Treatments of Severely Boron-Contaminated Soils for Phytorestoration

As a result of the boron refining process, the Rio Tinto Borax site in California has experienced boron contamination, leading to desertification and the loss of local vegetation. Increasing industrial demand for the energy reduction ability of boron promotes continued mining. As such, the development of an effective and low-cost method for long-term reclamation is important. To establish this method, I analyzed the effects of amendments on the growth of a boron tolerant plant. I tested the effectiveness of water and organic matter on the growth of weeping alkaligrass (Puccinellia distans) naturalized in the USA in severely boron-contaminated soil at the Rio Tinto Borax site. I conducted a multi-factorial experiment with 4 levels of dilution of contaminated soil with sands, 3 levels of organic matter (OM), and 3 concentrations of polyethylene glycol (PEG) with 3 replicates. This study aimed to determine if: (1) there is a significant difference in plant growth and water content in the soils and plants between the levels of each factor: (2) there are interaction effects between each combination of two factors: and (3) there is an interaction effect for all three factors. Sand and OM significantly affected the plant growth and water content. PEG significantly affected water in the soil, but did not affect the plant growth. The combination of sand and OM significantly affected the plant growth and water content. This study shows the importance of amendments for reclaiming boron-contaminated soils. Yuriko Kayama Phytorestoration Spring 2010 2 INTRODUCTION Boron contamination is a serious environmental problem affecting both ecosystems and human activities. There are many causes of boron accumulation in the surface soil and/or deep ground including irrigation, a dry local climate, fertilizer, industrial wastewater, and mining (Camacho-Cristobal et al. 2008, Col & Col 2003, Nable et al. 1997, Shani & Hanks 1993, Sommer & Sorokin 1928). Both irrigation and dry local environment cause the accumulation of salts in the ground which, because of insufficient water, can increase boron to a toxic level. Boron contamination caused by these factors degrades the vegetative quality and decreases the amount of crops supplied to people (Shani & Hanks 1993). In western Central California, the arid and semiarid environment and weathering of boron-containing parent rocks from California’s coastal ranges has led to boron accumulation in the subsurface and groundwater and has interrupted sustainable agriculture because excess boron degrades the physiological functions of plants (Banuelos et al. 1993). Other studies in Turkey have indicated that the wastewater containing excess boron (25-30 mg/L) discharged into a river from the power plants can decrease crop production, particularly for citrus trees (Akar 2007). Although many studies have reported the negative effects of boron accumulation, mining cannot be stopped because of increasing industrial demand for boron for its energy saving ability (Lyday 2000). For example, adding boron to certain chemical reactions can reduce the time and gas required to heat a reaction. Thus, because reduced energy requirements directly impact company sales in terms of efficiency of cost, time, and materials, the energy reduction ability of boron is significant (Lyday 2000). The United States and Turkey, the world’s largest boron producers, have the most significant boron contamination problems (Lyday 2000). In California, the Rio Tinto Borax Mining Company that refines borate (BO3) is now facing boron contamination problems as a result of boron refining process. The Rio Tinto Borax site has experienced desertification as local vegetation has died out and soils have lost a considerable amount of nutrients and water. As a result of substantial water loss in soils, boron concentration in the water system in the ground increases, which makes boron more available for plants to uptake. Through the process of desertification, this excess boron dissolved in water degrades several physiological functions of plant by retarding metabolism, reducing root cell division, decreasing leaf chlorophyll contents and photosynthetic rates, Yuriko Kayama Phytorestoration Spring 2010 3 decreasing lignin and suberin levels, and reducing growth of shoots and roots (CamachoCristobal et al. 2008, Nable et al. 1997). Another study also showed that boron toxicity could cause leaf burn that make plants unable to photosynthesize (Eaton 1944). Because most plants die from these kinds of boron toxicity, less organic matter from leaf litter falls to the ground and the soil loses its ability to provide nutrients and retain water (Parton et al. 1987). In addition, by the multiplier effect, a