiotic nitrogen-fixing Rhizobium in root nodules may be more protected from the inhibitory agents in oil shale retort water than are the free-living N-fixers studied by Hersman and Klein. Despite the fact that all soil parameters monitored in this study have returned to original levels, results from the field and bioassay indicate that something is continuing to inhibit the activity of VA fungi, Rhizobium-nodulation and the growth of M. oficinalis in the more heavily treated soils. Because Omega-9 retort water is high in total dissolved organic carbon (Stuber and Leenheer, 1978), which include toxic compounds such as phenols, Nheterocyclics and polynuclear aromatic hydrocarbons (Hilpert et al., 1979) that were not tested for by the soil analysis used in this study, it is possible these compounds are at least partly responsible for the persistent inhibitory effects of the 2RW: 1W and RW soils. Acknowledgements-Published
with approval of the Directar of the Wyoming Agricultural Experiment Station, UniVersitY of Wyoming, Laramie, WY 82071 as Journal Series Number JA 1373. This work was supported, in part, by a grant from the United States Department of Energy. REFERENCES
Allen E. B. and Allen M. F. (1980)Natural re-establishment of vesicular-arbuscular mycorrhizae following stripmine reclamation in Wyoming. Journal of Applied Ecology 17, 139-147. Allen M. F., Moore T. S. Jr, Christensen M. and Stanton N. (1979) Growth of vesicular-arbuscular mycorrhizal and non-mycorrhizal Bouieloua gracilis in a defined medium. Mycologia 71, 666-669.
Bergman H. L., DeGraeve G. M., Anderson A. D. and Farrier D. S. (1978) Effects of complex effluents from
Table 4. Results of bioassay. See Materials and Methods for explanation of labels Dry weight Treatment Control W lRW:2W 2RW:IW RW
Shoots (g) 1.16+0.78a’ 1.05 * 0.70a 1.06 I&0.72a 0.78 + 0.57b 0.68 k 0.53b
Roots (9) 0.50 0.53 0.44 0.43 0.36
_+0.30ab *0.29a k 0.28b +_0.29bc i 0.23~
Nodules (mg) 10.1 *7a 10.0 * 7a 6.9 f 4b 4.2 + 4bc 3.7 * 3c
Dry wt nodules (mg) g-’ dry roots 9 9 7 5 5
Moles C,H, produced mg-’ dry nodules h-’ 0.29 + 0.17a 0.33 +0.18a 0.27 + 0.18a 0.30 + 0.21a 0.15 +0.13b
‘Numerical entries within each column not followed by the same letter are statistically different at P .c 0.05.
Oil shale water affects VA fungi and Rhizobium in-situ fossil fuel processing on aquatic biota. In Synthetic Fossil Fuel Technology: Potential Health and Environmental Efects: Proceedings of the First Annual Oak Ridge National Laboratory Life Sciences Symposium (K. E. Cowser and C. R. Rjchmond, Eds), pp. X%21 1. Ann Arbor Science, Michigan. Daft M. J. and Nicolson T. H. (1974) Arbuscular mycorrhizas in plants colonizing coal wastes in Scotland. New Phytologist 73, 1129-1138. Davidson D. E. and Christensen M. (1977) Rootmicrofungal and mycorrhizal association in a shortgrass prairie. In The 3elo~ground Ecosystem: A Symhesis of Plant-Associafed Processes (J. K. Marshall, Ed.), pp. 279-287. Range Science Department, Science Series No. 26, Colorado State University, Ft Collins. Farrier D. S., Virgona J. E., Phillips T. E. and Poulson R. E. (1978) Environmental research for in-sifu oil shale processing. In Elevenrh Oil Shale Symposium Proceedings (J. II. Gary, Ed.) pp. 81-99. Colorado School of Mines Press, Golden. Fox J. P,, Farrier D. S. and Paulson R. E. (1978) Chemical characterization and analytical considerations for an in situ oil shale process water. Laramie Energy Technology Center Report of Investigations