Biology and Fertility of Soils

, Volume 52, Issue 2, pp 243–250 | Cite as

Fungal bioaugmentation of two rice husk-based biomixtures for the removal of carbofuran in on-farm biopurification systems

  • Kattia Madrigal-Zúñiga
  • Karla Ruiz-Hidalgo
  • Juan Salvador Chin-Pampillo
  • Mario Masís-Mora
  • Víctor Castro-Gutiérrez
  • Carlos E. Rodríguez-Rodríguez
Original Paper

Abstract

The ligninolytic fungus Trametes versicolor was employed in the bioaugmentation of compost- (GCS) and peat-based (GTS) biomixtures for the removal of the insecticide-nematicide carbofuran (CFN). Among several lignocellulosic substrates, fungal colonization was best supported in rice husk, and this pre-colonized substrate was used to prepare the biomixtures. Estimated half-lives for CFN were 3.4 and 8.1 days in the GTS and GCS biomixtures, respectively. The CFN transformation products 3-hydroxycarbofuran and 3-ketocarbofuran were detected at the moment of CFN application, but their concentration continuously decreased to complete removal in both biomixtures. Mineralization of 14C-radiolabeled CFN was faster in GTS (k = 0.00248 day−1) than in GCS (k = 0.00188 day−1). Complete elimination of the toxicity in the matrices was demonstrated after 48 days. Overall data suggest that the bioaugmentation improved the performance of the GTS rather than the GCS biomixture.

Keywords

Biopurification system Bioaugmentation Degradation Pesticides Fungi Toxicity 

Introduction

On-farm biopurification systems represent a biotechnological approach for the mitigation of point source contamination by pesticides, for example with wastewater derived from agricultural activities (Castillo et al. 2008). The main component of the biopurification systems is the biomixture, which acts as the biologically active core that accelerates the degradation of pesticides. The traditional biomixture developed in Sweden (Torstensson and Castillo 1997) consisted of straw, peat, and soil at a volumetric ratio of 2:1:1.

Components of the biomixture include soil, a humic material, and a lignocellulosic substrate. Soil should be preferably pre-exposed to the target pesticide as it contains important degrading microbiota. Peat is the most used humic component, employed to enhance the retention of the pesticides in the matrix; it can be substituted by compost, usually resulting in good removal performances (Coppola et al. 2007; Kravvariti et al. 2010). The lignocellulosic substrate promotes the colonization by ligninolytic fungi, which are involved in the degradation of diverse organic pollutants, thanks to their enzymatic systems including lignin-modifying enzymes (laccases and peroxidases) and intracellular complexes (cytochrome P450) (Doddapaneni and Yadav 2004; Pointing 2001). The organic contaminants degraded by ligninolytic fungi comprise polycyclic aromatic hydrocarbons, textile dyes, pharmaceuticals, brominated flame retardants and pesticides (Asgher et al. 2008; Cruz-Morató et al. 2013; Rodríguez-Rodríguez et al. 2012a), and their ability to remove pesticides in biomixture-like systems and other solid-matrices has been reviewed (Rodríguez-Rodríguez et al. 2013).

In this work, carbofuran (CFN) was employed as the model pesticide. It is a carbamate commonly used as a broad spectrum systemic nematicide, insecticide, and acaricide. It is highly toxic for mammals and aquatic life (Gupta 1994), and for this reason, it has been banned in agricultural activities in USA, EU, and in other countries; nonetheless, developing countries still use this pesticide and unregulated applications result in its presence in different environmental compartments (Otieno et al. 2010).

The aim of this work was to evaluate the performance of bioaugmented biomixtures in the removal of CFN, employing the ligninolytic fungus Trametes versicolor as the bioaugmentation agent. T. versicolor is a white rot fungus, capable of degrading diverse organic pollutants in several matrices (Tuomela et al. 1998; Rodríguez-Rodríguez et al. 2014). Biomixtures prepared using either compost or peat, soil, and rice husk as the lignocellulosic substrate were used to eliminate CFN and two of its transformation products; for this reason, we also monitored the mineralization of radiolabeled CFN and the toxicity reduction in the matrix.

