Re-vegetation of an Oil/Brine Spill: Interaction Between Plants and Mycorrhizal Fungi

Wes Colgan III, Milan C. Vavrek and Janine Bolton School of Biological Sciences

Louisiana Tech University

ABSTRACT Oil brine spills encompass multiple stresses to plants, including petroleum, heavy metals and salt. A substantial amount of research has been performed on salt-tolerant plants. The majority of these plants associate with mycorrhizal fungi. Little work has been done on this symbiosis as it applies to restoration of brine spills. To test whether fungi can enhance revegetation, plant species identified from the literature as tolerant to salt and/or oil were grown in a greenhouse, in 19L containers using an ebb and flow system. Treatments (n=10) included combinations of plants, NaCl (8 and 16 dS), oil brine (16 dS), crude oil (6 L/m^2), fungi and calcium chloride. Plant performance, mycorrhizal colonization and water stress was scored. Preliminary data indicate that plants of Cynodon dactyloides and Lolium perenne tolerate high salt concentrations and appear to benefit from fungal amendments. These species are also able to germinate in high salinities (16 dS).

1.0 INTRODUCTION

Drilling and production of petroleum result in a large quantity of waste, including brine (1,2). Salinity of brine may range from 1,000 to 400,000 mg L-1 (3). The produced water is considered a pollutant (4) and as a pollutant, brine disposal is regulated. However, historical inappropriate disposal and accidental spills have occurred. Bass (5) reported 567 accidental brine spills in Louisiana between 1990 and the first half of 1998 based on data from the LA Department of Environmental Quality. The resulting brine-contaminated sites require restoration. Excavation and replacement of contaminated soil is one approach used, but is expensive and impractical for large sites. Stratigies for in-situ remediation would be preferred. This process primarily involves natural leaching of salts, application of soil amendments to improve soil structure, biodegradation of oil and revegetation. Revegetation plays a large role in restoration as plants improve soil surficial chemistry, structure, organic content, water capacity, thermal regime and leaching of salt (6,7). Oil brine attenuation has been demonstrated to occur more rapidly in the presence of the plants than with a lack of vegetation (8).

A substantial quantity of research has been performed on plant species tolerance to salt and revegetation of highly saline sites. This research has been stimulated as a result of increasing salinity of irrigated lands, and the need to use alternative water supplies and marginal lands (9-12). A number of species, varieties and genotypes adapted to saline soil conditions have been identified (13,14) as well as mechanisms providing salt tolerance (15-19). Problems associated with revegetation have also been circumscribed including ion toxicity, water availability, plant establishment, differential plant responses as a function of developmental stage and environment, energy constraints and slow genetic improvement by breeding (20-32). Further, evaporation from the soil surface in conjunction with capillary action of water from below the surface often results in accumulation of salts at the soil surface (33,34). Thus, plants experience different concentrations of salt as a function of root length, time and space. Nevertheless, successful revegetation is possible (35-37). Although in cases of high salinity, plant establishment and growth may not occur until leaching reduces salt levels (38).

Brine spills associated with petroleum production include additional stresses to plants. Soils at oil brine spill sites often include high exchangeable sodium concentrations (after precipitation), suspended and dissolved hydrocarbons, and heavy metals (1,2,39). These additional contaminants may have significant impacts on soils and plant communities, particularly at low salt concentrations. Climate and competition also affect species composition and performance, in addition to osmotic potential, at moderate to low salinity (40-43). Salt potentially represents the greatest direct stress on plants while soil structure is affected by high percentages of exchangeable sodium (39,44). High percentage exchangeable sodium disperses soil clay, restricting water and gas movement through soils. If the excess sodium is not replaced by another cation before leaching, soil aggregates collapse damaging soil structure (45,46). Produced water contains limited quantities of hydrocarbons, generally not exceeding 3% by weight in the soil (39). This quantity of oil, however, may impact restoration. Hydrophobic soils associated with oil contamination reduce water availability to plant roots (47-49) and reduce salt leaching. Hydrocarbons and metals may persist longer in soils than salt because salt migrates through soil more rapidly than oil (50). In addition, biodegradation of oil by bacteria is slowed in the presence of salt, as many bacteria are sensitive to highly saline environments (51-53). Therefore, recovering plant communities at spill

sites are exposed to a complex, changing soil environment. At a minimum, oil brine environmental effects include high salinity, poor soil structure, and the presence of hydrocarbons.

The complex properties inherent to oil brine contaminated soils have resulted in little success of in situ remediation. Research has addressed the difficulties in re-vegetating saline soils or in restoring soil structure. But, little research has attempted to understand the multiple stresses experienced by plants at spill sites. The need remains therefore to examine these effects singly and in combination in order to economically restore oil brine spill sites via re-establishing self-sustaining plant communities.

