Approximately 20 g of Ottawa sand was spread across the soil and the lid was placed on the Petri dish and sealed with parafilm to maintain optimal growth conditions. A sandy loam control soil containing no PAH contamination was prepared in the same manner every 20 samples. The samples were placed in a growth chamber at 22±1 °C in darkness for 2 d and then 16/8 h day/night cycle for another 3 d. Afterwards, all germinated seeds were counted for the control, pre-, and post-remediation soils.Method blanks were included every 10 samples to determine any potential background contamination and no PAHs were detected.In addition, a deuterated PAH surrogate standard solution containing seven PAHs of various molecular weights was added to all samples prior to the PLE extraction to assess surrogate recoveries, and the surrogate recoveries were 91±11%. Least squares means for significant effects were determined using a protected least significant difference procedure at α = 0.05. All statistical analyses were performed using SAS® 9.4 . All treatments were conducted in quadruplicate and three non-vegetated mercuric-chloridesterilized control treatments were used to indicate any abiotic PAH loss.The initial soil parameters of the outdoor shooting range soil are given in Table 4.1. The heavy metal concentrations in the initial outdoor shooting range soil were substantially lower than levels in other outdoor shooting range soils.
This might be due to the fact that soil sampling was conducted at the location that contained the greatest concentration of clay target fragments and was relatively near the firing stand. Kajander and Parri suggested that shotgun ammunition travels approximately 200-250 m from the firing stand while clay target fragments typically land 20-80 m from the firing stand. Although the concentrations of PAHs in the initial outdoor shooting range soil were greater than typical background concentrations in urban soils,tower garden these levels were lower when compared to other outdoor shooting range soils contaminated with clay target fragments . For example, total soil PAH concentrations in other outdoor shooting ranges from clay targets in California and Florida were 2,431 and 1,324 mg/kg, respectively . Similar to other studies examining PAH contaminated soils from clay targets, acenaphthylene was not detected, and the majority of the total PAH concentrations consisted of HMW PAHs . This finding was to be expected due to the PAH composition of the binding agent pitch in clay targets as well as the increased recalcitrance of HMW PAHs in aged, field-contaminated soils . Based upon the individual PAH profiles in the clay target fragments on the soil surface, the binding material most likely consisted of coal tar pitch . Of the 16 priority PAHs, 5 PAH compounds in the initial soil exceeded U.S. EPA regional screening levels for industrial soils; these PAHs are also classified as probably or possibly carcinogenic to humans according to the IARC . Switch grass vegetation in the native treatments amended with Brij-35/SDS and rhamnolipid surfactants did not survive after thinning and initial surfactant addition at 4 weeks. Therefore, switch grass grown in soil without PAH contamination at similar heights to the other treatments were transplanted to the Brij-35/SDS- and rhamnolipidamended treatments to yield 8 plants/pot and the transplanted switch grass plants survived for the remainder of the 8-month experiment with surfactant reapplication.
At the end of the 8-month phytoremediation experiment, bermudagrass root, shoot, and total biomass was far greater than switch grass root, shoot, and total biomass amongst all vegetated treatments. Although bermudagrass and switch grass biomass did not differ between the surfactant-amended and the unamended control treatments, the bioaugmentation of M. vanbaalenii PYR-1 resulted in increased bermudagrass shoot biomass and switchgrass root biomass compared to the non-inoculated vegetated treatments . The positive effect of bioaugmentation on plant growth was potentially related to the increased dissipation of HMW PAHs due to M. vanbaalenii PYR-1 bioaugmentation . Rostami et al. also observed that the bioaugmentation of P. aeruginosa increased great millet [Sorghum bicolor] root biomass after a 90-d phytoremediation experiment in pyrene-contaminated soil due to increased pyrene biodegradation and reduced toxic effects of PAHs. Positive effects of P. aeruginosa bioaugmentation on alfalfa [Medicago sativa L.] root and shoot biomass were also demonstrated by Agnello et al. , who reported increased root and shoot biomass after a 90-d phytoremediation study of a co-contaminated soil containing high levels of heavy metals and petroleum hydrocarbons . Chen et al. also observed increased ryegrass [Lolium multiflorum Lam] and Seduce alfredii biomass following repeated inoculation with Microbacterium sp. KL5 and Candida tropicalis C10 in a 2-yr phytoremediation study in soil spiked with phenanthrene, fluoranthene, anthracene, and pyrene.The direct plant uptake of PAHs, especially HMW PAHs, from the soil has been previously shown to be negligible as soil microbial degradation is the primary process involved in effective PAH-contaminated site bio-remediation . However, the application of surfactant amendments to contaminated soils might increase the mass transfer of PAHs to the aqueous phase, thereby resulting in increased plant uptake of these compounds. For example, Gao et al. determined that ryegrass plant uptake of phenanthrene and pyrene in water was enhanced when amended with Brij-35 surfactant at concentrations lower than 74 mg/L.