Materials and methods

Chemicals and reagents

Commercial CFN formulation (Furadan® 48SC, 48 % w/v) was acquired from a local store. Analytical standards CFN (2,2-dimethyl-2,3-dihydro-1-benzofuran-7-ylmethylcarbamate, >99 % purity), 3-hydroxycarbofuran (99.5 %), and 3-ketocarbofuran (99.5 %) were obtained from Chemservice (West Chester, Pennsylvania, USA). Radio-labeled CFN (14C-CFN; [Ring-U-14C]-Carbofuran; 2.89 × 109 Bq g−1; radiochemical purity 100 %; chemical purity 99.5 %) was obtained from Izotop (Institute of Isotopes Co., Budapest, Hungary). Carbendazim-d3 (surrogate standard, 99.0 %) and carbofuran-d4 (internal standard, 99.5 %) were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Potassium hydroxide analytical grade was purchased from Merck (Darmstadt, Germany). Ultima Gold cocktail for Liquid Scintillation Counting was purchased from PerkinElmer (Waltham, Massachusetts, USA). Solvents and extraction chemicals are those reported by Ruiz-Hidalgo et al. (2014).

Fungal strain and biomixture components

T. versicolor (ATCC 42530) was obtained from the American Type Culture Collection and maintained by subculturing every 30 days on potato dextrose agar slants (pH 4.5) at 25 °C in the dark. T. versicolor-blended mycelial suspension was prepared according to a procedure by Font Segura et al. (1993), modified by using Sabouraud dextrose broth as culture medium. Clay loam soil (sand 40 %, silt 27 %, clay 33 %; 2.71 % C; 0.29 % N; pH 5.9) was collected from the upper soil layer (0–20 cm) of an onion field with history of CFN application, in Tierra Blanca, Cartago, Costa Rica. Soil was air-dried and sieved (<2 mm). Rice husk obtained from an agricultural input supplier from Tierra Blanca, Cartago, Costa Rica, was employed as the lignocellulosic substrate. Garden compost, employed as the humic component, was collected from a composting station located at Universidad de Costa Rica, dried and sieved (<2 mm). Peat moss (Berger, Saint-Modeste, Québec, Canada) was acquired from a local store.

Experimental procedures

Screening of fungal colonization on lignocellulosic substrates

Colonization of five lignocellulosic substrates by T. versicolor was evaluated before choosing the substrate to be used in the biomixtures. These substrates were newsprint paper, coconut fiber, rice husk, wood chips, and cane bagasse. The screening was performed in triplicate flasks (250 mL) containing the autoclaved lignocellulosic substrate (ratio 1:2, dry substrate/water, w/v), which was then inoculated with blended mycelial suspension (0.8 mL g−1 of dry substrate). Flasks were incubated at 25 °C (±1) for 24 days; colonization was visually monitored, and samples were periodically taken for extraction and determination of laccase activity.

Degradation assays

CFN degradation assays were performed with 5 g of biomixture. Plastic tubes (12 cm × 3.5 cm) containing the proper amount of humidified rice husk were sterilized at 121 °C for 15 min prior to the inoculation of blended T. versicolor mycelial suspension (0.35 mL g-1 of dry rice husk). After fungal colonization for 10 days at 25 °C (±1), biomixtures were prepared by adding pre-exposed soil and either peat or compost at a ratio colonized-rice husk/soil/peat (or compost) of 50:25:25 % (v/v), to obtain the biomixtures GTS (peat-based biomixture) and GCS (compost-based biomixture). The biomixtures were then spiked with Furadan® 48SC to a CFN final concentration of ∼10 mg kg−1 and incubated in static conditions at 25 °C (±1) until the end of the assay. Water was periodically added in order to keep a constant water content in the matrices. Two sets of triplicate biomixture samples were periodically sacrificed over a 62-day period to determine the concentration of CFN and its transformation products, ecotoxicological tests, and laccase activity.

Mineralization assays

The mineralization of 14C-CFN was determined by trapping the evolved 14CO2 in 0.1 M KOH in flasks containing 50 g of the biomixtures and spiked with commercial CFN (50 mg kg−1) and 14C-CFN (3000 dpm g−1) (Ruiz-Hidalgo et al. 2014). Each treatment was replicated three times. The flasks were incubated in the dark at 25 °C (±1) for 64 days; water content was kept constant as mentioned above. KOH solutions in the flasks were withdrawn at selected times and replaced with the same amount of fresh KOH. Flasks containing 50 g of non-bioaugmented soil were used as controls. 14C activity from the mineralized 14CO2 was determined in the KOH samples as described below.

