influence of organic and inorganic soil amendments on plant growth in crude oil‐contaminated soil
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Influence of organic and inorganic soil amendments onplant growth in crude oil‐contaminated soilP. M. White Jr. a , D. C. Wolf b , G. J. Thoma c & C. M. Reynolds da Department of Agronomy, 2004 Throckmorton Plant Sciences Center , Kansas StateUniversity , Manhattan, KS, 66506 Phone: 785–532–7106 Fax: 785–532–7106 E-mail:b Department of Crop, Soil, and Environmental Sciences, Plant Science 115 , University ofArkansas , Fayetteville, AR, 72701 Phone: 479–575–5739 Fax: 479–575–5739 E-mail:c Department of Chemical Engineering, Bell 3202 , University of Arkansas , Fayetteville, AR,72701 Phone: 479–575–4951 Fax: 479–575–4951 E-mail:d USACE Engineer Research and Development Center 72 Lyme Road , Cold RegionsResearch and Engineering Laboratory , Hanover, NH, 03755, USA Phone: 603–646–4394 Fax:603–646–4394 E-mail:Published online: 22 Sep 2008.
To cite this article: P. M. White Jr. , D. C. Wolf , G. J. Thoma & C. M. Reynolds (2003) Influence of organic and inorganic soilamendments on plant growth in crude oil‐contaminated soil, International Journal of Phytoremediation, 5:4, 381-397, DOI:10.1080/15226510309359044
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International Journal of Phytoremediation, 5(4):381-397 (2003)Copyright © 2003 Taylor and Francis Inc.ISSN: 1522-6514DOL 10.1080/16226510390268775
Influence of Organic and Inorganic Soil Amendmentson Plant Growth in Crude Oil-Contaminated Soil
P. M. White, Jr.,1 D. C. Wolf,2 G. J. Thoma,3 and C. M. Reynolds4
1 Department of Agronomy, Kansas State University, 2004 Throckmorton PlantSciences Center, Manhattan, KS 66506. Phone: 785-532-7106; Fax:785-532-6094; E-mail: [email protected]; 2Department of Crop, Soil, andEnvironmental Sciences, University of Arkansas, Plant Science 115, Fayetteville,AR 72701. Phone: 479-575-5739; Fax: 479-575-7465; E-mail:[email protected]; 3 Department of Chemical Engineering, University of Arkansas,Bell 3202, Fayetteville, AR 72701. Phone: 479-575-4951; Fax:479-575-7926; E-mail: [email protected]; 4USACE Engineer Research andDevelopment Center, Cold Regions Research and Engineering Laboratory, 72Lyme Road, Hanover, NH, USA 03755. Phone: 603-646-4394; Fax:603-646-4561 ; E-mail: [email protected]
ABSTRACT
Phytoremediation can be a viable alternative to traditional, more costly remedia-tion techniques. Three greenhouse studies were conducted to evaluate plant growthwith different soil amendments in crude oil-contaminated soil. Growth of alfalfa(Medicago sativa L., cultivar: Riley), bermudagrass (Cynodon dactylon L., cultivar:Common), crabgrass (Digitaria sanguinalis, cultivar: Large), fescue (Lolium arun-dinaceum Schreb., cultivar: Kentucky 31), and ryegrass (Lolium multiflorum Lam.,cultivar: Marshall) was determined in crude oil-contaminated soil amended with eitherinorganic fertilizer, hardwood sawdust, papermill sludge, broiler litter or unamended(control). In the first study, the addition of broiler litter reduced seed germination forryegrass, fescue, and alfalfa. In the second study, bermudagrass grown in broiler litter-amended soil produced the most shoot biomass, bermudagrass produced the most rootbiomass, and crabgrass and bermudagrass produced the most root length. In the thirdstudy, soil amended with broiler litter resulted in the greatest reduction in gravimetrictotal petroleum hydrocarbon (TPH) levels across the six plant treatments follow-ing the 14-wk study. Ryegrass produced more root biomass than any other specieswhen grown in inorganic fertilizer- or hardwood sawdust + inorganic fertilizer-amended soil. The studies demonstrated that soil amendments and plant species selec-tion were important considerations for phytoremediation of crude oil-contaminatedsoil.
KEY WORDS: phytoremediation, TPH, petroleum, broiler litter.
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I. INTRODUCTIONUsing phytoremediation to enhance petroleum hydrocarbon biodégradation can be
a cost effective, low-maintenance, environmentally acceptable method of remediat-ing crude oil-contaminated soil (Cunningham et al, 1996). The rhizosphere, or soilunder the influence of the plant root, often contains microbial populations that are5 to 100 times greater than numbers in non-rhizosphere bulk soil (Atlas and Bartha,1992). Additionally, the microbial populations in the rhizosphere can contain greaternumbers of hydrocarbon dégrader microorganisms. Nichols et al. (1997) found thatbacterial populations in general and hydrocarbon dégrader populations specificallywere stimulated by plant growth and by the addition of a mixture of organic contam-inants to the soil.
