Slow Delivery of a Nitrification Inhibitor to Soil Using Chitosan Hydrogel PDF

Chemosphere 93 (2013) 2854–2858 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier....

Chemosphere 93 (2013) 2854–2858 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere Technical Note Slow delivery of a nitrification inhibitor (dicyandiamide) to soil using a biodegradable hydrogel of chitosan E.P. Minet a,⇑, C. O’Carroll b, D. Rooney b, C. Breslin b, C.P. McCarthy b, L. Gallagher b, K.G. Richards a,⇑ a Teagasc Environment Research Centre, Johnstown Castle, Co. Wexford, Ireland b National University of Ireland Maynooth, Chemistry Department, Maynooth, Co. Kildare, Ireland h i g h l i g h t s  Dicyandiamide (DCD) was encapsulated in glyoxal-crosslinked chitosan hydrogel beads.  Chitosan delayed the release of nitrification inhibitor DCD in water and soil.  DCD release was controlled by glyoxal polymerisation inside chitosan.  The higher glyoxal polymerisation the more delayed DCD release in water or in soil.  The higher glyoxal polymerisation the less DCD encapsulated in the beads. a r t i c l e i n f o a b s t r a c t Article history: Using chemical inhibitors to reduce soil nitrification decreases emissions of environmental damaging Received 8 May 2013 nitrate and nitrous oxide and improves nitrogen use efficiency in agricultural systems. The efficacy of Received in revised form 29 July 2013 nitrification inhibitors such as dicyandiamide (DCD) is limited in soil due to biodegradation. This study Accepted 13 August 2013 investigated if the persistence of DCD could be sustained in soil by slow release from a chitosan hydrogel. Available online 12 September 2013 DCD was encapsulated in glyoxal-crosslinked chitosan beads where excess glyoxal was (i) partly removed (C beads) or (ii) allowed to dry (CG beads). The beads were tested in water and in soil. The beads con- Keywords: tained two fractions of DCD: one which was quickly released in water, and one which was not. A large Chitosan Hydrogel DCD fraction within C beads was readily available: 84% of total DCD bead content was released after Dicyandiamide 9 h immersion in water, while between 74% and 98% was released after 7 d in soil under low to high Slow release moisture conditions. A lower percentage of encapsulated DCD was readily released from CG beads: Nitrification inhibitor 19% after 9 h in water, and 33% after 7 d in soil under high rainfall conditions. Kinetic analysis indicated Nitrogen loss that the release in water occurred by quasi-Fickian diffusion. The results also suggest that DCD release was controlled by bead erosion and the leaching of glyoxal derivatives, predominantly a glyoxal-DCD adduct whose release was positively correlated with that of DCD (R2 = 0.99, p 6 0.0001). Therefore, novel chitosan/glyoxal composite beads show a promising slow-release potential in soil for agrochemicals like DCD. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction for these limitations, researchers have investigated complemen- tary N mitigation techniques, such as blocking NHþ 4 oxidation to Reactive N losses and greenhouse gas (GHG) emissions from NO3 using nitrification inhibitors (Yu et al., 2007; Watson et al., agricultural soils are an important source of water and air pollution 2009). Studies have revealed that the inhibitory potential of dic- (Stark and Richards, 2008a). In this contamination process, NHþ 4 yandiamide (DCD) can curb nitrate leaching by up to 76% (Di and plays a central role. To date, national and international green legis- Cameron, 2004) and N2O emissions by up to 70% (Di et al., 2007). lation has aimed to control such N losses and GHG emissions but Yet for all its effectiveness, degradation in soil limits the persis- with limited success (Stark and Richards, 2008b). To compensate tence of DCD (Estermaier et al., 1992), which has a half-life esti- mated between 110 d at 5 °C and approximately 20 d at 25 °C (Kelliher et al., 2008). Consequently, repeated soil applications ⇑ Corresponding authors. Tel.: +353 539171261; fax: +353 539142213 (K.G. are required to maintain efficacy. Richards). The ephemeral subsistence of agrochemicals applied to soils is E-mail addresses: [email protected] (E.P. Minet), [email protected] a recurrent problem that affects a large range of products. (K.G. Richards). 