Analytical procedures

Extraction and quantification of CFN and transformation products

Extraction was carried out as described by Ruiz-Hidalgo et al. (2014). Carbendazim-d4 and carbofuran-d3 were added as internal standards (type II and I, respectively) to samples (5 g) obtained from degradation experiments. CFN and its transformation products were analyzed by LC-MS/MS using ultra-high-performance liquid chromatography (UPLC 1290 Infinity LC, Agilent Technologies, CA, USA) coupled to a triple quadrupole mass spectrometer (6460 Agilent Technologies, CA, USA). Chromatographic separation was done at 40 °C by injecting 6-μL samples (2 μL loop) in a Poroshell 120 EC-C18 column (100 mm × 2.1 mm i.d., particle size 2.7 μm; Agilent Technologies, CA, USA), and using acidified water (formic acid 0.1 % v/v, A) and acidified methanol (formic acid 0.1 % v/v, B) as mobile phases. The mobile phase flow was 0.3 mL min−1 at the following conditions: 30 % B for 3 min, followed by a 15-min linear gradient to 100 % B, 4 min at 100 % B and 0.1-min gradient back to 30 % B, followed by 5 min at initial conditions. Selected transitions for the analytes are shown in Table 1. The mass spectrometer employed a jet stream (electrospray) ionization source operating at the following conditions: gas temperature 300 °C, gas flow 7 L min−1, nebulizer 45 psi, sheath gas temperature 250 °C, sheath gas flow 11 L min−1, capillary voltage 3500 V, and nozzle voltage 500 V; MS1 and MS2 were heated at 100 °C. Recovery was 91 % for CFN, 98 % for 3-hydroxycarbofuran and 95 % for 3-ketocarbofuran. Limit of detection (LOD) and limit of quantification (LOQ) were 13 and 26 μg kg−1 for CFN and 3-ketocarbofuran, respectively, and 16 and 32 μg kg−1 for 3-hydroxycarbofuran, respectively.
Table 1

Selected transitions and other parameters in the detection of CFN and its transformation products using the dynamic multiple reaction monitoring (MRM) method

Compound

Retention time (min)

Transition

Fragmentor (V)

Collision energy (V)

Type of transition

3-Hydroxycarbofuran

3.60

238 → 163

72

9

Q

238 → 107

33

q

3-Ketocarbofuran

6.10

236 → 161

82

17

Q

236 → 179

9

q

Carbofuran

7.92

222 → 165

82

9

Q

222 → 123

21

q

Carbendazim-d4

1.45

196 → 164

102

17

Q

196 → 136

34

q

Carbofurán-d3

7.92

225 → 165

86

9

Q

225 → 123

21

q

Q quantification transition, q qualifier transition

Determination of 14CO2

Scintillant liquid (8 mL) was added to 2-mL aliquots taken from the KOH solution, and the 14C activity was measured by liquid scintillation using a Beckman LS6000SC counter. The total cumulative 14CO2 activity and the initially added 14C-CFN activity were used to calculate the percentage of 14C-pesticide mineralized. Pesticide mineralization was modeled by a first-order model.

Laccase activity

Laccase was extracted from solid samples according to Rodríguez-Rodríguez et al. (2010); briefly, 30-mL acetate buffer (pH 5) was added to 3 g of homogenized biomixture, shaken for 30 min and centrifuged at 2862g for 15 min at 4 °C. Laccase activity of the supernatant was measured by a modified version of the method for the determination of manganese peroxidase activity (Kaal et al. 1993), using 2,6-dimethoxyphenol (DMP) as the substrate (extinction coefficient 24 800 M−1cm−1; λ = 468 nm) (Wariishi et al. 1992). Enzymatic activity was expressed as activity units (U) per kilogram of biomixture. One unit was defined as the number of micromoles of DMP oxidized per minute.

