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in reclaimed coastal tideland soils. ABSTRACT: We examined the effect of soil salinity on the deg- radation of chlorpyrifos and the residual effect of chlorpyrifos ...
Geosciences Journal Vol. 14, No. 4, p. 371 − 378, December 2010 DOI 10.1007/s12303-010-0032-2 ⓒ The Association of Korean Geoscience Societies and Springer 2010

Salinity effects on chlorpyrifos degradation and phosphorus fractionation in reclaimed coastal tideland soils Eui-Yong Yun

MONSANTO KOREA Inc. 331-3 Jeongjung-ri, Gangwoe-myeon, Cheongwon-gun, Chungbuk 363-955, Republic of Korea Hee-Myong Ro* Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea Goon-Taek Lee National Instrumentation Center for Environmental Management, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea Woo-Jung Choi Department of Biosystems and Agricultural Engineering and Institute of Agricultural Science and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea

ABSTRACT: We examined the effect of soil salinity on the degradation of chlorpyrifos and the residual effect of chlorpyrifos and its metabolites on soil P fractionation during 60-day aerobic incubation. A sandy loam soil (Typic Psammaquents) was collected from the Daeho reclaimed tideland and two-thirds of the soil was applied with Na salt to get three different soil salinity levels: 4.6 (low, EL), 9.7 (medium, EM), and 14.4 (high, EH) dS m–1. Estimated half-lives for chlorpyrifos degradation were 7.1 in EL, 10.0 in EM and 16.9 days in EH soils. During the degradation of chlorpyrifos in soil, microbial activity decreased by increasing soil salinity and its inhibitory effect increased with time. In contrast, the addition of chlorpyrifos did not inhibit soil alkaline phosphatase (SAP) activity, which was higher in EH than in control soils. Chlorpyrifos added at a rate of 5.0 mg a.i. kg–1 dry soil did not affect the distribution pattern of P fractions in control soils. Both an increase in soil salinity and soil sterilization increased the Ca-bound P fraction and decreased the occluded Fe + Al-bound P fraction with a significant interaction between soil salinity and sterilization. With time, the Ca-bound P fraction increased and organic- and occluded Fe + Al-bound P fractions decreased, while total-P, available-P, and non-occluded + adsorbed P fraction remained unchanged. Particularly, organic-P was mineralized more in EH than in control soils and the Ca-bound P fraction contained the highest inorganic P released. Mineralization of organic P and partitioning of released P in the recalcitrant Ca-bound P fraction increased by increasing soil salinity, while available P fraction remained unchanged, suggesting that the addition of chlorpyrifos at the currently recommended dosage level did not seem to considerably affect the available P fraction with low P leaching potential to waterways. Key words: chemical speciation, fluorescein diacetate, phosphatase, sterilization

1. INTRODUCTION Phosphorous has been recognized as a limiting nutrient for agricultural and aquatic productivity (Kaiserli et al., 2002), *Corresponding author: [email protected]

and its availability can increase by increasing salinity (Fox et al., 1986; House, 1999; Sundareshwar and Morris, 1999). However, cations (e.g., Al3+, Fe3+ or Ca2+) of the soil solution can precipitate phosphates, thus lowering the amount of available P (Ro and Cho, 2000). Therefore, the assimilation of P as orthophosphate by microorganisms and plants usually requires several phosphatases transforming organic P into inorganic P. As an extracellular enzyme, phosphatase activity in soil is mostly influenced by the interactions at solid-liquid interfaces (Burns, 1978), since they can be adsorbed by negatively charged clay minerals (Fusi et al., 1989). Chemical P speciation, P dynamics and hence P bioavailability in soil, particularly in a salt-affected soil, are poorly understood. Chlorpyrifos, O,O-diethyl-(O-3,5,6-trichloro-2-pyridyl) phosphorothioate, is a broad spectrum systemic organophosphorous insecticide with a low solubility in water and a high octanol-water coefficient (log10 Kow = 4.70). Two major toxic or potentially toxic metabolites, TCP (3,5,6-trichloropyridinol) and DETP (diethylthiophosphate), are found in soils, resulting from the hydroxylation of chlorpyrifos. Degradation of chlorpyrifos in soil can occur through chemical hydrolysis and microbial decomposition. Its persistence varies depending on several abiotic factors such as soil pH, temperature, organic carbon, moisture, and its concentration and chemical structure (Chapman and Chapman, 1986; Racke, 1993). Liu et al. (2001) suggested that Cu2+ concentration and soil salinity would be important factors in controlling chlorpyrifos hydrolysis in water, and Singh et al. (2003) showed that the degradation of chlorpyrifos occurred in soil is mediated by co-metabolic activities of aerobic bacteria. Chlorpyrifos has both positive and negative effects on soil microorganisms whose activity is important in controlling the availability and fate of some major plant nutrients