0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.08.043  E.P. Minet et al. / Chemosphere 93 (2013) 2854–2858 2855 Attempts to overcome this drawback have seen the emergence of 3. Results and discussions controlled release formulations (CRF) (Fernández-Pérez et al., 2001), whereby active compounds are encapsulated in a slow re- 3.1. Beads characterisation lease matrix prior to soil application. The expected advantages of this approach include: (i) prolonged activity in soil (protection 3.1.1. Bead morphology and surface analysis from microbial degradation until release), (ii) reduced number SEM micrographs of lightly washed C and CG beads are pre- of applications, (iii) reduced costs (due to single application) sented in Fig. 1. The beads are distorted spheres, with dimensions and (iv) reduced environmental loss (Akelah, 1996). A CRF with between 1.5 and 2 mm. Unlike CG beads, the surface morphology some interesting potential is chitosan, a polymeric hydrogel well of the C beads was quite wrinkled. Both bead types had a highly known for its slow drug delivery use in the pharmaceutical indus- compact interior showing no pores with diameters in the microm- try (Rinaudo, 2006). This non-toxic and biodegradable copolymer eter range. The interior of the CG beads appeared more fibrous in of b-(1 ? 4)-linked 2-acetamido-2-deoxy-D-glucopyranose and nature compared to that of the C beads. Despite all beads being 2-amino-2-deoxy-D-glucopyranose results from the partial made from the same amount of chitosan, the initial dry weight deacetylation of chitin, which is the main component of the of CG beads (4.408 ± 0.027 mg per bead) was 46% higher than that exoskeleton of crustaceans. Like all hydrogels, chitosan is highly of C beads (3.090 ± 0.009 mg per bead). The increased weight of the porous and hydrophilic which allows a high rate of wetting. This CG beads was attributed to the excess glyoxal, which can undergo a permits an encapsulated compound to diffuse out at a speed number of reactions within the beads. Glyoxal is known to undergo which is controlled by the crosslinking density and pore size (Ber- oligomerisation upon loss of water and it can easily polymerise in ger et al., 2004). To the best of our knowledge slow release of the solid state (Loeffler et al., 2006; Nakajima et al., 2007). In addi- DCD has only been attempted by Bishop (2010). Moreover, very tion, it is possible that glyoxal could react with the guanidine few studies have considered the use of chitosan-derived systems group of the DCD to form Schiff base glyoxal-DCD adducts (Panic- for the controlled delivery of agrochemicals. Some notable exam- ucci and McClelland, 1989). These excess glyoxal compounds were ples include the delivery of neem seed oil (Devi and Maji, 2009), partly removed from the C beads before the drying step by washing plant hormones (Quiñones et al., 2010; Fan et al., 2012) and fer- in a solution containing DCD. tilisers and herbicide (Teixeira et al., 1990). The objective of our study was to explore for the first time the effect of a slow release system (chitosan xerogel beads loaded with 3.1.2. DCD content within the beads dicyandiamide) on DCD soil release. An underlying purpose was to Immersing the beads in deionised or acidified water was used to assess the potential of chitosan encapsulation as a credible alterna- extract the DCD from the hydrogel and the quantity extracted was tive to repeated soil applications of DCD. measured by HPLC analysis. This study revealed that C beads contained significantly more (p 6 0.0001) DCD (about 0.2 mg DCD per bead depending on the extraction method) than CG beads 2. Experimental (less than 0.05 mg DCD per bead) (Fig. 2a). Acidification signifi- cantly increased (p 6 0.0001) the amount of DCD extracted when DCD was encapsulated in chitosan hydrogel beads formed by compared with immersion in deionised water. It is likely that the precipitation of an acidified chitosan gelling solution and cova- acidification causes an increase in DCD release from the CG beads lently crosslinked with