lack of vegetation and degraded soil quality promotes conditions in which plants cannot grow. Given these conditions, the most effective and low-cost method for long-term reclamation would be the improvement of soil quality by boron-tolerant plants (Nable et al. 1997). Borontolerant plants, which can survive exposure to boron without significant physiological damage, extract boron from the soil and eventually reduce the boron content in the ground. Although the mechanism of their tolerance to boron has not been determined in detail, there are some potential boron-tolerant species already reported: saltbush (Watson et al. 1994), wheat (Paull et al. 1988), and tall fescue (Festuca arundinacea) (Banuelos et al. 1995). These boron-tolerant plants provide organic matter that helps the soil retain moisture and acts as an excellent food source to support soil microbes as an initial vegetative cover when they die and decay (Reiley & Shry 2000). At the same time, boron tolerant plants can remediate other conditions caused by lack of vegetation and drought, such as erosion, increased concentration of heavy metals and salts, and reduced microbial population in soils (Skujins & Allen 1986). Therefore, by introducing borontolerant plants the soil would recover its natural conditions that can accommodate native vegetation again. Even for boron-tolerant plants, enhancing soil conditions by adding amendments is a necessary step of reclamation. According to a Chambers’ Group report (unpublished data), the soil at Rio Tinto Borax has high levels of boron, insufficient organic matter, high levels of salinity and alkalinity, possible potassium and phosphorus deficiencies, and insufficient soil moisture. These soil problems quickly kill plants even though they are tolerant to boron. To reduce these effects of those soil characteristics, amendments are usually utilized for restoration projects (Prather 1977). For example, organic matter enhances plant growth by increasing the capability of soil to hold moisture and providing nutrients (Reiley & Shry 2000). Adding sand to contaminated soil decrease the concentrations of toxic substances. Yuriko Kayama Phytorestoration Spring 2010 4 Among the potential boron-tolerant plants, weeping alkaligrass (Puccinellia distans) naturalized in the USA is expected to be highly effective for reclamation of boron. It is already known that Puccinellia distans native to Turkey (PDT), the same species as PDU, has high boron tolerance (unpublished data from Prof. Terry lab). Also, there are more PDU seeds available of lower cost in the United States. Once the feasibility of PDU as a boron-tolerant plant is determined, the possibility of low-cost reclamation by boron-tolerant plants in U.S.A is expected. However, there is a doubt that PDU and PDT share the same or similar characteristics in boron tolerance because their appearance is different. In this study, I analyzed the effects of adding sand, organic matter (OM), and polyethylene glycol (PEG) on the growth of weeping alkaligrass in severely boron-contaminated soil at the Rio Tinto Borax site by using a multi-factorial approach. I assessed all combinations of all three factors by measuring fresh and dry weight of shoots and by calculating shoot moisture content (SMC) and soil gravimetric water content (SGWC). Dilution of contaminated soil by adding sand should increase the rate of growth by lowering the concentration of boron contained in the water system. Organic matter should improve the condition of soils by increasing the capacity of water retention and providing nutrients. Because PEG reduces the amount of water taken up by plants by lowering the rate of transpiration (Michel and Kaufmann 1973), it should lower the amount of boron extracted by plants. Also, PEG is safe for environments. Working et al. confirmed the relative safety in PEG administration by reviewing the literatures about PEG (1997). The individual effects of these three factors are already known, but it has not been examined that each amendment actually helps plant growth in the severely boron-contaminated soil. Also, their combined effects are still unknown. This study will determine if: (1) there is a significant difference in plant growth and water content in the soils and plants between the levels of each factor: (2) there are interaction effects between each combination of two factors: and (3) there is an interaction effect for all three factors. Because PEG reduces both the amount of water and boron taken up by plants, it should have a negative effect on plant growth when boron is not abundant. As such, healthier plants should have higher biomass, less browning, less wilting, and be more greenish in color. Yuriko Kayama Phytorestoration Spring 2010 5 METHODS Study Organism and Germination