Plant establishment and productivity in saline, sodic, and saline-sodic soils have been enhanced by leaching via irrigation, improving drainage and amending soils. Greater leaching via irrigation and improved drainage may not be practical at oil brine spill sites however. Amendments include organic matter and a calcium substitute for sodium (e.g., calcium sulfate, calcium nitrate or calcium chloride; 54). Calcium substitution allows sodium to leach into deeper strata below the root zone and enables plants to maintain potassium transport and potassium/sodium selectivity (55). Calcium also enables plants to maintain high potassium to sodium absorption ratio (56) perhaps resulting in greater germination of seeds (57) and greater productivity (58). Further, calcium will help to prevent collapse of soil structure upon leaching of sodium (45,46). Several studies have avoided the use of soil amendments for restoration of an oil brine spill by relying on establishment of halophytes (D. Carty, personal communication). Avoiding chemical applications may be warranted to prevent further deterioration of groundwater or neighboring bodies of water (39). Eliminating soil amendments also reduces cost and limits perturbation of the spill site, as application of amendments may have to be continuous for long-term benefit.

The presence of symbiotic fungi may also enhance establishment of vascular plant species on oil brine sites and fungal spores can be considered a soil amendment. A number of studies have shown vesicular arbuscular (VA) fungi to be critical in re-developing soil structure after disturbance (59). Arbuscular mycorrhizae have been shown to improve survivorship, and above- and below-ground mass of Elaegnus commutata and Shepherdia canadensis grown on oil sand tailings (60). Similar results were found with Medicago sativa grown in hydrocarbon-polluted substrate (61) and Lactuca sativa under saline conditions (62). Lactuca grown in saline soils in the presence of mycorrhizal fungi showed improved rates of photosynthesis, transpiration, mineral nutrition, stomatal conductance, and water use efficiency (62,63). VA mycorrhizal fungi have also been found in association with salt tolerant Atriplex (64). Mycorrhizae may be the limiting factor in restoring ecosystem function in extremely arid or toxic sites (58). Therefore, symbiotic fungi may be essential for re-establishing plants on oil brine spill sites.

This project experimentally tested techniques to improve revegetation of oil brine contaminated sites. Specifically our objectives were to identify plant species tolerant of oil brine and quantify performance of these species under oil brine conditions in soils amended with arbuscular-mycorrhizal fungi and/or a calcium.

2.0 METHODS

2.1 Germination assay

To examine whether the germination stage of potential restoration species is tolerant to saline soil conditions, a germination assay was performed. Seeds (15) of a single plant species were placed in each of 40 - 100 x 15 mm petri dishes lined with filter paper. Species included Cynodon dactylon, Distichlis stricta, Festuca arundinacea, Lolium perenne, Lotus corniculatus, and Panicum virgatum. The filter paper was kept moist with one of four solutions: 0, 10 and 16 dS m-1 (electrical conductivity (EC) adjusted with NaCl) or oil brine (diluted to an EC of 16 dS m-1). Thus replicates consisted of 10 petri dishes. Seeds were exposed to 24 hr fluorescent lighting (50+3.1 mol m-2 s-1; 25–27 °C). Germination is reported for five days after plating. Analysis of variance was used to detect differences in mean number of seeds to germinate between treatments and species (JMP v.4, 2001, SAS Institute). 2.2.1 Experimental test of selected species

The experiment consisted of 280 individual ebb and flow units. Each unit was made from a 19 L plant pot placed in a 19 L plastic bucket. Each bucket had a spigot installed at the base. Pots contained oak-hickory-pine forest soil mixed with perlite at a ratio of 4:1 to prevent collapse of the soil structure after handling. Soils were collected from the Louisiana Tech University Arboretum (Ruston, LA). Each bucket was plumbed to one of 28 reservoirs containing the appropriate treatment solution. A marine bilge pump located in each reservoir was used to flood each bucket from above once per week. Once buckets were flooded, spigots were opened to allow the treatment solution to drain back into the reservoir. This system allowed for consistent maintenance of salt concentrations throughout the experiment.

The 28 treatments resulted from a full factorial design with salt, oil, calcium and symbiotic fungi as main effects (Table 1) and a second fully factorial design with brine, calcium and fungi as main effects (Table 1). NaCl was used to adjust the electrical conductivity (EC) to 10 and 16 dS m-1. These conductivities are equivalent to 8.0 and 12.8 ppt of salt, respectively. These concentrations were selected as relatively stressful to upland plants without causing complete mortality. Tap water was used in the control. For the oil treatment, a North Louisiana crude oil (Calumet Lubricants Co., Princeton, LA) was applied at the rate of 6 L m-2 to the soil surface. The application rate represented a substantial spill, but was not intended to eliminate all plant growth. Petroleum was allowed to volatilize for ca. seven days before the experiment was begun. Calcium chloride was applied at the rate of 0.48 g per m2, a low rate, because the calcium in the closed system would be re-circulated. Calcium chloride was used rather than gypsum because it would act more rapidly in the relatively short timeframe of this experiment. The calcium chloride was added directly to the treatment solution. Spores of the arbuscular mycorrhizal (AM) fungus, Glomus intraradices, in an inert clay carrier (Mycorrhiza Consultants, Grants Pass, OR) were also applied at the recommended rate to achieve a spore density of ca. 10,000 spores L-1 in the upper 10 cm of soil. The spores of Glomus intraradices were mixed into the soil at planting. This fungus has been demonstrated to be beneficial to a wide range of plant species and is commonly incorporated into nursery products to enhance plant establishment and growth. Brine was diluted at a ratio of 1:21 with tap water to reduce EC to 16 dS m-1.