glyoxal. In these beads, excess glyoxal due to two factors. Acidification caused the beads to swell consid- was (i) partly removed by washing with an aqueous solution of erably and therefore more DCD could diffuse out of the beads. DCD (60 mM) (C beads) or (ii) allowed to dry (CG beads) (see Moreover, it is possible that glyoxal-DCD adducts could be hydro- Method SM-1 for details). To estimate the amount of DCD encap- lysed under acidic conditions to re-form DCD. A study involving sulated in C and CG beads, DCD was extracted with acidified and mechanical destruction of the beads coupled with acidification unacidified deionised water (Method SM-2). DCD release from the confirmed that the total content of DCD in the beads was reliably beads, kinetics of that release and swelling ratio (Sw %) were obtained by simply incubating uncrushed beads in acidified water investigated in water over 2 wk incubations (15 min up to 14 d) for 2 wk (0.219 and 0.043 mg DCD per C and CG beads respec- (Method SM-3). DCD soil release from C beads was assessed un- tively). The incubation of DCD solutions under similar conditions der two treatments (rainfall, soil moisture expressed as water confirmed the stability of the molecule, so DCD extracted from holding capacity (WHC)), three rates of added water and three beads was assumed to truly reflect the amount encapsulated. The incubation times (up to 7 d) (Method SM-4). DCD soil release fact that not all the DCD was released from the beads after pro- from CG beads was assessed under the highest rate of rainfall longed immersion in deionised water implied the existence of only. two DCD fractions in the beads: available DCD which can easily dif- Bead morphology and surface analyses were carried out with a fuse out during water incubation, and locked DCD which remains scanning electron microscope (SEM). The DCD content in water trapped in the beads. samples and KCl soil extracts was quantified by HPLC analysis Besides DCD, a second compound with a HPLC retention time of according to a modified method by Turowski and Deshmukh 4.6 min was detected in beads incubated in water (no other mean- (2004) (Method SM-5). ingful peak was detected between 190 and 300 nm during the The effects of independent variables (bead type and DCD extrac- 10 min run). The peak did not correspond to those observed for tion method in Section 3.1.2; soil treatment, treatment rates and the starting glyoxal solution. It was detected only when both incubation time for DCD soil release from C beads in Section 3.2) DCD and glyoxal were present within the beads (no peak was and their interactions on response variables (amount of DCD detected when beads were prepared without glyoxal or without encapsulated in beads or released in soil in Sections 3.1.2 and 3.2 DCD). The destruction of beads by acidification released about respectively) were investigated by ANOVA after model assump- twice as much of this glyoxal derivative compared to incubation tions were met. When significant effects were found (p 6 0.05), in deionised water (Fig. 2b). This clearly indicates that this Bonferroni Post Hoc tests were used to make pairwise compari- compound, probably a glyoxal-DCD adduct, does not undergo facile sons. Parametric tests (correlations, linear and non-linear regres- acid hydrolysis to release more DCD. As can be seen from Fig. 2b, sions) were carried out to explore some relationships between CG beads released 54% more glyoxal-DCD adduct than C beads continuous variables. when acidified (53% with acidified crushed beads), which is 2856 E.P. Minet et al. / Chemosphere 93 (2013) 2854–2858 Fig. 1. SEM micrographs of (a) C beads (Chitosan) and (b) CG beads (Chitosan/Glyoxal). 0.250 consistent with the earlier observation that CG beads were 46% a uncrushed beads in deionised water uncrushed beads in acidified water heavier than C beads due to a higher glyoxal content. As it is only DCD extracted per bead (mg) after acidification that the locked fraction of DCD was also released 0.200 from the beads, it is likely that glyoxal derivatives (i.e. glyoxal-DCD adducts and glyoxal polymers/oligomers) trapped the locked DCD 0.150 within the beads. Glyoxal is a very commonly used covalent cross- linker for chitosan CRFs, but its ability to undergo polymerisation 0.100 and/or oligomerisation within chitosan beads has not been re- ported, nor the effect this may have on the release of the entrapped species within the polymeric matrix. 