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Eui-Yong Yun, Hee-Myong Ro, Goon-Taek Lee, and Woo-Jung Choi

(Sardar and Kole, 2005). Indeed, degradation byproducts can be assimilated by soil microorganisms, thus stimulating microbial growth and activities with positive effects on plant nutrient cycling (Das and Mukherjee, 1998; Jana et al., 1998), whereas some residues can have deleterious effects decreasing microbial biomass and activities (Martinez et al., 1992; Sardar and Kole, 2005). Particularly, the accumulation of TCP can inhibit proliferation of chlorpyrifos-degrading microorganisms (Racke et al., 1988). Soil salinity influences physical, chemical, and biological properties of the soil (Baldwin et al., 2006). Increases in ionic strength and changes in ionic composition can cause flocculation of particles, alter chemical equilibria affecting nutrient availability, and decrease microbial activity (Grace et al., 1997; House, 1999; Sardinha et al., 2003; Liang et al., 2005). To date, however, the effects of soil salinity on mineralization of organic P, chemical speciation and bioavailability of P are poorly known. As aforementioned, chlorpyrifos and its degradation byproducts can affect the concentration of available P to crops. Recently, Korea has dammed the Saemangeum (aka Ariul, an area of about 400 km2) estuarine tidal flat of the Yellow Sea, with the intention to develop agricultural (especially for commercially valuable horticulture crops), industrial or environmental sustainability. Therefore, it is needed to know how soil salinity affects P fractionations during the degradation of chlorpyrifos (a frequently-used organophosphate insecticide) in these saltaffected agricultural soils. Here, we have hypothesized that soil salinity would control chlorpyrifos degradation with effects on chemical speciation and bioavailability of P in saline soils through direct and indirect effects on composition and activity of microbial community. Therefore, we tested this hypothesis by examining the effect of soil salinity and the persistence of chlorpyrifos on the pattern of chemical P speciation through aerobic incubation. 2. MATERIALS AND METHODS 2.1. Preparation of Soil Samples A sandy loam soil having an ECe of 4.6 dS m–1 (low, EL), taxonomically classified as Typic Psammaquents, was collected from the surface layer (0–10 cm) at the Daeho reclaimed coastal tideland located in Chungnam Province, Korea (36°59'N, 126°28'E). Two-thirds of the soil was salinized with 0.07 and 0.15 M NaCl solutions to get two higher soil salinity levels: 9.7 (medium, EM) and 14.4 (high, EH) dS m–1. Each group of soil was air-dried, sieved to a 2.0-mm sieve, and mixed homogeneously for an incubation experiment. Each group of soil was incubated in the dark for 3 weeks at 25 ± 2 °C, and soil water content was adjusted to 50% of the saturated water content (0.29 kg kg–1). Some physicochemical soil properties are reported in Table 1.

Table 1. Physicochemical properties of the soils Soil salinity level Low Medium High ECe (dS m–1)a 4.6 9.7 14.4 b 8 7.8 7.7 pH (soil:water = 1:5) Organic carbon (g kg–1) 29 28 29 397 405 403 Total P (mg kg−1) Available Cu (mg kg–1) 1.4 1.5 1.6 Available P (mg kg–1) 5.7 5.4 5.2 271 246 250 Exchangeable Ca2+ (mg kg–1) Exchangeable Fe (mg kg–1) 82 76 70 Soil texturec Sandy loam Sandy loam Sandy loam a

Saturated soil pastes method Soil-to-suspension ratio of 1:5 c USDA classification scheme b