Plant species were selected based on an extensive literature review (Vavrek et al. 2002). Seeds of Cynodon dactylon, Distichlis stricta, Festuca arundinacea, Lolium perenne, Lotus corniculatus, and Panicum virgatum were previously sown onto sterile commercial potting mix (Promix BX; Premier Brands, Inc.) in SC-10 Super Cells (Stuewe and Sons, Inc.; 3.8 cm diameter x 21 cm depth; 164 ml volume). Super Cells were placed in a Louisiana Tech University greenhouse. Insufficient seeds of Distichlis germinated and thus this species was not used in the seedling portion of the experiment. When seedlings reached ca. 40 days of age, two seedlings of each species were transplanted into each pot. In addition to the seedlings, a rooted Myrica cerifera cutting was placed in all buckets except those receiving brine. Insufficient cuttings of similar size were available to fill all buckets. Myrica was cited as salt-tolerant only, but was included in this study because it is a common shrub in Louisiana and grows on the margins of brine spill sites (personal observation). At the time of planting, a slow release fertilizer (19-6-12, Osmocote, Scotts Co., Marysville, OH) was mixed into the soil of all pots at the manufacturer’s recommended application rate. Plants were allowed to grow for ca. 22 weeks. Temperatures ranged from a mean low of 19.5 °C to a mean high of 22.0 °C. Midway through the experiment, plants were sprayed with M-Pede (potassium salts of fatty acids; Mycogen Corp., San Diego, CA) to control pests. The spray did not soak into the soil. All plants were harvested at the end of the experiment. Plants were carefully removed from the pots and soil particles washed from the roots. Plants were divided into above- and belowground portions. Plant parts were dried in an oven at 70 °C and weighed. 2.2.2 Survival

Survival was censused upon completion of the study. Nominal logistic fit (-loglikelihood ratio) was used to assess differences between treatments (JMP Statistical Software). Salt, oil, calcium and fungi were used as main effects. Species-specific responses were examined with salt, oil and species as main effects. Tests using brine and species, and brine, calcium, fungi and species were also used. Lastly, survival in treatments with salt (NaCl used to adjust EC to 16 dS m-1), crude oil (6 L m-2), salt (16 dS m-1) with oil (6 L m-2), and brine (16 dS m-1) was compared directly in a separate test. 2.2.3 Pot yield

For pot yield, plant biomass (above- and belowground dry weight) was summed across all plants within a pot with the exception of Myrica cerifera. Many Myrica died early in the study although the woody stems and roots remained. To prevent confounding of treatment and transplant mortality, Myrica biomass was excluded. Treatment effects were tested in a multi-way analysis of variance using salt, oil, calcium and fungi as main effects. The effect of brine was examined with analysis of variance using brine, calcium and fungi as main effects. Impacts of brine were compared directly to salt, oil, and salt with oil using a one-way analysis of variance and Tukey-Kramer HSD test (JMP Statistical Software). 2.2.4 Biomass

Salt, oil, calcium, fungi and species were used as main effects in multi-way analyses of variance to compare plant biomass (sum of above- and belowground dry

weights), shoot biomass (aboveground dry weight), root biomass (belowground dry weight) and shoot per total biomass. Differences in biomass due to brine were examined with analyses of variance using brine, calcium, fungi and species as main effects. Brine effects were compared to salt and oil using a one-way analysis of variance and Tukey-Kramer HSD test (JMP Statistical Software). Normality was assessed with normal quantile plots. Normality of shoot per total biomass values was improved with an arcsine transformation. Mean shoot per total biomass values and associated standard errors presented have been back-transformed. Biomass of Myrica cerifera and Lotus corniculatus were excluded from these analyses because of high mortality. 2.2.5 Water potential

Leaf water potential measurements were made at midday to estimate maximum daily water stress (Lambers et al. 1998). Festuca arundinacea and Lolium perenne were measured because with high survival these species appeared to be present in the most pots. Measurements were made immediately after a leaf had been removed. Measurements were made with a Model 1000 Pressure Chamber Instrument (PMS Instrument Co., Corvallis, OR). Differences in water potential were analyzed with an analysis of variance using species, salt, oil, calcium and fungi as main effects.