0.050 3.1.3. DCD release into water 0.000 The incubation of chitosan beads in deionised water resulted in C beads CG beads a rapid release of DCD before it reached a plateau (Fig. 3a): C and 2500 CG beads lost 84 and 19% of their total DCD content respectively b uncrushed beads in deionised water after 9 h. These values correspond to available DCD fractions held uncrushed beads in acidified water within the beads (Table SM-1). After 14 d, the fractions of total Glyoxal-DCD adduct HPLC 2000 DCD content released slightly increased to 87% and 23% for C and CG beads respectively, highlighting that glyoxal-crosslinked chito- peak area (mV s) 1500 san can retain a substantial proportion of its DCD content for long periods of time. 1000 Conversely, the locked DCD fractions in C and CG beads repre- sented 16 and 81% of the total DCD content respectively. Despite large differences in their total DCD content, this translated into C 500 and CG beads containing similar amounts of locked DCD (Table SM-1). It should be noted that a brief final washing of the 0 beads with deionised water removed 32% and 17% of the total C beads CG beads DCD content of C and CG beads respectively, indicating that a sub- stantial proportion of the available DCD fraction simply lies close Fig. 2. Amount of (a) DCD (in mg ± one deviation of the standard error of the mean (SE), n = 3) and (b) glyoxal-DCD adduct (measured as a HPLC peak area in mV s) to the outer surface of the beads. extracted from C (Chitosan) and CG (Chitosan/Glyoxal) beads following incubation At each incubation time, beads were removed from the in deionised or acidified water (pH of 2.1). (All incubations carried out at 5 °C for deionised water, dried and weighed. A weight loss, determined 14 d with ten beads in 100 mL.) by comparison with the initial (pre-incubation) dry weight, was  E.P. Minet et al. / Chemosphere 93 (2013) 2854–2858 2857 observed. It was positively correlated with the HPLC peak area of Released fraction of total DCD bead content 1.0 0.9 a the glyoxal-DCD adduct for C and CG beads (R2 > 0.9, p 6 0.0001) (Fig. SM-1). The beads were therefore being eroded by releasing 0.8 quantifiable amounts of a glyoxal-DCD adduct. Quite significantly, 9h 0.7 the release of the glyoxal-DCD adduct was positively correlated 0.6 C beads (R2 = 0.99, p 6 0.0001) with that of DCD (Fig. 3b). It is not clear CG beads why both sets of beads behaved slightly differently (polynomial 0.5 fitting for C beads, linear fitting for CG beads). 0.4 0.3 3.1.4. Swelling ratio 0.2 The swelling ratio (Sw %) of CG and C beads was measured upon 0.1 9h immersion in water as a function of time over a 14 d incubation. It was observed that the CG beads had a higher swelling ratio com- 0.0 0 2 4 6 8 10 12 14 pared to the C beads by 27% (Fig. SM-2). In both cases, most of Incubation time (d) the swelling took place over the first 9 h of immersion. This was unexpected because it had been anticipated that the increased 1.0 amount of glyoxal polymers/oligomers in CG beads would block b y = -2E-05 x2 + 0.0045 x + 0.52 C beads some pores and make it harder for water to diffuse in. Released fraction of total DCD bead content CG beads 0.9 R2 = 0.99 Poly. (C beads) p 0.0001 Linear (CG beads) 0.8 3.1.5. DCD release kinetics 9h 0.7 Kinetic analysis of DCD bead release was carried out during incubation in water over nine time points between 15 min and 0.6 9 h (Table SM-2). The Korsmeyer–Peppas model displayed the best e tim fit among all four models tested for C (R2 = 0.97) and CG beads 0.5 n 15 min atio (R2 = 0.87). However, results should be read with caution as CG ub 0.4 inc beads contained small amounts of DCD. The diffusional exponent n values were lower than 0.5 suggesting that DCD was released 0.3 9h by quasi-Fickian diffusion. Deviations from ideal Fickian diffusion 0.2 could be explained by the existence of a bead erosion factor related y = 0.0004 x + 0.01 to the release of glyoxal derivatives. 