Successful phytoremediation first requires plant establishment in crude oil-contaminated soil. However, oil can pass through a seed coat and/or coat the seedrestricting H2O and O2 uptake, thereby killing the seed embryo (Baker, 1970). Lowmolecular weight aromatic compounds, such as naphthalene, present in crude oil canbe especially toxic to plants (Baker, 1970). Amadi et.al. (1993) found that 3% (v/w)crude oil significantly decreased maize germination in an agricultural soil and Plice(1948) found germination of seed was prevented when 2.4% (v/v) oil was mixed withsoil to a depth of 10 cm. Brown et al. (1982) found ryegrass (Lolium multiflorumLern.) seed emergence to be significantly reduced in a soil contaminated with 20%(w/w) petrochemical sludge with 9.1% aromatic compounds (w/w) as compared to auncontaminated control soil. However, alfalfa (Medicago sativa L.) seeds germinatedin soil containing 5% (w/w) crude oil (Wiltse et al, 1998). After fresh crude oil spills,processes such as adsorption and volatilization can result in reduction of volatile totalpetroleum hydrocarbon (TPH) components that can be toxic to seeds (Rogers et al.,1996).
Once established, plants must survive and grow for phytoremediation to be ef-fective. Grass species have been suggested as effective plants for phytoremediatingpetroleum-contaminated soils (Aprill and Sims, 1990). Grasses have fibrous root sys-tems with large root length and surface area measurements per unit volume of soil andmost roots are near the soil surface. The fibrous roots provide a larger surface for col-onization by soil microorganisms than a taproot (Anderson etal, 1993) and allow formore interaction between the rhizosphere microbial community and the contaminant(Schwab and Banks, 1994). A conceptual model proposed by Thoma et al. (2003a,b)shows that growing plant roots and their associated microbial community potentiallyincrease biodégradation rates for immobile contaminants such as crude oil. Since Ncan be a limiting nutrient in petroleum-contaminated soils, using leguminous plantsmay be advantageous due to their biological nitrogen fixation capability. Additionally,microbial biomass may increase as a result of increased soil N around legume roots(Anderson etal, 1993).
In order to extend the active period of phytoremediation during a growing sea-son, both warm- and cool-season plant species that germinate and grow in crudeoil-contaminated soil should be identified. While ryegrass has been widely grownin hydrocarbon-contaminated soils (Günther et al, 1996; Hutchinson et al, 2001;Olexa et al, 2000; Reynolds et al., 1999), many other warm- and cool-season grasseshave shown the ability to germinate and grow in hydrocarbon-contaminated soils
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Influence of Organic and Inorganic Soil Amendments
(Anderson et al, 1993; Aprill and Sims, 1990; Kulakow et al, 2000; Rogers et al,1996). However, temperature and moisture regimes along with soil physical andchemical parameters should be taken into account when choosing a specific plant forphytoremediation of a particular crude oil-contaminated soil.
Typically, crude oil-contaminated soils are hydrophobic. The growth of plant rootswould accentuate water infiltration by increasing macropore volume in the root zone.Molecular oxygen is important for rapid mineralization of petroleum hydrocarbonsand is not present in reduced or anaerobic conditions. Tillage and the use of bulkingagents such as sawdust and papermill sludge are often employed in land farmingoperations to promote adequate aeration of soil to increase oil biodégradation rates(Rhykerd et al, 1999). Papermill sludges are also added to soil to provide plants withnutrients (Dolar et al, 1972).
Three greenhouse studies investigated selection of plant species and soil amend-ments to be used for phytoremediation field projects. The objective of the first studywas to evaluate germination of five plant species in crude oil-contaminated soil ei-ther unamended or amended with inorganic fertilizer, broiler litter, papermill sludge,or hardwood sawdust. The objective of the second study was to evaluate survivaland growth of five plant species in crude oil-contaminated soil either unamended oramended with the materials above and all treatments covered with a 2.5-cm layer ofuncontaminated soil. The objective of the third study was to evaluate changes in TPHconcentrations and plant survival and growth in crude oil-contaminated soil eitherunamended or amended with inorganic fertilizer, broiler litter, papermill sludge, orhardwood sawdust + inorganic fertilizer.
II. MATERIALS AND METHODS
A. First Study
The crude oil-contaminated soil was collected from a storage tank area near ElDorado, AR where an oil spill occurred approximately 4 years earlier. The oil spilloccurred in an area that had not been contaminated with brine from the drillingoperation. The soil was sieved through a 2-mm stainless steel sieve and extensivelymixed using a cement mixer. A composite sample was collected and the plant availablenutrient content was determined using Mehlich 3 extradant and ICP analysis (Table 1)(Donahue, 1992). Total C and N were determined with a Leco CN 2000®. The pH andelectrical conductivity measurements were at 1:1 and 1:2 soil to water ratios (w/w),respectively.