Acute toxicity: Daphnia magna immobilization test

An acute test based on D. magna immobilization was employed to estimate the residual toxicity in the biomixtures. The test was conducted following the U.S. EPA guidelines (EPA 2002) modified as described by Ruiz-Hidalgo et al. (2014), using aqueous extracts from unitary biomixtures sampled during a 62-day period as analytical matrix. Aqueous extracts were obtained according to the protocol EPA-823-B-01-002 (2001). EC50, the concentration producing 50 % of immobilization in the daphnids, was determined using the TOXCALC (Toxicity Data Analysis Software from Tidepool Scientific Software). Toxicity was expressed as toxicity units (TU), calculated according to the expression: TU = (EC50)−1 × 100.

Results and discussion

The degradation of CFN was assayed in two fungal bioaugmented rice husk-based biomixtures employing T. versicolor as the bioaugmentation agent. The efficiency of the biomixtures was evaluated by considering the decrease in concentration of the parent compound, the production of transformation metabolites, the mineralization of 14C-CFN, and the decrease in the toxicity during the removal treatment.

The use of alternative lignocellulosic materials in biomixtures depends on the geographical area where the removal process occurs (Urrutia et al. 2013). In order to employ the most suitable lignocellulosic substrate for the growth and activity of T. versicolor in the biomixture, several lignocellulosic residues were tested by determining fungal colonization and laccase activity, which may be considered as an indicator of the oxidative power of the matrix, and it is also one of the most important enzymatic activities involved in the degradation of various organic pollutants by white rot fungi (Asgher et al. 2008; Pointing 2001). Laccase activity profiles during the growth of T. versicolor in five different lignocellulosic substrates are shown in Fig. 1. Enzymatic activity was negligible in cane bagasse during the 24-day period, but it was detected in the other substrates; this could be ascribed to the lowest lignin content of cane bagasse (21.1 %) respect to the other substrates and to potentially inhibitory compounds in this matrix, because visual colonization was not observed in the incubation period. Even though rice husk did not have the highest lignin content (36.8 %, lower than coconut fiber, 56.1 %), it showed a high laccase activity with a peak of 588 U kg−1 (±115) after 24 days. The maximum enzymatic activities of the other substrates were 338 U kg−1 (±59), 278 U kg−1 (±123), and 143 U kg−1 (±47) for newsprint paper, wood chips, and coconut fiber, respectively. On the other hand, visual fungal colonization was mainly observed in rice husk and wood chips, which correlated with high laccase activity values. These results, with the fact that the intrinsic toxicity of rice husk was very low (1 TU, lower than other substrates, as determined by acute toxicity tests with D. magna, Ruiz-Hidalgo et al. 2014), were at the basis of the choice of rice husk as the substrate in the bioaugmented biomixtures.
Fig. 1

Laccase activity during the colonization of several lignocellulosic substrates by T. versicolor: a coconut fiber (black circle), cane bagasse (white circle), and rice husk (black inverted triangle); b newsprint paper (black square) and wood chips (white square). Each value is the mean of three replicates ± standard deviation of the mean

Two rice husk-based biomixtures were prepared using two different humic components: compost or peat. The rice husk was colonized by T. versicolor before preparing the biomixtures. The profiles of CFN disappearance in the biomixtures are shown in Fig. 2a; this disappearance depended on both adsorption and biological processes. The elimination of CFN was higher in GTS (91.7 % elimination after only 6 days), than in GCS (33.8 %), with initial removal rates of 1.32 mg kg−1 day−1 (GTS) and 0.62 mg kg−1 day−1 (GCS). By day 13, over 98.5 % CFN was removed in GTS, and this percentage reached 99.5 % at the end of the incubation period after 62 days. On the contrary, 83.0 % CFN was eliminated from GCS in 13 days and 96.4 % at the end of the incubation period. Estimated CFN half-lives in the matrices were 3.4 days with GTS and 8.1 days with GCS and significantly lower than the half-life of 16.8 days obtained in the soil used to prepare the biomixtures (Chin-Pampillo et al. 2015a). Great differences were observed when these data were compared with those of non-bioaugmented biomixtures: In the case of GCS, when T. versicolor was added, there was an increase in the half-life respect to the non-bioaugmented GCS (3.8 days, Table 2); this could be ascribed to competition for growth substrates between T. versicolor and indigenous CFN degrading microorganisms, or to the presence of large amounts of fungal metabolites from the pre-colonized rice husk that could exert some inhibitory effect on other microbial communities (either bacteria or fungi) with degrading capacity in the biomixture. On the contrary, the bioaugmented GTS biomixture considerably reduced the CFN half-life respect to the non-bioaugmented GTS (10.3 days) (Chin-Pampillo et al. 2015b). There are several reasons that may explain the selective enhancement of the GTS biomixture by the fungus respect to GCS: (i) The content of lignin in GTS was higher than in GCS (6.91 vs 4.39 %), (ii) the ratio C/N was higher in GTS than in GCS (20.8 vs 14.7), and (iii) the pH in GTS was lower than in GCS (6.4 vs 6.9). Probably, these characteristics (higher lignin content and C/N ratio, and lower pH) favored the colonization of the ligninolytic fungus, as well as promoted the production of fungal degrading enzymes (Castillo and Torstensson 2007; Eggert et al. 1996; Tavares et al. 2006) in the GTS biomixture compared to the GCS biomixture.
Fig. 2