LETC/RI-78/7. Hardy R. W. F., Burns R. C. and Holsten R. D. (1973) Applications of the acetylene-ethylene assay for measurement of nitrogen fixation. Soit Biology BrBiochemistry 5, 47-81. Hersman L. E. and Klein D. A. (1979) Retorted oil shale effects on soil microbiological characteristics. Journal of Environmental Quality 8, 520-524. Hilpert L. R., Hertz H. S., May W. E., Chesler S. N., Wise S. A., Guenther F. R., Brown J. M. and Parris R. M. (1979) Quantification of individual organic compounds in shale oil. In Oil Shale Symposium: Sampling. Analysis and Quality Assurance (C. Gale, Ed.), pp, 355-362. U.S. Environmental Protection Agency, EPA-600/9-80-022. Jones J. B. (1971) Laboratory procedures for the analysis of soils, feed, water and plant tissue. Soil Testing and Plant Analysis Laboratory, Athens, Georgia. Klein D. A., Hersman L. E. and Wv 8. Y. (1979) Manitoring of retorted oil shale effects on surface soil nitrogen fixation processes: a resource for design and management of land reclamation programs. In Oil Shule Symposiums
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Sampling, Analysis and Quality Assurance (C. Gale, Ed.) pp. 546-554. Kuchier A. W. (1964) Potential natural vegetation of the conterminous United States (map and manual). American Geographic Society Special ~~lication No. 36. 116 pp. Lcbsack M. E., Anderson A. D., Nelson K. F. and Farrier D. S. (1979) Sub-lethal effects of an in-&u oil shale retort water on rainbow trout. Toxicology and Applied Pharmacology 54, 462-468. Olsen S. R. and Sommers L. E. (1982) Phosphorus soluble in sodium bicarbonate. In Methods of Soil Analysis, Part 2 (A. G. Page, Ed.), pp. 403430. American Society of A~onomy, Madison. Rao T. K., Epler J. L., Guerin M. R., Schmidt-Collerus J. J. and Leffler L. (1979) Biological monitoring of oil shale products and effluents using short-term genetic analysis. In Oil Shale Symposium: Sampling, Analysis and Qualify Assurance (C. Gale, Ed.), pp. 431-442. U.S. Environmental Protection Agency, EPA-6OOj9-80-022. Schwab S. and Reeves F. B. (1979) The effect of retorted oil shale on VA mycorrhiza formation in soil from the Piceance Basin of northwestern Colorado. In Oil Shale Symposium: Sampling, Analysis and Quality Assurance (C. Gale, Ed.), pp. 566-576. U.S. Environmental Protection Agency, EPA-600/9-80-022. Scott A. (1922) On the occupationa cancer of the paraffin and oil workers of the Scottish shale industry. British Medical Journal 2, 1108-l 109. Skinner Q. D., Moore T. S. Jr, Asphmd R. O., Sexton J. C. and Farrier D. S. (1979) Plant responses to aqueous effluents derived from in-situ fuel processing. I. Development of screening methods. Laramie Energy Technology Center Publication No. LETC/R-I-79/4. 25 pp. Stahl P. D. and Christensen M. (1982) Mycorrhizal fungi associated with Bou~eloua and Agropyron in Wyoming ~gebrush-grasslands. ~ycolog~a 74, 877-885. Stuber H. A. and Leenheer J. A. (1978) Fractionation of organic solutes in oil shale retort waters for sorption studies on processed shale. American Chemical Society, Division of Fuel Chemistry 23, 165-174. U.S. Department of Agriculture (1954) Diagnosis and improvement of saline and alkali soifs. Agriculture Handbook No, 60 (L. A. Richards, Ed.), U.S. Salinity Laboratory, Riverside, California.
Soil Bid.
1986
0038-0717/86 $3.00 + 0.00 Pergamon Journals Ltd
OIL SHALE PROCESS WATER AFFECTS ACTIVITY OF VESICULAR-ARBUSCULAR FUNGI AND RHIZOBIUM 4 YEARS AFTER APPLICATION TO SOIL PETER
D.
STAHL
and S. E.