Gravimetric total petroleum hydrocarbon (TPH) levels in the contaminated soilwere determined by adding ten grams anhydrous sodium sulfate (drying agent) to10 g soil and extracting the mixture with 100 mL méthylène chloride using a mod-ified version of the standard sonication method for extraction of organic chemicals(Sawhrey, 1996). The soil-solvent mixture was sonicated at a pulse rate of one sec-ond on, one second off, with a setting of 10 for 1.5 minutes with a Fisher Scientific550 Sonic Dismembrator™. Once the suspended particles present in the mixture hadsettled, the supernatant liquid was decanted through approximately 10 g of sodiumsulfate placed on #41 filter paper in a glass funnel. The extraction procedure wasrepeated three times and extracts combined. The méthylène chloride was removed
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TABLE 1. Chemical properties of the crude oil-contaminated and uncontaminated soils usedin the three greenhouse studies.
Total Mehlich 3 Extractable
Grav.Soil pH EC P K Ca Mg Na S Zn Cu C N TPH
1:1 dS/m% mg/kg
First and second greenhouse studiesCrude oil-contaminated 5.9 0.034 2 16 140 12 20 4 1.5 0.6 10.2 0.15 7.90Captina silt loam 6.1 0.048 19 59 533 30 5 10 1.0 0.9 1.2 0.06 nd
(uncontaminated soil)Third greenhouse study
Crude oil-contaminated 5.2 0.046 3 30 267 32 46 7 1.1 0.8 9.4 0.07 8.08
and the TPH level was determined on a gravimetric basis. Duplicate samples wereanalyzed and relative percent difference was maintained at less than 20%. Appropriateblanks were also included.
The soil amendments evaluated were inorganic fertilizer, broiler litter, papermillsludge, and hardwood sawdust. The amendment rates and chemical analyses aregiven in Table 2. The inorganic fertilizer (13-13-13) amendment rate was 90 kg/hafor N, P2O5, and KiO based upon soil test recommendations for pasture and forageestablishment (Chapman, 1999). Equal volumes of broiler litter, hardwood sawdust,or papermill sludge were uniformly mixed into the soil to normalize bulk density ofthe amended soil mixtures. The amendment volume was equivalent to incorporatinga 2.54-cm surface layer of the amendment into the soil to a depth of 20 cm. Broilerlitter was obtained from the Savoy Research Station near Fayetteville, AR, collectedafter five flock cycles over a bedding material of rice (Oryza L.) hulls. The papermillsludge was secondary biosolids and exhibited a typical C/N ratio for such materials(Dolar et al., 1972). The hardwood sawdust was derived from oak (Quercus sp.).
Four hundred grams of dry weight equivalent crude oil-contaminated soil wasmixed by hand with the appropriate amount of inorganic fertilizer, broiler litter, pa-permill sludge, or hardwood sawdust and added to polypropylene cone-tainers™.The cone-tainers™ had a 6.35-cm diameter at the top, 25.4-cm length, and 554-mLtotal volume. The bottom of each cone-tainer™ had 4 openings to allow drainage ofexcess water. Pyrex glass wool was placed in the bottom of each cone-tainer™ toprevent soil loss while allowing adequate drainage.
The plant species evaluated were alfalfa (Medicago sativa L., cultivar: Riley),bermudagrass (Cynodon dactylon L., cultivar: Common), crabgrass (Digitaria san-guinalis, cultivar: Large), fescue {Lolium arundinaceum Schreb., cultivar: Kentucky31), and ryegrass {Lolium multiflorum Lam., cultivar: Marshall).
Seed germination was measured by placing 10 seeds of each plant species in eachof four replications (40 seeds total) in appropriate cone-tainers™ at a depth of 0.5cm and counting the number of seeds that exhibited visible shoots within 2 wk. Seedgermination was also determined in the absence of contaminated soil by incubating
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TABLE 2. Application rates and nutrient contents of organic and inorganic soil amendments used in the greenhousestudies.
Amendment
Inorganic fertilizerBroiler litterPapermill sludgeHardwood sawdust
Inorganic fertilizerBroiler litterPapermill sludgeHardwood sawdust+In. lert.
Density*g/cm3
na0.430.620.18
na0.380.460.24na
g/kg"
0.3571.555.826.4
4.437.917.223.6
+4.4
Rate
cm37kg"Moisture
content* (%)
Total (%**)
P
First and second greenhouse studiesna
200200200
na18.255.429.5
Third greenhouse studyna160160160na
na36.976.337.2na
5.71.70.10.0
5.71.70.10.0
+5.7
K
10.82.80.00.1
10.82.80.20.1
+ 10.8
Total (
N
13.04.20.60.1
13.04.23.30.1
+13.0
%**)
C
0.034.224.144.9
0.034.233.044.9+0.0
TPHC:TN ratio*
148020
2002540
12040
120120
'Values on wet weight basis."Values on a dry weight basis.'Ratio of (olal petroleum hydrocarbon carbon: Total nitrogen added in the amendment on a dry weight basis.na = not applicable.
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40 seeds of each plant species on filter paper saturated with deionized water in sterilepetri dishes. The germinated seeds were counted at 1 wk. No supplemental lightingwas used during the spring greenhouse study where temperatures ranged from 21°Cto 30°C. Plants were watered daily by hand using tap water.