15 min 0.1 R2 = 0.99 p 0.0001 0.0 3.2. DCD release in soil 0 100 200 300 400 500 600 Glyoxal-DCD adduct HPLC peak area (mV s) The incubation of lightly washed C beads (0.148 mg DCD per Fig. 3. Fraction of total DCD bead content (±SE, n = 3) released from C (Chitosan) bead) in soil under simulated rainfall and soil moisture conditions and CG (Chitosan/Glyoxal) beads during incubation in deionised water, plotted resulted in the delayed release of DCD over 7 d (Fig. 4). DCD release against (a) incubation time (15 min up to 14 d) and (b) glyoxal-DCD adduct HPLC in soil significantly increased with time (p 6 0.0001): the amount peak area (in mV s ± SE, n = 3) (All incubations carried out at 5 °C for 15 min up to of DCD recovered in soil was the lowest after 1 d, followed by 4 d 14 d with ten beads in 100 mL of deionised water; polynomial and linear regression and then 7 d (p 6 0.0001 for all pairwise comparisons). Treatment lines fitted for C and CG beads respectively in (B)). had a significant effect on DCD soil release (p 6 0.0001) as it was 3.2 100% release 98 1 day 3.0 2.8 88 4 days 85 2.6 80 80 7 days 80 80 80 2.4 74 DCD extracted from soil (mg) 71 2.2 68 67 2.0 63 59 1.8 56 1.6 45 1.4 39 1.2 35 1.0 0.8 100% release 0.6 0.4 33 15 22 0.2 0.0 Low WHC Medium WHC High WHC Low Rain Medium Rain High Rain High Rain (41 %) (48 %) (64 %) (0.1 mm) (1.0 mm) (3.1 mm) (3.1 mm) C beads CG beads (15.1 kg DCD ha -1) (3.6 kg DCD ha-1) Fig. 4. Amount of DCD extracted from soil (in mg ± SE, n = 3) after incubation with twenty C (Chitosan) and CG (Chitosan/Glyoxal) beads at 5 °C for 1 up to 7 d under varying rates of rainfall or % water holding capacity (WHC) (C and CG beads underwent a preliminary light washing step with deionised water; figures over bars represent the percentages of total DCD bead content released and recovered in soil). 2858 E.P. Minet et al. / Chemosphere 93 (2013) 2854–2858 observed that rainfall caused more release than soil moisture (i.e. Acknowledgements WHC treatment) alone. This most likely reflected the fact that the beads were wet more thoroughly and quickly when water The authors thank the Irish Department of Agriculture, Food was applied from above (rain) than when moisture had to diffuse and the Marine (DAFM) for funding this study (Research Stimulus up from the soil surface into the hydrogel. The treatment rate also Fund Programme – RSF 07545) and Dr. Jim Grant (Teagasc) for ad- showed a significant effect on DCD soil release (p 6 0.0001): the vice on statistics. The DAFM had no other role in this study. amount of DCD in soil was greatest at the high levels of rainfall or WHC, followed by medium and then low levels (p 6 0.0005 for Appendix A. Supplementary material all pairwise comparisons). The interaction between treatment rate and time was also significant (p = 0.0187). Supplementary data associated with this article can be found, in The fractions of total DCD bead content released in soil by C the online version, at http://dx.doi.org/10.1016/j.chemosphere. beads after 7 d were close to values of the available DCD fraction 2013.08.043. (i.e. 84%), except in the high rain treatment where almost all the encapsulated DCD was released. This indicates that under low or References moderate moisture, C beads can quickly release a large portion if not all their available DCD fraction without depleting a significant Akelah, A., 1996. Novel utilisation of conventional agrochemicals by controlled proportion of the locked reservoir. release formulation. Mater. Sci. Eng. C – Mater. 4, 83–98. Berger, J., Reist, M., Mayer, J.M., Felt, O., Peppas, N.A., Gurny, R., 2004. Structure and The behaviour of lightly washed CG beads (0.035 mg DCD per interactions in covalently and ionically crosslinked chitosan hydrogels for bead) was different. The amount of DCD released in soil under biomedical applications. Eur. J. Pharm. Biopharm. 57, 19–34. the high rainfall treatment was much lower than with C beads Bishop, P.A., 2010. Polymer Coated Controlled Release Agrichemicals as Mitigation Tools in Pastoral Farming. PhD thesis, Massey University, Palmerston North, (Fig. 4). 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