Removal of CFN (a) and transformation products (b) in bioaugmented biomixtures GCS and GTS. a CFN in GCS (black circle) and GTS (white circle). b 3-ketocarbofuran (black circle) in GCS, 3-hydroxycarbofuran (white circle) in GCS, 3-ketocarbofuran (black inverted triangle) in GTS, and 3-hydroxycarbofuran (white triangle) in GTS. Each value is the mean of three replicates ± standard deviation of the mean. GCS is the biomixture containing rice husk, compost, and soil; GTS is the biomixture containing rice husk, peat, and soil

Table 2

Removal and mineralization of CFN and transformation products in different matrices related to the design of bioaugmented biomixtures

Matrix

CFN estimated half-life (day)

Mineralization rate constant, k (day−1)

Transformation products from CFN

Reference

Bioaugmented GCS

8.1

0.00188

3-Hydroxycarbofuran (initial concentration of 0.244 mg kg−1 to <LOD after 13 days); 3-ketocarbofuran (initial concentration of 0.163 mg kg−1 to <LOD after 34 days)

This work

Bioaugmented GTS

3.4

0.00248

3-Hydroxycarbofuran (initial concentration of 0.193 mg kg−1 to <LOD after 6 days); 3-ketocarbofuran (initial concentration of 0.105 mg kg−1 to <LOD after 20 days)

 

Soil

16.8

0.00203

Not determined

Chin-Pampillo et al. (2015a)/This work

Non-bioaugmented GCS

3.8

0.00158

3-Hydroxycarbofuran (<LOQ, 0.021 mg kg−1); 3-ketocarbofuran (<LOQ, 0.164 mg kg−1)

Chin-Pampillo et al. (2015b)

Non-bioaugmented GTS

10.3

0.00047

3-Hydroxycarbofuran (peak of 0.0569 mg kg−1 after 4 days); 3-ketocarbofuran (peak of 0.805 mg kg−1 after 8 days)

 

Rice husk + T. versicolor

29.9

0.00126

3-Hydroxycarbofuran (peak of 1.56 mg kg−1 after 15 days)

Ruiz-Hidalgo et al. (2014)

LOD limit of detection, LOQ limit of quantification

Typically biomixtures used in biopurification systems have employed peat as the humic component; nonetheless, given that peat is not easily available, it can be replaced by compost (or even spent mushroom substrate, Gao et al. (2015)). These compost-based biomixtures can be more efficient in degrading pesticides than peat-based biomixtures (Coppola et al. 2007, 2011; Vischetti et al. 2008). These reports contrast with the findings of the present work, since the bioaugmentation only enhanced the performance of the peat-based biomixture.

The elimination of CFN by T. versicolor in rice husk was slow, with a half-life of 29.9 days (Ruiz-Hidalgo et al. 2014), almost ninefold higher than that obtained with the bioaugmented GTS; in addition, the CFN removal was only 55 % after 60 days. Therefore, the use of this lignocellulosic substrate within a biomixture can significantly increase the removal performance.