WILLIAMS
Division of Plant Science, University of Wyoming, Laramie, WY 82071, U.S.A. (Accepted 25 January 1986) Summary-The effects of different concentrations of water from oil shale processing on the vesicular-arbuscular (VA) fungal and Rhizobium activity of an arid land soil were investigated 4 yr after contamination. Effects were assessed in field plots and by a greenhouse bioassay. Addition of process water had marked effects on the chemistry of the soil and increased concentrations of Ca, Mg, Na, NO, and NH, as well as raising the electrical conductivity. VA infection analysis of roots from field plots and the bioassay combined with counts of VA fungal spores indicate reduced mycorrhizal activity in treated soils. The individual species of VA fungi were found to be affected differently by the process water. Roots of the legume yellow sweetclover developed fewer nodules in soils treated with retort water. An acetylene reduction assay indicated that nitrogenase activity was reduced in nodules from soils treated with undiluted process water.
lNTRODLJCTION
Water from oil shale processing, a by-product of in-situ retorting, is generated in substantial amounts during oil extraction and is considered to be biologically hazardous (Scott, 1922; Rao et al., 1979). This
effluent originates from three sources during the retorting process: combustion, dehydration of minerals and intrusion of groundwater (Farrier et al., 1978). These waters typically contain organic compounds in concentrations up to 2% and inorganics to 5% (w/w). The process water used in this study, from in-situ retort site Omega-9, has toxic effects on plants (Skinner et al., 1979), fish (Lebsack et al., 1979) and other aquatic biota (Bergman et al., 1978). Hersman and Klein (1979) have shown that retorted Paraho oil shale has varied and significant effects on several important microbially-mediated soil processes and on the major groups of soil micro-organisms. The importance of VA mycorrhizal fungi and Rhizobium to plant-soil systems is well documented and it is known that the distribution and activity of these micro-organisms are influenced by soil conditions. Klein et al. (1979) found that N, fixation, both by free-living and legume-Rhizobium associations, is sensitive to oil shale process water. Schwab and Reeves (1979) reported that retorted oil shale added to soil reduced the formation of mycorrhizae on plants growing in that soil. Because it is probable that oil shale retort water will contact soil through unintentional exposure or waste disposal, it is important to obtain more information on the effects of this waste water on symbiotic soil micro-organisms. Our objective was to investigate the effects, after 4 yr, of application of oil shale process water to soil on the VA fungi and Rhizobium in an otherwise undisturbed soil. This was accomplished by assessing the VA fungal activity in field treatment plots and by using treated soils in a bioassay to determine VA infection and Rhizobium-nodulation potentials 4 yr after process water application.
MATERIALS
AND METHODS
Small plots in a native sagebrush-grassland were treated with different concentrations of Omega-9 retort water. Four years after treatment, soil and roots from the plots were collected and analyzed for VA fungal spore populations. A bioassay determined VA fungal and Rhizobium infectivities, and soil physical and chemical characteristics were ascertained. Roots were examined for frequency of VA mycorrhizal infection. Omega-9 oil shale process water has high pH (9.4) total dissolved organic C (1000 mg l-‘), bicarbonateC (15,940 mg l-l), thiosulfate (2740 mg I-‘), sulfate (1990 mg I-‘), NH:-N (3470 mg l-t), Kjeldahl N (3420 mg l-r), soluble salts (20,400 mhos cm-’ specific conductance), sodium (4333 mg 1-t) and low total phosphorus (3.2 mg 1-l) (Fox et al., 1978). The field site, located in an oil shale region, is approximately half way between Rock Springs and Green River, Wyoming, at the United States Department of Energy’s retort site (Omega-9). This semi-arid area (245cm precipitation yr-‘) was classified by Kuchler (1964) as a sagebrush steppe. The dominant plant species on the study site is Artemisia tridentata; subdominant species include Atriplex confertifolia, Oryzopsis hymenoides, Sitanion hystrix, Ceratoides lanata and Pascopyron smithii.