The first greenhouse study was a randomized complete block design with fourreplications. The analysis of variance was conducted with SAS software (SASInstitute, Cary, N.C.). Means were separated using a LSD with p = 0.05 level ofsignificance.
B. Second Study
For the second greenhouse study, all treatments used in the first greenhouse studywere covered with 2.54 cm of uncontaminated Captina silt loam (fine-silty, siliceous,mesic Typic Fragiudults) that was collected from the University of Arkansas MainAgricultural Experiment Station farm. The Captina soil was sieved through a 2-mmstainless steel sieve and soil chemical properties analyzed as previously described(Table 1).
Bermudagrass (Cynodon dactylon L. cultivan Tifway) springs were separated fromsod obtained from Razorback Sod and Turf in Robinson, AR. Two sprigs were washedwith distilled water and blotted dry, then transplanted into each cone-tainer™. Tenseeds of alfalfa, crabgrass, fescue, or ryegrass were placed into appropriately labeledcone-tainers™.
Upon completion of seeding and sprigging, all cone-tainers™ were watered withtap water via a sprinkler system. No supplemental lighting was used during the summergreenhouse study where temperatures ranged from 22°C to 29°C.
The plants were harvested after 7 wk. The number of individual plants survivingin each cone-tainer™ was recorded, the shoots were cut at the soil surface, washed,and dried at 60°C to a constant weight. The roots were removed and attached soil waswashed off with distilled water. The roots were blotted dry and the fresh root weightdetermined.
The fresh roots were stained with 0.1 g méthylène blue/L 10% ethanôl (95%)(v/v) and stored at 4°C until root length analysis. Root length was determinedby a line intersect method (Tennant, 1975). The roots were placed in a 30-cm x20-cm x 5-cm transparent dish that was placed over a grid of 1.0-cm x 1.0-cmsquares. With the aid of a magnifying glass, counts of root intersections with the gridwere taken to obtain root length. The roots were then dried to a constant weight at60°C.
Following the 7-wk greenhouse study, the crude oil-contaminated soil remain-ing in the cone-tainers™ was analyzed for soil chemical properties as previouslydescribed. Due to the short duration of the experiment and assumed limited plantimpact on soil chemical properties, the soil was combined across plant species foranalysis.
The second greenhouse study was a randomized complete block design withthree replications. The analysis of variance was conducted with SAS software (SASInstitute, Cary, N.C.). Means were separated using a LSD with p = 0.05 level ofsignificance.
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C. Third Study
A different crude oil-contaminated soil was collected from within a similar bermedstorage tank area near El Dorado, AR. The soil was sieved through a 2-mm stainlesssteel sieve and thoroughly mixed using a cement mixer. A composite sample wascollected and soil chemical properties determined as previously described (Table 1).Soil TPH levels were measured gravimetrically by EPA method 3540C (USEPA,1998). Briefly, approximately 10 g soil was mixed with 10 g anhydrous sodiumsulfate and extracted with 300 mL of méthylène chloride using a soxhlet extractor for18 h. The extracts were allowed to dry under forced ventilation in pre-tared beakersand the dry extracts were then weighed.
Alfalfa, bermudagrass, crabgrass, fescue, and ryegrass were grown with or with-out papermill sludge, hardwood sawdust + inorganic fertilizer (13-13-13), inorganicfertilizer, or broiler litter as amendments. The application rates and chemical anal-yses of each amendment are given in Table 2. Due to the high C content in crudeoil (85%), soils contaminated with high concentrations of crude oil typically havehigh C:N ratios. As a result, soil N is immobilized by microbes decomposing theoil. Due to this immobilization, more effective prediction of N needs during phytore-mediation could be made by basing N addition rates on TPH-C levels in the soil.Broiler litter was added at a rate to yield a 40:1 TPH-C:Total added N (TN) ratio.Papermill sludge and hardwood sawdust were added at volumetric rates equivalentto the broiler litter to normalize the effect of bulking agents. The inorganic fertilizer(13-13-13) amendment rate was adjusted to be equivalent to the total N containedin the papermill sludge amendment. Inorganic fertilizer was also added with thehardwood sawdust to provide a TPH-C:TN ratio of 120. The papermill sludge ex-hibited a low C/N ratio atypical for such materials because it was primary biosolidsand not composted (Dolar et al, 1972). The hardwood sawdust was derived fromoak.
For each cone-tainer™, 400 g dry weight equivalent of crude oil-contaminatedsoil was uniformly mixed with the appropriate amount of inorganic fertilizer, broilerlitter, papermill sludge, or hardwood sawdust -f inorganic fertilizer and placed inpolypropylene cone-tainers™. Four bermudagrass sprigs or 10 alfalfa, crabgrass,fescue, or ryegrass seeds were planted into appropriately labeled cone-tainers™.Percent plant survival at 11 d after seedling emergence was recorded for each plantspecies after which time the cone-tainers™ were thinned to 4 plants. Upon completionof seeding and sprigging, all cone-tainers™ received tap water via a sprinkler system.No supplemental lighting was used during the fall study where temperatures rangedfroml8°Cto29DC.