The transformation products 3-hydroxycarbofuran and 3-ketocarbofuran were detected at concentrations below 0.25 mg kg−1 in the bioaugmented biomixtures at the moment of spiking with the commercial formulation of CFN. Concentration profiles revealed a continuous removal of both metabolites in the biomixtures to levels below detection limits (Fig. 2b) suggesting that their removal was faster than their potential production in these matrices. A less efficient performance was achieved in the non-bioaugmented GTS, where some accumulation of these products (particularly 3-ketocarbofuran, up to 0.8 mg kg−1) occurred within the first days (Chin-Pampillo et al. 2015b). The product 3-hydroxycarbofuran accumulated during the first 15 days of CFN removal (peaks of around 1.5 mg kg−1) in the rice husk bioaugmented with T. versicolor, whereas 3-ketocarbofuran was not detected (Ruiz-Hidalgo et al. 2014).

In order to estimate the complete oxidation of CFN to CO2, mineralization assays with 14C-CFN were performed, as shown in Fig. 3. As observed by monitoring the removal of the parent compound, mineralization rate was higher in the GTS than in the GCS biomixture (k = 0.0025 vs k = 0.0019 day−1). When compared to the mineralization achieved in the soil alone (k = 0.0020 day−1), only the GTS biomixture produced a higher mineralization rate. Lower removal efficiencies in biomixtures than in soil have been reported, for example in the case of chlorpyrifos (Coppola et al. 2007). By using non-bioaugmented biomixtures, the 14C-CFN mineralization rate values were inverted, higher in GCS (k = 0.00158 day−1) than GTS (k = 0.00047 day−1) (Chin-Pampillo et al. 2015b). Comparison of bioaugmented versus non-bioaugmented biomixtures revealed that in the case of GCS, the effect of the bioaugmentation was almost negligible, whereas in the case of GTS, the mineralization rate was fivefold higher when the fungal bioaugmentation was performed. These findings could be explained as already discussed in the case of CFN removal, due to lignin content, C/N ratio, and pH values that might favor fungal activity in GTS but not in GCS.
Fig. 3

Cumulative 14CO2 produced from 14C-CFN (3000 dpm g−1) in soil (×) and the bioaugmented biomixtures GCS (white circle) and GTS (white triangle). Each value is the mean of three replicates ± standard deviation of the mean. GCS is the biomixture containing rice husk, compost, and soil; GTS is the biomixture containing rice husk, peat, and soil

Although laccase activity is not involved in the initial degradation of CFN by T. versicolor (Mir-Tutusaus et al. 2014), it has been related to the degradation of a broad spectrum of organic pollutants (Asgher et al. 2008). Initial laccase activities were 117.8 and 79.6 U kg−1 in GTS and GCS biomixtures, respectively. Nonetheless, this enzymatic activity was negligible during the rest of the incubation period, which may depend on either the competition effect of the microbial communities present in the soil and the compost (McErlean et al. 2006) or laccase inactivation in the matrix (Botterweck et al. 2014). The removal of CFN by T. versicolor in rice husk alone produced a maximum in laccase activity of 62.9 U kg−1 (Ruiz-Hidalgo et al. 2014). Biopiles containing T. versicolor showed laccase activity during the removal of pharmaceuticals in sewage sludge in non-sterile conditions (Rodríguez-Rodríguez et al. 2012b). Nonetheless, the conditions of humidity and the amount of indigenous microbiota were presumably lower in the sludge systems with respect to the biomixtures, thus promoting the better fungal colonization and activity in the former. Laccase activity also plays a role in the formation of non-extractable residues of pesticides, by favoring the formation of strong covalent bonds between these molecules and the soil humic substances; this phenomenon has been described for metalaxyl and its major transformation product, metalaxyl acid, in the presence of immobilized laccase (Botterweck et al. 2014). This indicates that if this enzyme is active in the matrix, it may contribute to the apparent elimination of CFN in the biomixtures, rather than to produce derivative metabolites.