Within the 25 x 50 m study site, 25 1 x 1 m treatment plots were randomly established and soil samples from each were obtained prior to treatment. Five plots were treated on 28 June 1978 with a 5 cm depth of retort water (RW), five plots with a 5 cm depth of a 2:l mixture of retort water to deionized water (2RW: lW), five plots with a 5 cm depth of I :2 mixture of retort water to deionized water (lRW:2W), five plots with deionized water (W) and five plots were not treated (Control). Soil samples were collected again on 14 September 1978 for physical and chemical analysis. On 11 October 1982, 15 kg of the upper 20 cm of
452
PETER
D. STAHL and S. E. WILLIAMS
soil was removed from each plot and roots of Oryzopsis hymenoides were collected from as many of the plots as possible. Samples were refrigerated at 4°C until used and the roots were stained in lactophenolcotton blue (Davidson and Christensen, 1977). Most of the soil collected was used in the bioassay with the remainder being analyzed for VA spore populations and soil physical and chemical characteristics. Analyses of VA spore populations of treated and control soils were as described by Stahl and Christensen (1982). Spores were extracted from four 100 g soil subsamples by the sucrose flotation method (Allen et al., 1979). Four subsamples were extracted per field plot. Species present and their spore densities were determined. Absolute density is defined as the mean number of spores per 100 g dry soil and relative density as the number of spores of a particular species as a percentage of the total number of spores. Roots of Oryzopsis hymenoides were assayed for VA mycorrhizal infection using the technique of Allen and Allen (1980). A bioassay was designed to evaluate the VA mycorrhizal and Rhizobium infectivity potentials of the soils treated in this study. Melilotus ojicinalis (yellow blossom sweetclover) was grown in soil from the field site in the greenhouse. Soil collected from each treatment plot was sieved (~4.8 mm) and used to fill five 20 cm (2068 cm’) pots which were planted with 10 sweetclover seeds. Two weeks after planting, seedlings were thinned to five per pot. After 100 days of growth, entire plants were harvested and roots and shoots were separated. Roots were immediately assayed for nitrogenase by the acetylene reduction method (Hardy et al., 1973). Soon afterwards, root systems were weighed and examined for Rhizobiuminduced nodules which were then removed; also, a sample of fine roots was removed, weighed and inspected for VA infection. The roots, shoots and nodules were then dried in a forced air oven at 60°C for 24 h and weighed. Soil (100 g) from each pot was collected at the time of harvest for analysis of its VA spore population. Soil physical and chemical analyses were made by the Soils Testing Laboratory at the University of Wyoming. RESULTS
Soil characteristics
Results of physical-chemical analyses of soil from field treatment plots both before and after process water application are given in Table 1. All soil from the field study site had a fine clay texture. Addition of the process water to the test plots had marked effects on the chemistry of the soil solution as evidenced by the results of the September 1978 soil analyses. Soils to which process water was applied showed substantial increases in concentrations of Ca, Mg, Na, N03, NH, and electrical conductivity. Other analyses which showed no statistical differences either within a sampling period or within treatment levels include organic matter percentage, cation exchange capacity and extractable K. By October 1982, the examined chemical characteristics of treated soils appear to have returned to a condition similar to those of the pretreatment and control soils (Table 1).
Field site VAM analysis
In October 1982, soils at the field site to which oil shale process water had been added had VA fungal spore populations which differed in several ways from the Control and W treatments (Table 2). Retort water-treated soils had consistently lower total spore numbers and different absolute and relative densities of the individual species than the soils to which no process water had been added. VA spore populations from the W treatments were very similar to those from the control plots. Densities of Glomusfasciculatum and G. mosseae were similar at all sites except the IRW :2W treatments where there was an increase in G. mosseae. Absolute densities of G. microcarpum and Entrophospora infrequens were significantly less in all retort-treated soils. The most concentrated process water applications had the most significant effects on VA spore populations. VAM infection levels in roots of Oryzopsis hymenoides from retort water-treated plots were lower than in roots from the controls (Table 3). Bioassay