3.0 RESULTS 3.1 Germination assay

Treatment (p<0.0001) affected germination. The number of germinated seeds declined with increasing salt concentration and was most affected by exposure to brine (Fig. 1). Summed across treatments, species also exhibited different germination under laboratory conditions (p<0.0001). Seeds of Lolium perenne germinated most (55.3%), while seeds of Distichlis stricta germinated the least (3.1%). Species germination also differed as a function of treatment (treatment x species interaction, p<0.0001). Lotus corniculatus and Panicum virgatum appeared to be relatively sensitive to salinity (Fig. 2). Cynodon dactylon, Distichlis, Festuca arundinacea and Lolium were more tolerant of salinity. No species was tolerant of brine, although germination of Lolium in brine was 4.7%. 3.2 Survival

Preliminary results indicated that soils contaminated with salt (p=0.0363) and crude oil (p<0.0001) reduced seedling survival (Fig. 3). Survival tended not to be altered in the presence of salt with oil more than expected in salt or oil alone (p=0.0885). Amending salt-contaminated soils with calcium chloride (p=0.9344), symbiotic fungi (p=0.8848), or calcium and symbiotic fungi (p=0.6681) did not improve seedling survival. Calcium (p=0.6948), fungi (p=0.5409) and calcium with fungi (p=0.3313) also did not improve survival in crude oil-contaminated soils.

Seedling survival also depended upon species (p<0.0001). Lotus corniculatus and Myrica cerifera exhibited high mortality. In contrast, greater than 70% of Cynodon dactylon, Festuca arundinacea and Lolium perenne seedlings survived. With exposure to salt (p<0.0001) or oil (p<0.0001), survival varied differentially as a function of species.

Survival of Cynodon and Panicum virgatum increased with salt concentration (Fig. 4). The remainder of the species exhibited declining survival with increased salt concentration, although the declines were moderate in Festuca and Lolium. Survival of Myrica and Panicum was unaffected or improved in the presence of crude oil (Fig. 5), while the other species were sensitive to oil. These species-specific responses were also apparent with exposure to combined salt and oil treatments (p=0.0208). In terms of survival, Panicum tolerated exposure to salt and oil (Fig. 7f). Cynodon, Lolium, and to a lesser extent Festuca also tolerated salt and oil contamination, although treatments with oil reduced survival (Fig. 6).

Species responses to brine exposure were similar to salt and oil treatments (p<0.0001; Fig. 7); Lotus exhibited little tolerance to brine. Summed across species and amendments, brine did not affect survival (p=0.4617) (Fig. 8). Amending soils with calcium chloride and symbiotic fungi did not alter survival (p=0.2462). 3.3 Pot yield

Salt (p<0.0001; Fig. 9) and oil (p<0.0001; Fig. 10) reduced pot yield (sum of above and belowground dry weights of all plants within a pot) based on preliminary results. Combinations of salt and oil also reduced pot yield, but to a lesser extent than expected based on the additive effects of salt and oil (p<0.0154; Fig. 11). Symbiotic fungal soil amendments improved pot yield with exposure to oil (p=0.0140; Fig. 12), but did not improve yield of plants in salt-contaminated soil (p=0.0998; Fig. 13). Similarly pot yield was reduced by brine (p<0.0001; Fig. 14) and was improved by the addition of fungi (p=0.0179; Fig. 15). The reduction in pot yield by brine was not different from the reduction as a result of salt (EC of 16 dS m-1). Oil singly or in combination with salt (16 dS m-1) reduced yield to a greater extent than brine or salt alone (p<0.05; Fig. 16). 3.4 Biomass

Preliminary results demonstrated that mean biomass per plant (sum of above and belowground dry weight) was less than controls when exposed to salt (p<0.0001; Fig. 17) or oil (p<0.0001; Fig. 18). Lolium grew to the largest size followed by Cynodon, Festuca and Panicum (p<0.0001; Fig. 19). Responses in plant size to contaminants were species specific (salt x species interaction, p<0.0001; oil x species interaction, p<0.0001). Similar to survival, performance of Cynodon improved with exposure to salt. Panicum and Festuca tolerated salinity. Lolium’s size was reduced by salt, but mean plant size was consistently remained larger than the other species. Panicum tolerated exposure to crude oil while Lolium was the most sensitive (Fig. 20). These species responses were also detected upon exposure to combinations of salt and crude oil (p=0.0125). Cynodon, Festuca and Panicum tolerated exposure to both salt and oil; Lolium was intolerant (Fig. 21).