However, this was not the case in a surfactant-enhanced phytoremediation experiment of PAHcontaminated soils where pyrene concentrations in plant tissues accounted for less than 0.1% when amended with Tween 80 or Brij-35 surfactants . At the end of the 8-month phytoremediation experiment, plant uptake of PAHs was negligible with the maximum PAH plant uptake occurring in the native rhamnolipid-amended bermudagrass treatment and total PAH plant accumulation accounting for only 0.8% of the initial PAH amount. These results were similar to Reilley et al. that conducted a 24-wk phytoremediation study of anthracene- and pyrene-spiked soil at 100 mg/kg using four plant species . They estimated that the total accumulation of both PAHs in the four plants accounted for less than 0.03% of the initial PAH concentrations. Gao and Zhu also demonstrated that plant-promoted biodegradation was the dominant contribution to phenanthrene and pyrene dissipation compared to the contribution of direct plant uptake after a 45-d phytoremediation study evaluating 12 plant species. Although bermudagrass produced significantly more root and shoot biomass than switchgrass after the 8-month phytoremediation experiment,stacking flower pot tower the use of both grasses increased dissipation of low-molecular-weight and HMW PAHs compared to the non-vegetated treatments . Bermudagrass has been recognized as a hydrocarbon-tolerant plant and switch grass has been previously utilized in PAH phytoremediation studies . Hutchinson et al. compared the effectiveness of bermudagrass and tall fescue during a 1-year phytoremediation study of total petroleum hydrocarbons and observed that bermudagrass generated almost twice as much root and shoot biomass as tall fescue in all treatments and bermudagrass growth resulted in a 68% reduction in total petroleum hydrocarbons. Thompson et al. examined the effects of fertilizer rate on bermudagrass growth and subsequent dissipation of pyrene-contaminated soils at 1,000 mg/kg and determined that at a C:N ratio of 4.5:1, bermudagrass increased pyrene biodegradation from 31% in the non-vegetated treatment to 62% in the bermudagrass treatment after 100 d. Another study by Krutz et al. examined pyrene biodegradation spiked at 500 mg/kg in a 63-d bermudagrass phytoremediation experiment and showed that pyrene degradation was significantly greater in the bermudagrass rhizosphere soil compared to the bermudagrass bulk soil or non-vegetated bulk soil due to the presence of bermudagrass root exudates and possible selective PAHdegrader population enrichment. Reilley et al. examined the effects of switch grass on PAH biodegradation in aged, MGP-contaminated soil and reported that switch grass vegetation resulted in a total PAH reduction to 2,053 mg/kg with substantial biodegradation occurring for 4- and 5-ring PAHs after 1 yr. Pradhan et al. reported a 57% PAH reduction in MGP-contaminated soil vegetated with switch grass following a 6-month phytoremediation experiment. Bermudagrass and switch grass significantly reduced some PAH levels in the outdoor shooting range soil compared to the non-vegetated treatments . However, the application of Brij-35/SDS surfactant mixture did not result in a significant difference between the non-vegetated and vegetated treatments for some PAHs . The mixed surfactant amendment treatments were not significantly different from the Brij-35- and rhamnolipid-amended non-vegetated treatments; however, the Brij- 35/SDS-amended treatments resulted in a significantly greater dissipation for phenanthrene, anthracene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene, dibenzo[a,h]anthracene, and benzo[g,h,i]perylene compared to the unamended, nonvegetated treatment . In most practical applications of surfactant-enhanced PAH bio-remediation, a mixture of anionic and nonionic surfactants is utilized because the mixture typically increases the effective surfactant concentration in soil due to the reduction in nonionic surfactant soil sorption, and a decrease of the surface and interfacial tension at a lower CMC, resulting in increased PAH bio-availability .