The greenhouse bioassay conducted using soil from the field plots provided data to compare with those from the field site (Tables 2 and 3). VA fungal spore populations in these soils were changed markedly by the bioassay (Table 2). Total spore density was increased in all soils by the growth of M. oficinalis. Spore populations from the control and W treatments substantially increased in total spore density but the relative densities of the component species remained similar with the exception of an increase in the relative density of G. mosseae. In contrast, the spore populations of the retort water-treated soils not only increased in total density but there were also important changes in the absolute and relative densities of the individual VA species. Most noticeably affected were G. microcarpum and G. mosseae. Their densities were greatly increased by the conditions of the bioassay. These two species, along with E. infrequens, were also most affected by retort water application in the field. Spores of E. infrequens were not recovered from retort water-treated plots in the field but were recovered from the soils after the bioassay. Biomass data of the M. ojicinalis is given in Table 4. Plants grew significantly less in the soils treated with undiluted and 2RW: 1W oil shale retort water. Root biomass appeared to be more affected by retort water treatment than did shoot biomass. M. ojkinalis roots were nodulated in all soil treatments tested in the bioassay (Table 4). Sweet clover plants grown in the retort water-treated soils had fewer nodules than plants grown in control and W treated soils. The acetylene reduction assay indicates that nitrogenase activity of nodules was similar in all treatments except RW which had a significantly lower mean value for C2H4 produced mg-’ dry nodule h-‘. DISCUSSION
The variation in response (spore production) by the different endomycorrhizal fungi to the retort water treatments suggests that the individual species are affected disparately by the retort water. G. micro -
O.lOaA O.llaA O.lOaA 0.09a A 0.12aA
0.14aA 0.16aB 0.30b B 0.44c B 0.47~ B
0.16a bA 0.12aA 0.08a A 0.12a bA 0.20b C
8.2a7A* 8.5~1A 8.3a A 8.4a A 8.3a A
7.9a B 8.0a B 7.9a A 1.9a B 7.8a B
8.Oa AB 8.2a AB 8.IaAB 8.2aA 8.3a A
EC? (S m-‘)
0.9a BA 0.9a A I.OaA I.OaA
0.9a A
1.2aAB 1.2aAB 0.9a A I.laA 5.0b B
2.laB 2.2a A 3.8bB 4.9b B 5.5b B
6.5a B
6.5a B 9.6abB 9.9b B 11SbB
I.laA 1.3aA 1.4aA I.laA 0.8a A
Mg’ (m-equiv I-‘)
1.4aA 2.IaA 1.9a A I.4aA 1.2aA
Ca3 (m-equiv I-‘)
Enrrophospora infiequens Glomns fascicularum G. microcarpwn G. mosseae Total number of spores
Species of VA fungi
I7 -+ 5 128 &32 118+28 21&4 284+61
Field
Control
20+8 208-131 142234 54+ 18 424 + 86
Bioassay 8+4 154+23 130521 16&8 308 + 51
__---____ FieId
W
22.Oa B 22.0a B 34.Oa B 29.Oa C 22Sa C
16+8 168 + 35 106+23 132+31 422 & 102
0 108k46 850 28+8 144+45
0 75f8 1016 22 + 9 107k42 0 98227 20+ II 74+ 19 192 +42
5k3 151 f32 65+21 85 + 30 306_+86 15+6 2CQ+45 160+_21 87k21 462 _t 15
Field
eO.05.
0.03a A O.OOaA 0.03a A 0.03a B 0.03a A
38.8 39.5 38.8 37.8 30.6
1O.OaB 9.9a B I3.2a B 9.9a B
7.9a B 8.laB 8.0a B 8.IaB 8.ia B
41.4 42.3 46.7 44.8 44.5
12.0aB
0.02aA
0.03a 0.02a 0.02a 0.04a
B A A A
43.7 50.6 48.2 46.4 48.2
A A A A A
Saturation (%)
3.9a 3.8a 3.0a 3.8a 3.5a
PO,@ tPRg-‘)
O.OIaA O.Ola A 0.02a A O.OlaA 0.02a A
Total’ N (%)
RW
II +7 I63 +27 46rt16 98 + 19 318 +77
-I___ Bioassay
and NH,+ determined using autoanalyzer.
13.9a AB 7.IaA 41.8b B 63.9b B 105.8~ B
8.9a A 9.6a A 9.2a A 9.9a A 9.6a A
NH,-N4 (&?a-‘)
Mean number of spores 100 g soil-’ + standard deviation IRW:2W 2RW:lW _ --___ Bioassay Field Bioassay FieId Bioassay
Table 2. VA spore populations
9.3aB 11.8bA 9.3a A 9.3a B 7.4a B
I5.4a A 17.9a A 36.7b A 43.3b A 44.2b A
16.2a A0 24.la A 18.9aA 27Sa AB 27.3a AB
NO,-N4 (PRg-‘f
HCI added per 20mI solution. NO;
9.0a bA IO&A 8.IbB 16.5~ AB 9.6abAB
7SaA 9.laA i 5.9bcA 20.9~ 8 23.Obc B
9.9a A IO.laA 15.7aAB 12.3a A 8.4a A
Na’ (m-equiv I-‘)
‘Each reported analysis is an average of five subsamples. PH and electrical conductivity ofpaste and saturation extract, respectively (USDA, 1954). ‘Soluble cations according to USDA (1954). Final analysis by inductively coupled plasma. ‘5g soil (wet weight) shaken in 2~ KCI. Flasks sealed and allowed to settle 12 h. One drop Ultrex concentrated ‘Jones (1971). bOIsen and Sommers (1982). ‘Numbers within each sampling period not followed by the same lower case letter are statistically different at P ‘Numbers within each treatment not followed by the same upper case letter are statistically different at P ~0.05.