The plants were harvested 14 wk after planting. The shoots were cut at the soilsurface and dried at 60"C to a constant weight. Roots were removed from the soil,washed with distilled water, and dried to a constant weight at 60cC. Remaining soilsamples were then combined by soil amendment across plant species and analyzedfor plant nutrients and TPH as described in this section.
The study design was a randomized complete block design with four replications.The analysis of variance was conducted with SAS software (SAS Institute, Cary,N.C.). Means were separated using a LSD with p = 0.05 level of significance.
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III. RESULTS AND DISCUSSION
A. First Study
7. Initial Soil and Amendment Chemical Analysis
Soil used in the greenhouse study had a gravimetric TPH level of 7.90 ± 0.47% andlow levels of plant available nutrients (Table 1). The C/N ratio of the oil-contaminatedsoil was approximately an order of magnitude greater than typical uncontaminatedsoils. The soil pH was slightly acidic, but not unacceptable for plant growth. Thelow Na and low electrical conductivity (EC) of the soil indicated that salt brinecontamination of the soil was not a major concern as saline soils are defined by an ECvalue of >4 dS/m (U.S. Salinity Laboratory, 1954). Analysis of the papermill sludgeand broiler litter amendments (Table 2) showed that the nutrient levels were similar toreported values for such materials (Dolar et ai, 1972; Sims and Wolf, 1994; Zibilske,1987).
2. Seed Germination
Percentage germination for the seeds in water was >93% (Table 3). Seed germi-nation percentages in the crude oil-contaminated soil with or without amendmentsshowed that the plant by treatment interaction was significant. Bermudagrass germi-nation was significantly lower than alfalfa, crabgrass, fescue, or ryegrass when grownin the unamended control soil and soil amended with papermill sludge or hardwoodsawdust (Table 3). Additionally, all plants except bermudagrass had >75% germina-tion in the uamended control, papermill sludge-, inorganic fertilizer-, and hardwoodsawdust-amended soil. Germination in broiler litter-amended soil for alfalfa, fescue,and ryegrass was lower than with any other soil amendment including the unamendedcontrol. Germination of crabgrass in broiler litter-amended soil was lower than inpapermill sludge-, inorganic fertilizer-, or hardwood sawdust-amended soil, but notthe unamended control soil. Uric acid in the broiler litter was most likely hydrolyzedto urea, which in turn was hydrolyzed to ammoniacal-N (Sims and Wolf, 1994). Urea
TABLE 3. Seed germination in crude oil-contaminated soil either unamended or amendedwith inorganic or organic materials in the first greenhouse study. The plant speciesby amendment interaction was significant.
Germination (%)
Plant species
AlfalfaBermudagrassCrabgrassFescueRyegrass
Water
98*1009395
100
Unamendedcontrol
77abcde~30h75bcde95ab90ab
Papermillsludge
93ab60cdef90ab93ab
100a
Inorganicfertilizer
83abc58def80abcd95ab
100a
Hardwoodsawdust
90ab30h90ab90ab97ab
Broilerlitter
33gh45fgh55efg55efg43fgh
'Germination in water was a seed viability test and was not included in the statistical analysis."Seed germination means followed by the same letter are not significantly different at the p = 0.05 level.
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hydrolysis and the concomitant soil pH increase in microsites surrounding seedlingscould result in NH3 levels that were toxic to seedlings (Goyal and Huffaker, 1984).The NH3 toxicity problem could be avoided by adding the broiler litter and waitingapproximately 2 to 3 weeks before planting.
B. Second Study
1. Plant Survival
Due to the negative effect of broiler litter on seed germination for several plantspecies evaluated, the crude oil-contaminated soil used for the first greenhouse studywas covered with a 2.54-cm layer (50 g dry weight) of uncontaminated Captinasilt loam soil to provided a more suitable environment for seed germination andearly growth. Similarly, Günther et al. (1996) used an uncontaminated soil layer toreduce the toxic effects of a hydrocarbon-contaminated soil on early plant growthand reported that ryegrass {Lolium perenne L.) grown in the layered soil producedthe same shoot dry biomass as when grown in a non-hydrocarbon-contaminated soil.By covering the crude oil-contaminated soil with uncontaminated Captina soil, thegermination and early growth of plants would be less affected by the crude oil-contaminated soil and soil amendments, and allow for better evaluation of longer-termplant growth.
Because of the low germination in soil of bermudagrass seed in the first greenhousestudy, bermudagrass sprigs were used for the second greenhouse study and were notincluded in the 7-wk plant survival analysis. Fescue and ryegrass had significantlyhigher 7-wk survival percentages than alfalfa and crabgrass (Table 4). None of the soilamendments affected plant survival differently than the unamended control (Table 5).While the broiler litter-amended soil exhibited lower germination in the first study,no impacts on 7-wk plant survival were observed in the second study, most likely dueto the uncontaminated Captina silt loam layer acting as a buffer.