The effect of soil amendments (calcium chloride and symbiotic fungi) on mean plant biomass was not consistent. In salt-contaminated soils (16 dS m-1), Panicum appeared to benefit by the presence of calcium and fungi (species x salt x calcium x fungal interaction, p=0.0305; Fig. 22). Fungal amendment to crude oil-contaminated soil improved plant biomass in the absence of calcium (oil x calcium x fungal interaction, p=0.0343). The fungal amendment was not beneficial in the presence of calcium however (Fig. 23).

Soil amendments did not improve plant performance in oil brine-contaminated soils. While mean plant biomass was reduced by brine contamination (p<0.0001; Fig. 24), the reduction was not as large as the reduction in size after exposure to salt (16 dS m-

1) and crude oil (p<0.05, Fig. 25). 3.6 Water potential

Salt-contaminated soils increased water stress (p<0.0001, Fig. 26). Oil contamination (p<0.0001) and the calcium amendment (p=0.0071) also affected water stress, but significantly reduced stress (Figs. 27 and 28, respectively). Oil combined with calcium particularly reduced water stress at an EC 10 dS m-1 adjusted with NaCl (salt x oil x calcium interaction, p<0.0001; Fig. 29). Oil was expected to elevate water stress similar to salinity as oil-contaminated soils tend to be hydrophobic. The oil applied to the soil surface may have reduced evaporation from the soil maintaining greater soil moisture. The application of the symbiotic fungal amendment did not improve water stress unless oil contaminated the soils as well (salt x oil x fungi interaction, p=0.0016; Fig. 30). Festuca arundinacea was slightly more responsive to calcium amendments than Lolium perenne at ECs of 10 and 16 dS m-1. Festuca also responded more favorably to the fungal amendment at an EC of 10 dS m-1 (species x salt x calcium x fungi interaction, p=0.0322; Fig. 31).

4.0 Discussion The experimental tests of six species identified in the Vavrek et al.(2002) literature review indicated that Cynodon dactylon, Festuca arundinacea and Lolium perenne are viable species for revegetating oil brine spill sites. These species germinated, survived and grew in salt, oil and brine contaminated soils. Lolium in particular exhibited germination and substantial growth. The lower percent germination of Cynodon and Festuca suggests that a relatively high density of seeds would have to be sown. Growth of Panicum virgatum appeared to be tolerant of soil contamination, but survival was low. The greater allocation to root biomass may have contributed to Panicum’s tolerance of salt and oil contamination. The use of larger (older) plants may have improved establishment of Panicum as more mature plants would possess larger root systems. Lotus corniculatus and Myrica cerifera are not considered potential species for revegetation under the experimental conditions because of low survival. The differences in germination and growth responses emphasize the need to screen species for tolerance throughout their lifecycle or match tolerances with application techniques.

The relative success of Lolium does not imply that Lolium should be sown in monoculture. Lolium is a cool season grass with a limited growing season in the South. The rapid establishment and growth would be beneficial if planted in winter and early spring. To continue primary productivity in warm months, however, a warm season grass or forb would have to be included in the seed mix. Planting a mix of species has additional advantages including expanded rhizosphere volume, reduced direct competition for resources, greater diversity for ecosystem function, and enhanced cover across a heterogeneous environment. Planting both shallow and deeply rooting species may maximize rhizosphere volume. In this study, mean root length differed among the four established species (p<0.0001; mean root length+SE for Cynodon, Festuca, Lolium and Panicum = 35.7+0.87, 25.1+0.88, 30.8+0.75 and 21.1+0.98, respectively). Further, species such as Cynodon produce adventitious roots at leaf nodes resulting in rapid

expansion of the rhizosphere. The success of the different species with different traits such as root length and root biomass is consistent with conclusions from previous work (Vavrek and Campbell 2000), that no single species possesses all the different traits that contribute to tolerance and enhancement of bioremediation.

Brine represented additional stress beyond salinity for seed germination, resulting in lower germination than salt alone. The data do not provide an indication of what the additional contaminants is in brine that reduced germination, e.g., oil or heavy metal. Crude oil may be the additional stress because in terms of growth, oil reduced biomass more than the brine used in this study. Oil content of the brine used in this experiment was likely to be low as brine was removed from the bottom of a storage tank. The quantity of crude oil contaminating the soil (6 L m-2) in this study was equivalent to the maximum soil contamination from a brine spill (3.16% by weight). Carty et al. (1997) estimates that oil generally does not exceed 3% by weight in the soil from spilled produced water.

Other than germination, brine did not affect survival or productivity to a greater extent than salinity. Thus, depending on oil concentration in produced water, species selection for revegetation will require tolerance of salt and oil. These results may differ from the field because brine was diluted to an EC of 16 dS m-1 to allow for direct comparison with salt treatments. Brine effects may be more pronounced initially at actual spill sites although leaching will reduce salt concentrations over time. In this experiment, electrical conductivities (NaCl concentrations) were maintained, as treatments were applied weekly to a closed system without the opportunity for leaching of salts.