June 1978’ Control Water (W) IRW:ZW 2RW:IW Retort Water (RW) Post-treatment September 1978 Control W IRW:ZW 2RW: IW RW Post-treatment September 1982 Control W IRW:ZW 2RWIW RW
pH2 (paste)
Table I. Pretreatment and two most-treatment soil analvses from exoerimental olots from near Rock Snrinas. Wvominn. See Materials and Methods for exolanation of labels
PETER D. STAHL and S. E. WILLIAMS
454
offcinalis, although
Table 3. VA infection levels in roots from field plots and bioassay’. See Materials and Methods for explanation of labels
Oryzopsis roots Treatment
in field
Control W IRW:ZW 2RW:lW RW
89 +_8a2 91 +_8a 64+31b 38+26c 36+2Oc
Melilorus roots in bioassay 12 +_22a 67 _t 19a 64 + 22ab 49 + 24b 54 + 19b
‘Values indicate percentage of observed 1 mm root segments inhabited by VA fungi. 2Numerical entries within each column not followed by the same letter are statistically different at P < 0.05.
carpum and E. infreqwens were sensitive to the retort water while G. fasciculatwm appeared to be more tolerant and G. mosseae spores actually increased in density at the treated sites. At the field study site, absolute densities of G. fasciculatum and G. mosseae were much less changed by retort water-treatment than were the densities of G. microcarpum and E. injiiequens, which were lowered significantly. The response of G. mosseae to both retort watertreatment and the bioassay is interesting and unique. This species was recovered in similar absolute densities (17-28 spores per 100 g soil) from all field soil treatments except lRW:2W where it had substantially higher density (74 spores per 100 g soil) and it increased in all bioassayed soils. G. mosseae may have benefited from the lack of competition from the sensitive species in the retort water-treated soils. Daft and Nicolson (1974) investigated VA mycorrhizas of plants colonizing coal waste in Scotland and report an interesting distribution of the three endophytes present. These authors found an endophyte with narrow hyphae to be more prevalent on the most recently colonized spoils but it was gradually replaced by other, more typical, VA mycorrhizal endophytes as plant colonization progressed (and, presumably, soil conditions improved). The substantial changes in spore populations in retort water-treated soils during the bioassay suggest that these soils may lose some of their inhibitive properties as the bioassay progresses. It is possible that the biweekly watering of plants caused leaching of toxic compounds through the soil. This is supported by the doubling and tripling of total spore numbers and increases in the densities of the sensitive species G. microcarpum and E. infrequens in the retort water-treated soils during the bioassay. No leguminous plants were found growing at any of the contaminated field plots 4 yr after treatment. Nevertheless, soil from these sites was shown to support Rhizobium-induced root nodulation of M.
it does appear that nodulation was suppressed in process water treated soils. When nodulation is expressed as dry weight nodules produced g-’ dry weight root (Table 4) it is clear that less nodule tissue was formed on root systems grown in the retort water-treated soils. Nitrogenase activity of the M. oficinalis-Rhizobium nodules, as determined by acetylene reduction, was significantly reduced only in soil treated with full-strength retort water. Hersman and Klein (1979) report that of all the soil microbiological characteristics they investigated nitrogen fixation (acetylene reduction) rate of soil was most affected by the presence of retorted oil shale. Although nodulation was inhibited in retorttreated soils in this study, lesser amounts of nodules were still produced and nitrogenase activity of these nodules was not affected in the IRW :2W and 2RW: IW soils. The symbiotic nitrogen-fixing Rhizobium in root nodules may be more protected from the inhibitory agents in oil shale retort water than are the free-living N-fixers studied by Hersman and Klein. Despite the fact that all soil parameters monitored in this study have returned to original levels, results from the field and bioassay indicate that something is continuing to inhibit the activity of VA fungi, Rhizobium-nodulation and the growth of M. oficinalis in the more heavily treated soils. Because Omega-9 retort water is high in total dissolved organic carbon (Stuber and Leenheer, 1978), which include toxic compounds such as phenols, Nheterocyclics and polynuclear aromatic hydrocarbons (Hilpert et al., 1979) that were not tested for by the soil analysis used in this study, it is possible these compounds are at least partly responsible for the persistent inhibitory effects of the 2RW: 1W and RW soils. Acknowledgements-Published
with approval of the Directar of the Wyoming Agricultural Experiment Station, UniVersitY of Wyoming, Laramie, WY 82071 as Journal Series Number JA 1373. This work was supported, in part, by a grant from the United States Department of Energy. REFERENCES
Allen E. B. and Allen M. F. (1980)Natural re-establishment of vesicular-arbuscular mycorrhizae following stripmine reclamation in Wyoming. Journal of Applied Ecology 17, 139-147. Allen M. F., Moore T. S. Jr, Christensen M. and Stanton N. (1979) Growth of vesicular-arbuscular mycorrhizal and non-mycorrhizal Bouieloua gracilis in a defined medium. Mycologia 71, 666-669.