Ni et al. evaluated the effects of sodium dodecyl benzene sulfonate and Tween 80 anionic-nonionic surfactant mixture at different surfactant ratios with ryegrass phytoremediation in phenanthrene- and pyrene-contaminated soils and concluded that the 1:1 surfactant mixture at less than 150 mg/kg showed the best remediation efficiency and was more effective than individual surfactants in promoting plant-microbe associated bio-remediation. Even though rhamnolipid bio-surfactants are a promising alternative to synthetic surfactants, the application of rhamnolipid biosurfactant was not significantly different from the unamended control in the nonvegetated treatments . Szulc et al. also reported that the addition of rhamnolipids did not contribute to diesel-oil removal at the end of a 1-yr bioaugmentation experiment using a PAH-degrading consortium. Additionally, Lin et al. observed that the biodegradation rate of rhamnolipid-amended treatments in the latter stage of diesel remediation were similar to unamended treatments. Currently, there are limited phytoremediation studies evaluating the effects of vegetation on PAH levels in outdoor shooting range soils contaminated with clay target fragments. Wawra et al. concluded that only the combined treatment of black lotus [Robinia pseudoacacia Nyirsegi] and ferrihydrite-bearing material, gravel sludge, and green waste biochar amendment significantly decreased ∑16PAH from an initial concentration of 200 mg/kg. Specifically, the amendment-enhanced black lotus treatment was the only treatment that appreciably decreased phenanthrene, benzo[a]anthracene, chrysene, benzo[a]pyrene, fluoranthene, benzo[b]fluoranthene, benzo[k]fluoranthene, and indeno[1,2,3-cd]pyrene concentrations after 1 yr .Four applications of M. vanbaalenii PYR-1 during the 8-month study significantly reduced PAH levels in contaminated outdoor shooting range soil. Bioaugmentation did not reduce levels of benzo[g,h,i]perylene and the LMW PAHs that are more easily biodegradable and were initially at lower concentrations than the HMW PAHs . Mycobacterium vanbaalenii PYR-1 bioaugmentation has been extensively studied in pure culture settings . Mycobacterium vanbaalenii PYR-1 is an effective HMW PAH-degrading microbe, partially attributable to the production of surface-active trehalose-containing glycolipids that has previously been reported to be aseffective as external surfactants in the biodegradation of pyrene . Child et al. evaluated the growth of M. vanbaalenii PYR-1 and four other Mycobacterium isolates associated with barley [Hordeum vulgare] root surfaces after growth of the seedlings from inoculated seeds and concluded that M. vanbaalenii PYR-1remained associated with the root as it grew from the inoculated seed and followed the root tip as it traveled throughout the growth matrix. In addition, M. vanbaalenii PYR-1 had one of the lowest contact angles of ethylene glycol on confluent layers of cells on agar, likely due to glycolipid biosurfactant production, indicating that this microorganism had a high potential to colonize the barley root tip. This is a beneficial trait for PAH remediation potential as the microbe would be distributed through contaminated soils as the roots grow . In another study, Child et al. evaluated the effects on 14C-pyrene mineralization using a Mycobacterium sp. KMS in barley rhizosphere and concluded that roots inoculated with the microbe mineralized 14C-pyrene to a greater extent than treatments with solely bioaugmentation or sterile barley because the microbe was dispersed throughout the entire soil matrix as it traveled with the roots. Ma et al. reported that the bioaugmentation of M. gilvum CP13 in combination with mustard [Brassica juncea] resulted in a significant reduction of total PAH levels after 183 d. The most significant increases in PAH biodegradation rate were for 4-6 ringed PAHs, indicating that the bioaugmentation of M. gilvum CP13 improved the degradation of recalcitrant HMW PAHs as the microbe could readily employ pyrene as a sole carbon source, similar to M. vanbaalenii PYR-1 . Additionally, dehydrogenase activity of the soil was examined to reflect the degree of PAH biodegradation and it was observed that dehydrogenase activity was significantly higher in the bioaugmented treatments compared to the control group, suggesting that PAHs or their metabolites were likely used as substrates by M. gilvum CP13, thus increasing the activity of dehydrogenase .