Soil amendments, arbuscular-mycorrhizal fungi and calcium chloride, improved pot yield, plant biomass and water stress, in part. Fungal amendments increased pot yield in oil and brine contaminated soils. Plant biomass was improved in oil, and in an EC of 16 dS m-1 adjusted with salt in conjunction with calcium. Water stress was reduced by calcium at an EC of 10 dS m-1 combined with oil; calcium with fungi lowered water stress in high salt and oil-contaminated soils. While no consistent benefit of soil amendments was apparent with the different treatment combinations, the enhanced productivity detected after only 22 weeks in a closed system suggests that amendments may be useful under actual spill conditions. Other studies (e.g., Raloff 1985; Barzegar et al. 1996) have demonstrated improvement in soil structure with an application of calcium to salt-contaminated soils when calcium was applied before the soil structure collapsed. Thus, adjustment in the time and rate of calcium application would probably improve the effectiveness of the calcium amendment. Further, the addition of chloride in the closed experimental system may have had a negative effect. Presumably, field results would be better than the experimental results also because of leaching of salts in the field. No studies have examined the benefit of applying symbiotic fungi to brine spill sites. Our results suggest that amending soils with fungi may be beneficial, but would not replace calcium. Additional research involving strain selection and improved application techniques is predicted to enhance fungal performance. For example symbiotic fungal spores were stirred into the soil surface during transplanting, mixing spores with soils used to grow outplanted seedlings before transplanting would concentrate spores in the rhizosphere and provide for increased colonization prior to exposure to salt-, oil- and brine-contaminated soils. Additionally, increased time under the experimental conditions may have allowed for further expansion of the symbiotic fungi within the rhizosphere of the plants. Lastly, plant growth responses, e.g., water potential, to soil amendments

appeared to be species specific. Future plant species selection may include response to soil amendments. Other methods to further enhance revegetation and restoration of brine spill sites remain to be researched. Many studies (e.g., Amakiri and Onofeghara 1984; Seliskar and Gallagher 2000) have demonstrated intra-specific variation in salt and oil tolerance. Therefore, selection for plant varieties or ecotypes must be considered, as well as adaptation to regional environmental conditions. A second restoration technique to be evaluated is hydroseeding in which seed, fertilizer, mulch and water are applied by spraying. The mulch and water may control water stress caused by osmotic pressure. A third method with potential advantages, considering the low germination found in this study, is hydrosprigging. Hydrosprigging involves the application of grass sprigs in a mix of mulch, fertilizer, tactifier and water under pressure in the same manner as hydroseeding. Cynodon dactylon and Festuca are currently being applied via hydrosprigging (Hydro Turf Planters Association). Ultimately, restoring oil brine spill sites involves assisting natural recovery processes. The recovering site should contain diverse, reproducing plant species, the appropriate animal, bacteria and fungal species, and should encompass re-initiated ecosystem functions. Successional change helps to determine community makeup and structure, and should be considered; i.e., species’ tolerance, competitiveness and response to disturbance will contribute to community composition. Therefore, expanding the database of tolerant species may allow for the selection of early successional species, enhancing recovery. Allowing successional change to direct restoration would require a long timeframe. The severity of disturbance as a result of oil brine contamination suggests that natural recovery without assistance would be too slow however, allowing nutrient loss, erosion and collapse of the soil structure. Alternatively, excess engineering adds cost and may be less successful. Long-term field studies will be required to define to what extent revegetation and soil amendments are needed to accelerate natural recovery processes and whether the cost of these techniques outweighs the benefits.

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Figure 1. The effect of salinity (NaCl) and oil brine on the mean number of seeds germinated per 15 seeds plated under laboratory conditions. Bars represent +SE.

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Figure 2. Species-specific germination responses to salinity (NaCl) and oil brine indicated by the mean number of seeds germinated per 15 seeds planted. Bars represent +SE.

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Figure 3. Proportion of seedlings surviving three EC levels adjusted with NaCl and two levels of North Louisiana crude oil.

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Figure 4. Species-specific responses to three levels of electrical conductivity adjusted with NaCl in a greenhouse experiment.

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Figure 5. Species-specific responses to two levels of North Louisiana crude oil contamination in a greenhouse study.

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Figure 6. Survival of (a) Cynodon dactylon, (b) Festuca arundinacea, (c) Lolium perenne, (d) Lotus corniculatus, (e) Myrica cerifera and (f) Panicum virgatum in salt (NaCl) and crude oil contaminated soils in a greenhouse experiment.

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Figure 7. Survival of species grown in oil brine contaminated soils in a greenhouse experiment.

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Figure 8. Survival of plants grown in brine (diluted to an EC of 16 dS m-1), salt (NaCl), crude oil or salt with crude oil contaminated soils in a greenhouse experiment.