Bergman H. L., DeGraeve G. M., Anderson A. D. and Farrier D. S. (1978) Effects of complex effluents from
Table 4. Results of bioassay. See Materials and Methods for explanation of labels Dry weight Treatment Control W lRW:2W 2RW:IW RW
Shoots (g) 1.16+0.78a’ 1.05 * 0.70a 1.06 I&0.72a 0.78 + 0.57b 0.68 k 0.53b
Roots (9) 0.50 0.53 0.44 0.43 0.36
_+0.30ab *0.29a k 0.28b +_0.29bc i 0.23~
Nodules (mg) 10.1 *7a 10.0 * 7a 6.9 f 4b 4.2 + 4bc 3.7 * 3c
Dry wt nodules (mg) g-’ dry roots 9 9 7 5 5
Moles C,H, produced mg-’ dry nodules h-’ 0.29 + 0.17a 0.33 +0.18a 0.27 + 0.18a 0.30 + 0.21a 0.15 +0.13b
‘Numerical entries within each column not followed by the same letter are statistically different at P .c 0.05.
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Sampling, Analysis and Quality Assurance (C. Gale, Ed.) pp. 546-554. Kuchier A. W. (1964) Potential natural vegetation of the conterminous United States (map and manual). American Geographic Society Special ~~lication No. 36. 116 pp. Lcbsack M. E., Anderson A. D., Nelson K. F. and Farrier D. S. (1979) Sub-lethal effects of an in-&u oil shale retort water on rainbow trout. Toxicology and Applied Pharmacology 54, 462-468. Olsen S. R. and Sommers L. E. (1982) Phosphorus soluble in sodium bicarbonate. In Methods of Soil Analysis, Part 2 (A. G. Page, Ed.), pp. 403430. American Society of A~onomy, Madison. Rao T. K., Epler J. L., Guerin M. R., Schmidt-Collerus J. J. and Leffler L. (1979) Biological monitoring of oil shale products and effluents using short-term genetic analysis. In Oil Shale Symposium: Sampling, Analysis and Qualify Assurance (C. Gale, Ed.), pp. 431-442. U.S. Environmental Protection Agency, EPA-6OOj9-80-022. Schwab S. and Reeves F. B. (1979) The effect of retorted oil shale on VA mycorrhiza formation in soil from the Piceance Basin of northwestern Colorado. In Oil Shale Symposium: Sampling, Analysis and Quality Assurance (C. Gale, Ed.), pp. 566-576. U.S. Environmental Protection Agency, EPA-600/9-80-022. Scott A. (1922) On the occupationa cancer of the paraffin and oil workers of the Scottish shale industry. British Medical Journal 2, 1108-l 109. Skinner Q. D., Moore T. S. Jr, Asphmd R. O., Sexton J. C. and Farrier D. S. (1979) Plant responses to aqueous effluents derived from in-situ fuel processing. I. Development of screening methods. Laramie Energy Technology Center Publication No. LETC/R-I-79/4. 25 pp. Stahl P. D. and Christensen M. (1982) Mycorrhizal fungi associated with Bou~eloua and Agropyron in Wyoming ~gebrush-grasslands. ~ycolog~a 74, 877-885. Stuber H. A. and Leenheer J. A. (1978) Fractionation of organic solutes in oil shale retort waters for sorption studies on processed shale. American Chemical Society, Division of Fuel Chemistry 23, 165-174. U.S. Department of Agriculture (1954) Diagnosis and improvement of saline and alkali soifs. Agriculture Handbook No, 60 (L. A. Richards, Ed.), U.S. Salinity Laboratory, Riverside, California.