TABLE 4. The main effect of plant species on percentage plant sur-vival, root biomass, and root length in the second green-house study. The cone-tainers™ had uncontaminated soilcovering the crude oil-contaminated soil either unamendedor amended with inorganic or organic materials.
Plant species
AlfalfaBermudagrassCrabgrassFescueRyegrass
Survival(%)
56b*100**
61b81a85a
Root biomassmg/plant
3c39a14b4c
9bc
Root lengthcm/plant
99b623a774a147b328b
'Means in a column followed by the same letter are not significantly different atthe p = 0.05 level.**Bermudagrass was transplanted and thus not included in the survival statisticalanalysis.
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TABLE 5. The main effect of soil amendment on percentage plantsurvival, root biomass, and root length in the second green-house study. The cone-tainers™ had uncontaminated soilcovering the crude oil-contaminated soil either unamendedor amended with inorganic or organic materials.
Soil amendment
Unamended controlPapennill sludgeInorganic fertilizerHardwood sawdustBroiler litter
Survival(%)
63a*78a"74a77a63a
Root biomassmg/plant
6c18ab13bc6c
25a
Root lengthcm/plant
179c679a327bc200c586ab
'Means in a column followed by the same letter are not significanlty differentat the p = 0.05 level.** Bermudagrass was transplanted and thus not included in the survival statisticalanalysis.
2. Soil Chemical Analysis
Chemical analysis of soil samples collected from the contaminated soil indicatedthat the addition of broiler litter significantly increased the total N and plant availableP in the crude oil-contaminated soil compared to the other amendments (data notshown). Hardwood sawdust did not affect soil nutrient levels during the greenhousestudy. Values for EC were #0.249 dS/m and pH values ranged from 5.4 to 5.9.
3. Plant Shoot Growth
For shoot biomass production, the plant species by soil amendment interaction wassignificant (Table 6). Bermudagrass grown in broiler litter-amended soil produced the
TABLE 6. Shoot biomass production in crude oil-contaminated soil either una-mended or amended with inorganic or organic materials in the secondgreenhouse study. The plant species by amendment interaction wassignificant.
Plant species
AlfalfaBermudagrassCrabgrassFescueRyegrass
Unamendedcontrol
0c*26c12c4c7c
mg biomass/plant
Papennillsludge
9c88b86b
5c16c
Inorganicfertilizer
4c63bc12c0c8c
Hardwoodsawdust
6c33bc13c4c6c
Broilerlitter
6c191a88b14c48bc
*Plant dry shoot biomass means followed by the same letter are not significantly differentat the p = 0.05.
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highest shoot biomass compared to other plant species and soil amendment combina-tions. Bermudagrass and crabgrass both produced more shoot biomass when grownin broiler litter-amended soil than when grown in hardwood sawdust- or inorganicfertilizer-amended soil, or the unamended control soil. The increased shoot biomassproduction for the bermudagrass grown in broiler litter-amended soil could be theresult of the higher levels of plant available nutrients present and the effects of sprig-ging versus seeding. Bermudagrass and crabgrass also produced more shoot biomassthan any other plant species when grown in papermill sludge-amended soil. Papermillsludges can be low in plant available nutrients, but may improve soil tilth (Zibilske,1987), defined as a soil's physical condition as it affects plant growth (Brady andWeil, 2002).
4. Plant Root Growth
. Plant species and soil amendment main effects were significant for dry root biomassyield. Bermudagrass produced more root dry biomass than any other plant speciesacross all soil amendments (Table 4), most likely due to the accelerated growth of thesprigs as compared to the seeds. Crabgrass produced more root biomass than fescueor alfalfa, but was not different than ryegrass. Broiler litter-amended soil producedmore root biomass than the unamended control, inorganic fertilizer, or hardwoodsawdust, likely due to increases in soil nutrients (Table 5). Papermill sludge-amendedsoil outperformed the unamended control and the hardwood sawdust-amended soilin terms of dry root biomass production possibly due to enhanced soil tilth character-istics, which can involve enhancing soil aeration, moisture content, and bulk densityto more optimum levels for plant growth (Brady and Weil, 2002). Of the total rootbiomass produced in the contaminated and uncontaminated layers combined, rootbiomass in the contaminated layer was 31, 33, 37, 58, and 64% for the inorganicfertilizer, hardwood sawdust, unamended control, papermill sludge, and broiler litter,respectively.
Root length can be important in determining the volume of rhizosphere soil. Asroots grow, they can bring active microbial populations into contact with a largerportion of contaminated soil, possibly resulting in faster degradation of crude oil.Plant species and soil amendment main effects were significant for root length. Rootlength values were significantly greater for bermudagrass and crabgrass across allsoil amendments (Table 4). Papermill sludge produced more root length than anyother soil amendment except broiler litter (Table 5). Broiler litter produced more rootlength than hardwood sawdust or the unamended control, but not inorganic fertilizer.
Amendment of the crude oil-contaminated soil with broiler litter and papermillsludge increased soil nutrient levels and most likely improved tilth. Both factorswould enhance plant growth. However, bermudagrass and crabgrass are warm- seasongrasses, therefore their growth was favored by the higher greenhouse temperatures.