After incubation, 0.37% acid-free formaldehyde (Merck, Germany) buffered with CaCO3 was applied at a rate of 0.1 L kg–1 (dry basis) to half of the incubated soil at each salinity level to inhibit bacterial activity (Tuominen et al., 1994) and then all soil samples were incubated for an additional week. Therefore, we have the following 6 treatments: SEL, SEM and SEH in sterilized soils, and NSEL, NSEM and NSEH in unsterilized soils, respectively. The control was soil without application of chlorpyrifos and it was also split in sterilized (C-SEL) or unsterilized (C-NSEL) low-salinity level soils. One kg (dry basis) of each above incubated soil was placed into 1 L amber glass bottle and treated with 20 mL of a reagent-grade chlorpyrifos (10 kg a.i. ha–1), purchased from Chem Service (West Chester, USA). This product contains 99.5% of the pesticide and then applied at a rate of 5.0 µg a.i. g–1 dry soil. The application rate of chlorpyrifos was chosen on the basis of the recommended dose for pest control (Racke et al., 1994). Uniform mixing of soil was achieved by shaking the glass bottle for 1 hour and checked by analyzing the chlorpyrifos residues of each sample. For each salinity level, triplicate 30 g (dry basis) portions of each soil mixture were individually placed into 168 plastic bottles (each volume is 100 mL) and covered with perforated aluminum foil to ensure gas exchange. Mixtures were incubated at 25 ± 2 °C in darkness for 0 (5 hours), 3, 6, 12, 20, 40, and 60 days. Soil water content was kept constant throughout the incubation by adding deionized water. Chlorpyrifos residue was analyzed at every sampling time. Microbial activity and soil alkaline phosphatase (SAP) activity were analyzed at each sampling time, and the chemical P speciation was measured at the beginning and at end of incubation. 2.2. Measurements of Chlorpyrifos Residue, Enzyme Activity, and Phosphorus Speciation Ten grams of each soil sample were placed into a 250 mL shaking flask and 10 glass beads were added to facilitate the destruction of aggregates. The mixture was extracted with

Chlorpyrifos degradation and P fractionation in coastal saline soils

100 mL of acetone (HPLC-grade, Fisher Scientific) by shaking for 2 hours on a horizontal shaker (DS-300L, DASOL, Korea), and filtered by a vacuum manifold with a funnel covered with foil to prevent losses due to evaporation. The filtrate was evaporated in a vacuum rotary evaporator (N1000, EYELA, Japan) at 39 ± 1 °C until the disappearance of acetone. The resulting extract was mixed with 40 mL dichloromethane, and poured into a 500 mL separatory flask with 20 mL of 1% Na2CO3 for the partition of chlorpyrifos. The lower solution (dichloromethane with chlorpyrifos) was passed through the funnel filled with anhydrous sodium sulphate (Analytical grade, JUNSEI, Japan). This procedure was repeated three times. The resulting filtrate was transferred to a 500 round-bottom flask and evaporated to dryness by a rotary evaporator, and the residue was redissolved in 10 mL acetone for residue analysis of chlorpyrifos. Chlorpyrifos residue was analyzed on an Agilent 6890N gas chromatograph equipped with HP-5 capillary column (30 m × 0.25 m × 0.25 µm, Agilent Technologies, Wilmington, DE), with the inlet kept at 260 °C and operated on a split mode (20 mL min–1) with a sample injection volume of 2 µL. The oven temperature was initially set at 200 °C with a hold of 1 min, and then raised to 300 °C at 10 °C min–1 with a final hold of 1 min. The µ-ECD was kept at 300 °C. Under these conditions, the retention time was 5.70 ± 0.08 min for chlorpyrifos and recovery was 92 ± 2.9%. Microbial activity was determined by a fluorescein diacetate hydrolysis assay (Adam and Duncan, 2001), and SAP activity as reported by Tabatabai (1994). An iron oxidecoated filter paper method was used to measure the concentration of available P (Chardon et al., 1996). Organic P was determined by the ignition method (Saunders and Williams, 1955) and total P by the HNO3/H2SO4 digestion method (Kuo, 1996). Inorganic P of calcareous soils was partitioned into three following chemical forms according to the fractionation procedure reported by Kuo (1996): (i) NaOH extractable P (Al-bound P + occluded Fe-bound P) obtained by mixing soil with 0.1 M NaOH + 1 M NaCl and shaking for 17 h, (ii) Citrate-Dithionite-Bicarbonate extractable P (non-occluded Fe-bound P + adsorbed-P) obtained by adding 0.3 M Na3C6H5O7 + 1 M NaHCO3 to the residue and heating the suspension in a water bath after adding Na2S2O4, and (iii) HCl extractable P (Ca-bound P) obtained by adding 0.5 M HCl to the residue and shaking for another 1 h. Saturated NaCl was used to wash the residue after each extraction step. Concentrations of P of various fractions were determined by the ascorbic acid method (Murphy and Riley, 1962).