The plant species used in this study is weeping alkaligrass (Puccinellia distans) naturalized to the USA. This species was introduced from Eurasia to North America and naturalized in the USA where the Rio Tinto Borax site is located. Weeping alkalgrass is highly expected for restoration because it is adapted to sites with high salt concentrations as a facultative halophyte (Tarasoff 206). Also, PDU is adapted to high pH and low available water, which are soil characteristics at Rio Tinro Borax site. The soil used for the germination of the seeds was a normal potting soil for gardening, Scott Potting Medium (Scotts-Sierra Horticultural Products Co, Marysville, OH, USA) provided from a greenhouse at University of California, Berkeley. I sowed the seeds of weeping alkaligrass and watered them with tap water regularly. Approximately 5 weeks from sowing PDU seeds, they established a suitable size (about 15 cm) to be used in experiments. Experimental Design for Multi-factorial Analysis To determine how each factor affects the plant growth and water in the soils and the plants in severely boron-contaminated soils, I conducted a multi-factorial analysis by using sand, peat moss as organic matter, and PEG as soil amendments. In this study, there were 36 treatments with 3 replicates: all combinations of 4 levels of dilution by adding sands (0 %, 30 %, 60 %, and 90 % by weight), 3 levels of peat moss (0 %, 25 %, and 50 % by volume in a pot), and 3 concentrations of PEG (0, 45, and 90 g/L). In total, there were 108 pots. Severely boron-contaminated soils were collected at the Rio Tinto Borax site (870 mg of boron per litter). I put all pots into a clear bag that holds leaching from pots. Then, I placed an additional larger pot under the pot with the bag and rotated smaller pots 45 degrees, which makes enough space to hold leaching between smaller and larger pots and prevent smaller pots to soak into leaching (Fig. 1). If there is no space under smaller pots, then the pots are submerged into water for a long time and the roots of plants may die because of too much water. Yuriko Kayama Phytorestoration Spring 2010 6 Figure 1. Bag System. A bag holds leached water from a smaller pot. There is a space created between two pots by rotating the smaller one 45 degrees. To prepare the soil, I first added dry gypsum powder at the concentration of 1.2 g per 100g of contaminated soil to reduce the effects of boron. Then, I diluted the contaminated soils with the proper amounts of sands to 0, 30, 60, and 90 % by weight. Each pot held 300 g of soil and/or sands or 40 g of peat moss. I mixed 0, 25, and 50 % of peat moss by volume in each pot with the proper amount of mixed soil to make them 100 % in total. I poured these soils into each pot and gave them 200 ml of half strength Hoagland solution containing 0, 45, or 90 g of PEG per litter. After about 3 days, these solutions spread throughout the pots and made a homogeneous environment. I transferred seedlings to pots without removing potting soil, so that potting soils acts as a buffer and prevent transplant shock. Twice a week, I watered each plant with 50 ml of distilled water. I first poured distilled water into a clear bag catching the leached water and returned it to the pots after shaking it, so that constant environments were created by pouring back all leached water containing boron, nutrients, and PEG to pots. Throughout the study, I monitored plant health in terms of browning, leaf color, and wilting. Fresh and Dry Weight Measurements To determine the effects of addition of sand, organic matter, and PEG and their interaction effects on plant growth, I measured the fresh and dry weight of plant shoots harvested 3 weeks after transplanting. I cut the above ground part of shoots and measured their weight. For dry weight measurements, shoots were placed in an Yuriko Kayama Phytorestoration Spring 2010 7 incubator oven at 70°C for 72 hours, and subsequently weighted (unpublished data from Prof. Norman’s lab). Calculations To determine the effects of sand addition, OM, and PEG on the water in the soils and the plants in severely boron-contaminated soils, I calculated the shoot moisture content (SMC) and Soil Gravimetric Water Content (SGWC). SMC is the ratio of weight of water in shoots to shoot fresh weight and expressed on a percent basis. SGWC is the ratio of weight of water to soil dry weight and expressed on a percent basis. Data and Statistical Analysis To determine if: (1) there is a significant difference of plant growth and water content in soil and plant between the levels of each factor: (2) there are interaction effects between two factors: and (3) there is an interaction effect of all three factors, I analyzed the data by using factorial ANOVA (R 2.10.1 for Mac OS X, Rcmdr XQuartz 2.3.4).