C. Third Study
1. Plant Survival
Results from the 14-wk greenhouse study showed significant plant species andsoil amendment main effects. For the plant species main effect, fescue exhibited a
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TABLE 7. Plant survival 11 dafteremergence in soil containing 8.08% TPHby weight in the third greenhouse study.
Plant species
AlfalfaCrabgrassFescueRyegrass
Plant survival
(%)
53b*38c80a57b
Soil amendment
Unamended controlPapermill sludgeInorganic fertilizerHardwood sawdustBroiler litter
Plant survival(%)
72a64a65a63a21b
'Percentage plant survival means in a column followed by the same letter are notsignificantly different at the p = 0.05 level.
higher survival rate l i d after seedling emergence than alfalfa or ryegrass, whichhad higher survival rates than crabgrass (Table 7). Seed viability was determinedto be >85% for the 4 plant species evaluated. For the soil amendment main effect,broiler litter significantly reduced plant survival, but the papermill sludge, inorganicfertilizer, and hardwood sawdust + inorganic fertilizer were not different than theunamended control (Table 7). Similar to the first greenhouse experiment, ammoniatoxicity in microsites surrounding seedlings could have reduced the survival of plantsin broiler litter-amended soil.
2. Soil TPH Levels
The initial soil TPH value was 8.08 ± 0.01 % by weight and significant differencesin gravimetric TPH were measured following the 14-wk study. Broiler litter-amendedsoil had significantly lower gravimetric TPH compared to the other amended soilsand the unamended control after the 14-wk study (Table 8). The maximum dilutionof soil TPH for any treatment was <3.8% based upon amendment mass and wouldhave minimal influence on final TPH concentrations.
TABLE 8. Gravimetric TPH in crude oil-contaminated soil fol-lowing the third greenhouse experiment conducted for14 wk. Samples were combined across plant speciesfor each amendment prior to analysis.
Soil amendment
Unamended controlPapermill sludgeInorganic fertilizerHardwood sawdust 4- inorganic fertilizerBroiler litter
Gravimetric TPHconcentration (%)
7.88a*7.93a7.12b6.61c6.01d
"Gravimetric TPH means followed by the same letter are not significantlydifferent at the p = 0.05 level. Values reported are the mean of four replica-tions and the LSD to compare values = 0.26.
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Broiler litter-amended soil had higher levels of total N and plant available P thanany other treatment or the control. The effect of broiler litter on TPH level maybe explained by noting that, of the nutrients needed for microbial decomposition oforganic compounds, N is usually needed in the highest concentration (Alexander,1994). Also, Chang et al. (1996) found that CO2 evolution in a soil contaminatedwith 9% by weight crude oil was increased with the addition of N and P as opposedto N alone. Hardwood sawdust + inorganic fertilizer-amended soil had a lower TPHvalue than the inorganic fertilizer- and papermill sludge-amended and unamendedcontrol soil. Plant tissues such as the hardwood sawdust used in the experimenttypically contain mono- and polyphenolic compounds (Schubert, 1965), some ofwhich resemble the recalcitrant aromatic compounds present in crude oil. Thesephenolic compounds in the hardwood sawdust may have acted as analogs to morerecalcitrant crude oil compounds and increased the biodégradation rate of TPH. Plantproduced phenolic compounds were shown to be acceptable substitutes for biphenylas growth substrates for aPCB degrading bacterium, possibly leading to increases inthe rates of PCB degradation (Leigh et al, 2002). Inorganic fertilizer-amended soilhad lower TPH values than the papermill sludge-amended or unamended control soil,most likely due to the more available forms of N present in the fertilizer (inorganicvs. organic) and higher P values for the inorganic fertilizer-amended soil.
3. Soil Chemical Analysis
Soil chemical analysis following the third greenhouse study demonstrated theeffect of adding the soil amendments on plant available nutrients. Similar to thesecond greenhouse study, broiler litter-amended soil had significantly higher valuesfor total N and plant available P than any other soil amendment (data not shown).The hardwood sawdust + inorganic fertilizer- and inorganic fertilizer-amended soilhad higher values for P than the papermill sludge-amended or the unamended controlsoil. Soil salinity levels in all treatments were low with EC values for <0.075 dS/m.The pH values ranged from 4.9 to 6.3 following the study.
4. Plant Shoot Growth
For shoot dry biomass production, the plant by soil amendment interaction wassignificant (Table 9). Similar to the second greenhouse experiment, bermudagrassgrown in crude oil-contaminated soil amended with broiler litter produced signifi-cantly higher shoot biomass than any other plant-amendment combination. Ryegrassand crabgrass grown in broiler litter- or inorganic fertilizer-amended soil producedmore shoot biomass than any plant species grown in papermill sludge-amended orunamended control soil.