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time (day), Ct and C0 are the concentration of chlorpyrifos at time t and time 0 (mg kg−1), respectively, and k is a firstorder rate constant. The calculated values were fit to the degradation data using SigmaPlot 10.0 (SPSS Inc., 2006). Then the half-life (t1/2) was calculated. Because R2, the coefficient of determination, is not a legitimate descriptive statistic for nonlinear regression analyses (Neter et al., 1996), we have used the Pseudo-R2. The experimental design was a two-way randomized complete block using salinity and bacterial activity as variables and time as a block. The PROC GLM procedure was used to carry out analysis of variance (ANOVA) for comparing the rate constants (k) of chlorpyrifos degradation, microbial activity, SAP activity, and chemical P speciation among treatments (SAS Institute, 2002). 3. RESULTS 3.1. Degradation of Chlorpyrifos in Soils Degradation of chlorpyrifos followed the first-order

2.3. Statistical Analysis To compare the effect of salinization and sterilization on the degradation of chlorpyrifos in soil, we used the following nonlinear regression equation Ct = C0e−kt, where t is

Fig. 1. Degradation of chlorpyrifos in (a) sterilized and (b) unsterilized soils. Error bars are standard errors of three replicates and k is the first-order rate constant.

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kinetics. Rate constants (k) decreased by increasing soil salinity and were further lowered by soil sterilization (Fig. 1). The estimated half-lives (t1/2) for chlorpyrifos degradation in unsterilized soils were 7.1 at EL, 10.0 at EM and 16.9 days at EH soils, and their counterparts in sterilized soils were 9.6, 13.3 and 18.2 days, respectively. The result of PROC GLM showed that the rate constants differed significantly at p < 0.001 (n = 3), and the estimated half-life was longer in sterilized soils than in unsterilized soils, but the difference between them decreased by increasing soil salinity. 3.2. Soil Enzyme Activities The application of chlorpyrifos decreased microbial activity and the extent of this decrease increased with salinity and time (Fig. 2), resulting in a decrease in microbial activity by 51.5% in sterilized soils and by 34.6% in unsterilized soils after 60 days of incubation compared to their respective control soils. However, there was no significant interactive effect between soil sterilization and soil salinity.

Fig. 3. Soil alkaline phosphatase activities (SAP) in (a) sterilized and (b) unsterilized soils. Error bars are standard errors of three replicates.

In contrast, soil alkaline phosphatase activity (SAP) was not inhibited by the addition of chlorpyrifos, and the decrease due to soil sterilization was not as great as that of microbial activity (Fig. 3). The SAP activity was maintained higher in EH soils than in control soils. 3.3. Phosphorous (P) Fractionation

Fig. 2. Total microbial activities in (a) sterilized and (b) unsterilized soils. Error bars are standard errors of three replicates.

Chlorpyrifos addition at a rate of 5.0 mg a.i. kg–1 dry soil did not affect the initial P distribution in the control soils (Table 2). Both the increase in soil salinity and soil sterilization significantly increased the HCl-P fraction and decreased the NaOH-P fraction, with a significant interactive effect between soil salinity and sterilization on both P fractions. Overall, the mineralization of organic-P fraction was greater in EH than in the control soils, and the released inorganic P was partitioned more into the HCl-P fraction than in the other fractions. Soil sterilization and time increased the HCl-P fraction but decreased the organic-P and NaOHP fractions whereas the concentrations of total-P, availableP and CDB-P fractions remained unchanged.