Reports on the bioaugmentation of biomixtures for biopurification systems are scarce. Biomixtures containing T. versicolor and barley straw removed 36–58 % of pesticides such as metalaxyl, terbuthylazine, atrazine, diuron, iprodione, and chlorpyrifos (Bending et al. 2002); however, these biomixtures were previously sterilized, and the effect of indigenous microbial communities was suppressed. Similarly, isoproturon was completely removed after 100 days in biomixtures containing straw when they were bioaugmented with Phanerochaete chrysosporium, another white rot fungus, whereas the removal accounted for around 80 % without bioaugmentation (von Wirén-Lehr et al. 2001). In the case of bacterial bioaugmentation, Verhagen et al. (2013) could not find significant differences in the removal rate of chloropropham between the non-bioaugmented and the bioaugmented biomixtures with a chloropropham-degrading strain of Delftia acidovorans or with a chloropropham-degrading enrichment culture.

The use of biopurification systems aims to reduce the impact of pesticides in the environment. Therefore, the toxicological analysis of the matrix during the degradation process is a good indicator to evaluate the impact of the biomixture. Few studies report the ecotoxicological changes during the removal of pesticides in biopurification systems. A marked toxicity reduction was achieved in the elimination of CFN in ten different biomixtures after a 60-day period (Chin-Pampillo et al. 2015b). Here, toxicity tests with D. magna performed in aqueous extracts from the bioaugmented biomixtures showed a complete reduction in the residual toxicity of both GCS and GTS after 48 days (Fig. 4). Nonetheless, the decrease in the toxicity was higher in the GCS than in the GTS biomixture due to the high toxicity at the time of spiking (211.7 TU) which dropped to 8.8 TU after 13 days. In the case of GTS, initial toxicity was lower (16.0 TU) than in GCS and remained between 11 and 20 TU after 34 days. Non-bioaugmented soil achieved a similar toxicity reduction (only 1.7 TU after 34 days); however, this residual value was also present at the end of the incubation (62 days), whereas the elimination of toxicity was complete in the biomixtures. Variations in the toxicity during the degradation of CFN by T. versicolor in rice husk showed a similar trend: accelerated degradation in the first 15 days (over 80 % reduction), with a final residual toxicity of 11 TU after 60 days (Ruiz-Hidalgo et al. 2014); these values were higher than those obtained in the bioaugmented biomixtures. Toxicological results suggest that these bioaugmented biomixtures may also detoxify CFN in agricultural wastewaters. Further research should focus on the optimization of the biomixture formulation and on monitoring the persistence of the fungus within the biomixture, so as to better estimate the fungal effect in the removal process. Moreover, reinoculations of fungal biomass (Rodríguez-Rodríguez et al. 2014) might be necessary during long-time operation of biopurification systems.
Fig. 4

Residual acute toxicity obtained in aqueous extracts from soil (black inverted triangle) and bioaugmented biomixtures GCS (black circle) and GTS (white circle) during the removal of CFN. Results are based on an immobilization test on D. magna. GCS is the biomixture containing rice husk, compost, and soil; GTS is the biomixture containing rice husk, peat, and soil

Conclusions

The fungal bioaugmentation of rice husk-based biomixtures represents a suitable biotechnological option for the removal of CFN. A clear enhancement in the performance of the biomixtures occurred when T. versicolor was employed in the bioaugmentation of GTS, the biomixture employing peat as a humic component; in this case, the removal of CFN and its transformation products was faster than in the non-bioaugmented biomixtures or in the pre-exposed soil. A complete removal of toxicity was achieved in the biomixtures after 48 days of treatment, which supports the ecological relevance of the biomixture. The fact that the bioaugmentation as a strategy for the enhancement of biomixture efficiency was effective for only one of the two tested biomixtures highlights the importance of a careful design and evaluation of biomixtures before their large-scale use.

Notes

Acknowledgments

The authors acknowledge Vicerrectoría de Investigación, Universidad de Costa Rica (projects 802-B2-046, 802-B4-503 and 802-B4-609), the Costa Rican Ministry of Science, Technology and Telecommunications, MICITT (project FI-093-13), and the Joint FAO/IAEA project TC COS5/029 for supporting this work.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Kattia Madrigal-Zúñiga
    • 1
  • Karla Ruiz-Hidalgo
    • 1
  • Juan Salvador Chin-Pampillo
    • 1
  • Mario Masís-Mora
    • 1
  • Víctor Castro-Gutiérrez
    • 1
  • Carlos E. Rodríguez-Rodríguez
    • 1
  1. 1.Centro de Investigación en Contaminación Ambiental (CICA)Universidad de Costa RicaSan JoséCosta Rica

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