01020304050607080

0 10 16E C (d S /m )

Pot y

ield

(g)

Figure 9. Mean pot yield (sum dry weights of all plants within a pot) in response to three levels of NaCl contaminated soils. Bars represent +SE.

01 02 03 04 05 06 07 08 0

0 6C r u d e o i l (L /m 2 )

Pot y

ield

(g)

Figure 10. Mean pot yield (sum of dry weights of all plants within a pot) in response to two levels of North Louisiana crude oil contaminated soils. Bars represent +SE.

0

20

40

60

80

100

0 10 16E C (d S /m )

Pot y

ield

(g)

0 L /m 2 o il6 L /m 2 o il

Figure 11. Mean pot yield (sum of dry weights of all plants within a pot) in response to salt (NaCl) and crude oil contaminated soils. Bars represent +SE.

0102030405060708090

0 0 with

calcium

6 6 with

calcium

C rude o il (L /m 2)

Pot y

ield

(g) w ith fu n g i

w ith o u t fun g i

Figure 12. Mean pot yield (sum of dry weights of all plants within a pot) in response to soils contaminated with North Louisiana crude oil, and treated with calcium chloride and symbiotic fungi. Bars represent +SE.

01 02 03 04 05 06 07 08 09 0

0 0 with calcium

10 10 with calcium

16 16 with calcium

E C ( d S / m )

Pot y

ield

(g)

w i t h f u n g iw i t h o u t f u n g i

Figure 13. Mean pot yield (sum of dry weights of all plants within a pot) in response to soils contaminated NaCl and treated with calcium chloride and symbiotic fungi. Bars represent +SE.

0

20

40

60

80

100

0 16Oil brine (EC dS/m )

Tota

l pot

yie

ld (g

)

Figure 14. Mean total pot yield (sum of dry weights of all plants within a pot) in response to oil brine contaminated soils. Bars represent +SE.

020406080

100120

0 16Brine (E C dS /m )

Tota

l pot

yie

ld (g

)

w ith fungiw ithout fungi

Figure 15. Mean total pot yield (sum of dry weights of all plants within a pot) in response to oil brine contaminated soils amended with symbiotic fungi. Bars represent +SE.

0

10

2030

40

50

Brine (16 dS /m ) Salt (16 dS /m ) Oil (6 L /m 2) Salt (16 dS /m ) &Oil (6 L /m 2)

T reatm ent

Tota

l pot

yie

ld (g

)

Figure 16. A comparison of total pot yield (sum of dry weights of all plants within a pot) responding to oil brine, salt (NaCl), crude oil or salt and crude oil. Bars represent +SE.

02468

1012

0 10 16E C (d S /m )

Bio

mas

s (g

)

Figure 17. Mean plant biomass for plants exposed to salt (NaCl) contaminated soil in a greenhouse experiment. Bars indicate +SE.

02468

1012

0 6C ru d e o il (L /m 2)

Bio

mas

s (g

)

Figure 18. Mean plant biomass for plants exposed to North Louisiana crude oil contaminated soil in a greenhouse experiment. Bars indicate +SE.

05

1 01 52 02 53 03 5

0 1 0 1 6

E C (d S /m )

Bio

mas

s (g

) C y n o d o n d a c ty lo nF e s tu c a a r u n d in a c e aL o liu m p e r e n n eP a n ic u m v ir g a tu m

Figure 19. Mean plant biomass (dry weight) for species exposed to NaCl contaminated soil in a greenhouse experiment. Bars represent +SE.

05

1 01 52 02 53 0

0 6

C ru d e o il (L /m -2 )

Bio

mas

s (g

) C y n o d o n d a c ty lo nF e s tu c a a ru n d in a c e aL o liu m p e re n n eP a n ic u m v irg a tu m

Figure 20. Mean plant biomass (dry weight) for species exposed to North Louisiana crude oil contaminated soil in a greenhouse experiment. Bars represent +SE.

a

02468

1012

0 10 16

Bio

mas

s (g

)

b

0

2

4

6

0 10 16E C (d S /m )

Bio

mas

s (g

)

c

0

10

20

30

40

50

0 10 16

d

0.0

0 .1

0 .2

0 .3

0 10 16E C (d S /m )

0 L /m 2 o il

6 L /m 2 o il

Figure 21. The effects of salt (NaCl) and North Louisiana crude oil on mean plant biomass for (a) Cynodon dactylon, (b) Festuca arundinacea, (c) Lolium perenne, and (d) Panicum virgatum. Bars indicate +SE.

a

0

2

4

6

8

1 0

1 2

0 0 with

calcium

10 10 with

calcium

16 16 with

calcium

Bio

mas

s (g

)

b

012345

0 0 with

calcium

10 10 with

calcium

16 16 with

calcium

EC (dS/m)

Bio

mas

s (g

)

c

01020304050

0 0 with

calcium

10 10 with

calcium

16 16 with

calcium

d

0.0

0.1

0.2

0.3

0 0 with

calcium

10 10 with

calcium

16 16 with

calcium

EC (dS/m)

Bio

mas

s (g

)

w ith fungi without fungi

Figure 22. Effect of salinity (NaCl), calcium chloride and symbiotic fungi on mean plant biomass (dry weight) for (a) Cynodon dactylon, (b) Festuca arundinacea, (c) Lolium perenne and (d) Panicum virgatum. Bars indicate +SE.