Chlorpyrifos degradation and P fractionation in coastal saline soils

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Table 2. Concentrations of P fractions in soils at 0 and 60 days after the application of chlorpyrifos

C-SEL SEL SEM SEH C-NSEL NSEL NSEM NSEH ANOVA Time Salinity Sterilization Sterilization x Salinity

Total P Organic P (mg kg–1) (mg kg–1) 401 (5)e 72 (3) 388 (9) 77 (9) 402 (7) 76 (7) 399 (2) 70 (9) 397 (8) 73 (3) 406 (5) 72 (4) 407 (5) 70 (0) 406 (5) 67 (4) nsf ns ns ns

*** ns ** ns

0 day Inorganic P (mg kg–1) Total P Organic P a Av. P CDB-Pb NaOH-Pc HCl-Pd (mg kg–1) (mg kg–1) 6 (2) 16 (1) 87 (7) 212 (5) 402 (6) 61 (4) 7 (1) 15 (0) 82 (3) 216 (2) 389 (4) 59 (3) 6 (1) 16 (0) 78 (4) 233 (7) 404 (4) 64 (7) 6 (1) 17 (2) 69 (2) 245 (6) 403 (0) 53(1) 11 (2) 16 (1) 94 (3) 200 (4) 400 (7) 55 (2) 7 (1) 16 (0) 94 (5) 198 (3) 408 (5) 57 (9) 7 (1) 15 (1) 90 (3) 205 (3) 406 (2) 60 (4) 6 (1) 17 (0) 77 (6) 226 (1) 404 (12) 38 (1) ns ns ns ns

ns ns ns ns

** *** *** *

60 day Inorganic P (mg kg–1) Av. P CDB-P NaOH-P HCl-P 9 (1) 15 (2) 87 (6) 230 (1) 9 (1) 11 (1) 83 (6) 237 (1) 7 (0) 16 (2) 61 (4) 258 (16) 8 (1) 14 (1) 30 (3) 309 (1) 9 (1) 16 (2) 92 (3) 231 (5) 8 (2) 14 (0) 90 (3) 223 (2) 8 (1) 13 (2) 87 (3) 237 (2) 8 (1) 15 (1) 78 (3) 251 (6)

*** *** *** *

a

Av. P: Available P CDB-P: Citrate-Dithionite-Bicarbonate Extractable P (non-occluded Fe-bound P + adsorbed-P) c NaOH-P: NaOH Extractable P (Al-bound + occluded Fe-bound P) d HCl-P: HCl Extractable P (Ca-bound P, calcium phosphates) e Mean and standard deviation of three replicates in parentheses f ns: not significant; *: 0.05 ≥ p > 0.01; **; 0.01 ≥ p > 0.001; ***: 0.001 > p b

4. DISCUSSION Degradation of chlorpyrifos in soils was affected by both soil salinity (ionic strength) and soil sterilization (bacterial activity). A decrease in bacterial activity due to soil sterilization decreased the degradation of chlorpyrifos (Fig. 1), and this observation is consistent with the results of Singh and Walker (2006) suggesting that soil bacteria are involved in chlorpyrifos degradation. The effect on the degradation of chlorpyrifos by bacterial activity decreased by increasing ionic strength probably because soil salinity negatively affected bacterial activity and this could be attributed to higher osmolarity outside the bacterial cell than inside (Singh et al., 2003). In this case, intracellular water flows out until the water potential inside the cells equals that outside, with decreased degradation of chlorpyrifos in the solution phase of saline soils via the inhibition of cellular processes (Csonka, 1989). The reduced degradation of chlorpyrifos by increasing soil salinity agreed with the results of Liu et al. (2001). However, the inhibition of the degradation of chlorpyrifos by increasing ionic strength may also be due to chemical properties of chlorpyrifos and conformational changes of extracellular enzymes during adsorption to soil mineral surfaces. Chlorpyrifos has low solubility in distilled water (1.39 mg L–1) and even lower in seawater (73 µg L–1) (Racke, 1993), and thus only a small percentage (