5. Plant Root Growth
For root dry biomass production, the plant by soil amendment interaction wassignificant (Table 10). Ryegrass grown in crude oil-contaminated soil amended withinorganic fertilizer or broiler litter produced significantly higher dry root biomassyields than alfalfa, bermudagrass, or crabgrass with any soil amendment. Ryegrassgrown in soil amended with hardwood sawdust + inorganic fertilizer, fescue and
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TABLE 9. Shoot biomass production in crude oil-contaminated soil either unamended oramended with inorganic or organic materials in the third greenhouse study. Theplant species by amendment interaction was significant
Plantspecies
AlfalfaBermudagrassCrabgrassFescueRyegrass
Unamendedcontrol
10c*l ie3d9c9c
Papermillsludge
7dl ie4d
12c33c
mg biomass/plant
Inorganic 1fertilizer
37c24c
113b17c77b
Hardwood sawdusi+in. fert.
16c15c35c15c64b
t Broilerlitter
nd179a91b62bc92b
'Plant shoot dry biomass means followed by the same letter are not significantly different at the p = 0.05level.
bermudagrass grown in soil amended with broiler litter, and crabgrass grown in soilamended with inorganic fertilizer also produced high yields. No plant species grownin papermill sludge-amended soil produced more root biomass than plants grown inthe unamended control soil.
6. Plant Growth and Soil Amendment Assessment
Plant growth in the unamended control soil was limited by low nutrient levels,especially N and P levels. In amended soils, a TPH-C:TN ratio of 120:1 should haveresulted in net N immobilization and minimal plant available N (Table 2). However,ryegrass and crabgrass grown in inorganic fertilizer-amended soil produced highyields. The positive plant response observed suggests that only a fraction of theTPH-C was bioavailable. The hardwood sawdust + inorganic fertilizer amendmentproduced low yields despite nutrient levels similar to those in the inorganic fertilizer-amended soil. However, at the end of the 14-wk study, the hardwood sawdust +
TABLE 10. Root biomass production in crude oil-contaminated soil either unamended oramended with inorganic or organic materials in the third greenhouse study. Theplant species by amendment interaction was significant.
Plantspecies
AlfalfaBermudagrassCrabgrassFescueRyegrass
Unamendedcontrol
llf*12fIf
13f14f
Papermillsludge
9f13f2f
18f34def
mg biomass/plant
Inorganic ]fertilizer
19f17f62bcd29def
108a
hardwood sawdusi+in. fert.
8f15f17f22ef90ab
: Broilerlitter
nd55bcde35def73abc
101a
•Root diy biomass means followed by the same letter are not significantly different at the p = 0.05 level.
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inorganic fertilizer-amended soil had lower TPH values than the inorganic fertilizer-or papermill sludge-amended soil or the unamended control. The nutrients present inthe hardwood sawdust + inorganic fertilizer amended soil may have been immobilizedby the soil microbes degrading the oil. Additionally, phenolic compounds releasedfrom the hardwood sawdust could have had a toxic effect on the plants.
In the third greenhouse study, the broiler litter- and the papermill sludge-amendedsoils had TPH-C:TN ratios of 40:1 and 120:1, respectively. Plants grown in broilerlitter-amended soil produced high yields, while plants grown in papermill sludge-amended soil produced low yields. This could be related to the net N mineralization ofthese two organic materials. Using published methods, the calculated plant available Nvalues (inorganic + mineralized organic N) for broiler litter and papermill sludge wereabout 1200 and 200 mg N/kg soil, respectively (Gale and Gilmour, 1986; Gilmour,1998).
The higher N mineralized in conjunction with the added plant available P in thebroiler litter resulted in high yields and may have contributed to the lower TPHvalues present in the broiler litter-amended soil. The low levels of mineralized Nand available P in the papermill sludge-amended soil resulted in low yields. In thesecond experiment, the papermill sludge addition resulted in increased plant growth.However, the rate used in the third greenhouse experiment was approximately one-third of the rate used in the second greenhouse experiment, and any effects relatingto enhanced soil tilth were probably reduced.
IV. CONCLUSIONSAlfalfa, crabgrass, fescue, and ryegrass all exhibited higher germination rates than
bermudagrass in the crude oil-contaminated soil across all amendments. Broiler littersignificantly reduced germination when compared to the other soil amendments,but increased plant growth for surviving plants. When the crude oil-contaminatedsoil was overlaid with uncontaminated soil, ryegrass, fescue, crabgrass, and alfalfagerminated, survived, and grew, and the soil amendments did not affect survival ofthe plant species. Broiler litter resulted in increased soil N and plant available P in thecrude oil-contaminated soil and greater reductions in TPH levels compared to the othersoil amendments and the unamended control. If a cool-season species was needed,ryegrass would be an acceptable selection; a suitable warm-season species wouldinclude bermudagrass or crabgrass. These studies demonstrate the importance of thedevelopment of appropriate management strategies for effective phy toremediation.
ACKNOWLEDGEMENTThis research is supported in part by the Integrated Petroleum Environmental
Consortium (IPEC); U.S. Army Research Office (ARO) contract/grant numberDACA89-97-K-005/DAAG55-98-4-0379; and the Army Environmental QualityTechnology Program, work unit EC-B06 BT25 "Biodegradation Processes ofExplosives/Organics Using Cold Adapted Soil Systems."
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