02468

1 01 2

0 0 with

calcium

6 6 with

calcium

C ru d e o il (L /m 2)

Bio

mas

s (g

)w ith fu n g iw ith o u t fu n g i

Figure 23. Effect of North Louisiana crude oil, calcium chloride and symbiotic fungi on mean plant biomass. Bars indicate +SE.

02468

1 01 21 41 6

0 1 6B r in e (E C d S /m )

Bio

mas

s (g

)

Figure 24. The effect of brine (adjusted to an EC of dS m-1) on mean plant biomass in a greenhouse experiment. Bars indicate +SE.

0

2

46

8

10

Brine (16 dS /m ) S alt (16 dS /m ) O il (6 L /m 2) S alt (16 dS /m ) &O il (6 L /m 2)

T reatm ent

Bio

mas

s (g

)

Figure 25. Mean plant biomass (dry weight) in response to brine, salt (NaCl), crude oil, and salt with crude oil. Bars indicate +SE.

-5

-4

-3

-2

-1

0

0 1 0 1 6E C (d S /m )

Wat

er p

oten

tial (

MPa

)

F e s tu c aa r u n d in a c e aL o liu mp e r e n n e

Figure 26. Effect of salinity (NaCl) on leaf water potential. Bars indicate +SE.

-4

-3

-2

-1

0

0 6Crude oil (L/m 2)

Wat

er p

oten

tial (

MPa

)

FestucaarundinaceaLoliumperenne

Figure 27. Effect of North Louisiana crude oil on leaf water potential. Bars indicate +SE.

- 4

- 3

- 2

- 1

0

w i t h o u t c a l c i u m w i t h o u t c a l c i u mT r e a t m e n t

Wat

er p

oten

tial (

MPa

)

Figure 28. Effect of amending soils with calcium chloride on leaf water potential. Bars indicate +SE.

-5

-4

-3

-2

-1

0

0 1 0 1 6E C (d S /m )

Wat

er p

oten

tial (

MPa

)

0 L /m 2 o il

0 L /m 2 o il w ithc a lc iu m6 L /m 2 o il

6 L /m 2 o il w ithc a lc iu m

Figure 29. The effect of salinity (NaCl), North Louisiana crude oil and calcium chloride on leaf water potential. Bars indicate +SE.

-5

-4

-3

-2

-1

0

0 1 0 1 6S a lin ity (d S /m )

Wat

er p

oten

tial (

MPa

)

0 L /m 2 o il

0 L /m 2 o il w ithfu n g i6 L /m 2 o il

6 L /m 2 o il w ithfu n g i

Figure 30. Effect of salinity (NaCl), North Louisiana crude oil and symbiotic fungi on leaf water potential. Bars indicate +SE.

a

-5

-4-3

-2-1

0

0 10 16EC (dS/m)

b

-5

-4-3

-2-1

0

0 10 16EC (dS/m)

without calciumor fungiwith calcium

with fungi

with calciumand fungi

Figure 31. Effect of salinity (NaCl), calcium chloride and symbiotic fungi on leaf water potentials of (a) Festuca arundinacea and (b) Lolium perenne. Bars indicate +SE. Table 1. Treatments applied in a greenhouse study involving re-vegetation of oil brine spill sites (n=10). EC adjusted with NaCl (dS m-2)

Crude oil (L m-2)

EC of brine (dS m-2)

CaCl2 (g m-2)

Symbiotic fungi (spores per pot)

0 0 0 0.00 0 0 0 0 0.00 13,000 0 0 0 0.48 0 0 0 0 0.48 13,000 0 6 0 0.00 0 0 6 0 0.00 13,000 0 6 0 0.48 0 0 6 0 0.48 13,000 10 0 0 0.00 0 10 0 0 0.00 13,000 10 0 0 0.48 0 10 0 0 0.48 13,000 10 6 0 0.00 0 10 6 0 0.00 13,000 10 6 0 0.48 0 10 6 0 0.48 13,000 16 0 0 0.00 0 16 0 0 0.00 13,000 16 0 0 0.48 0 16 0 0 0.48 13,000 16 6 0 0.00 0 16 6 0 0.00 13,000 16 6 0 0.48 0 16 6 0 0.48 13,000 0 0 16 0.00 0 0 0 16 0.00 13,000

0 0 16 0.48 0 0 0 16 0.48 13,000

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