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Environ Sci Pollut Res (2015) 22:19184–19193

DOI 10.1007/s11356-015-5093-3

RESEARCH ARTICLE

Design of an optimized biomixture for the degradation


of carbofuran based on pesticide removal and toxicity reduction
of the matrix
Juan Salvador Chin-Pampillo 1 & Karla Ruiz-Hidalgo 1 & Mario Masís-Mora 1 &
Elizabeth Carazo-Rojas 1 & Carlos E. Rodríguez-Rodríguez 1

Received: 8 April 2015 / Accepted: 16 July 2015 / Published online: 7 August 2015
# Springer-Verlag Berlin Heidelberg 2015

Abstract Pesticide biopurification systems contain a biolog- Keywords Biopurification system . Pesticides .
ically active matrix (biomixture) responsible for the accelerat- Degradation . Coconut fiber . Optimization
ed elimination of pesticides in wastewaters derived from pest
control in crop fields. Biomixtures have been typically pre-
pared using the volumetric composition 50:25:25 (lignocellu- Introduction
losic substrate/humic component/soil); nonetheless, formal
composition optimization has not been performed so far. Improper use of pesticides in agricultural activities results in a
Carbofuran is an insecticide/nematicide of high toxicity wide- negative impact on surface water bodies (Chin-Pampillo et al.
ly employed in developing countries. Therefore, the compo- 2012). The point source release due to intentional or acciden-
sition of a highly efficient biomixture (composed of coconut tal spills, or incorrect disposal of wastewater from pesticide
fiber, compost, and soil, FCS) for the removal of carbofuran applications is one of the main causes of the arrival of pesti-
was optimized by means of a central composite design and cides to rivers and other water environmental compartments
response surface methodology. The volumetric content of soil (Carter 2000; Wenneker et al. 2010).
and the ratio coconut fiber/compost were used as the design The use of biopurification systems (BPSs) arises as an eco-
variables. The performance of the biomixture was assayed by friendly approach for the treatment of pesticide-containing
considering the elimination of carbofuran, the mineralization wastewaters derived from field pesticide application, mainly
of 14C-carbofuran, and the residual toxicity of the matrix, as present in application equipment or in the preparation con-
response variables. Based on the models, the optimal volumet- tainers. The use of BPS, initially named as biobeds, is a prac-
ric composition of the FCS biomixture consists of 45:13:42 tice implemented in the 1990s in Sweden and later dissemi-
(coconut fiber/compost/soil), which resulted in minimal resid- nated to some countries of Europe and America. BPSs are
ual toxicity and ∼99 % carbofuran elimination after 3 days. low-cost devices used for the accelerated degradation of pes-
This optimized biomixture considerably differs from the stan- ticides; they contain an active core called biomixture, com-
dard 50:25:25 composition, which remarks the importance of posed by a mixture of solid substrates. The biomixture con-
assessing the performance of newly developed biomixtures sists of a lignin-rich substrate, a humic component, and soil
during the design of biopurification systems. previously exposed to pesticides (Castillo et al. 2008;
Torstensson and Castillo 1997). The driving force in the
BPS is the biomixture activity of microorganisms mainly con-
tributed by the soil, and the action of lignin-modifying en-
Responsible editor: Gerald Thouand zymes produced by white rot fungi; these enzymes are capable
of degrading lignocellulosic substrates and are widely known
* Carlos E. Rodríguez-Rodríguez to be involved in the transformation of several organic pollut-
carlos.rodriguezrodriguez@ucr.ac.cr ants (Asgher et al. 2008). The presence of lignin-rich sub-
strates promotes the growth of these fungi, whereas the humic
1
Centro de Investigación en Contaminación Ambiental (CICA), component helps to maintain favorable conditions of moisture
Universidad de Costa Rica, 2060 San José, Costa Rica for the development of microbial communities, plus retention
Environ Sci Pollut Res (2015) 22:19184–19193 19185

of the pesticides while the biological activity takes place comprises not only the removal of the pesticide but also the
(Castillo et al. 2008). mineralization rate and the ability of the biomixture to reduce
The original biomixture developed in Sweden was com- the residual toxicity in the matrix.
posed of straw as the lignocellulosic substrate, peat as the
humic component, and local soil, all agricultural by-products
or readily available materials in that country. Various adapta-
tions of the biomixture have been elaborated with alternative Materials and methods
materials in other latitudes according to local availability
(Castillo et al. 2008; Chin-Pampillo et al. 2015; Fogg et al. Chemicals, radiolabeled pesticide standards,
2004; Pussemier et al. 2004; Spliid et al. 2006). Peat has been and formulates
usually replaced by compost of different origins (Coppola
et al. 2007; Kravvariti et al. 2010), and the use of diverse Commercial CFN (Furadan® 48SC, 48 g active ingredient
lignocellulosic substrates such as bagasse, coconut chips, cit- (a.i.)/100 mL formulate) was purchased from a local market.
rus peel, vine branches, coconut fiber, and rice husks has been Analytical standards CFN (2,2-dimethyl-2,3-dihydro-1-
implemented (Castillo and Torstensson 2007; Chin-Pampillo benzofuran-7-yl methylcarbamate, >99 % purity), 3-
et al. 2015; Coppola et al. 2007; de Roffignac et al. 2008; de hydroxycarbofuran (99.5 %), and 3-ketocarbofuran (99.5 %)
Wilde et al. 2009a, b; Karanasios et al. 2010a; Spanoghe et al. were obtained from Chemservice (West Chester, Pennsylva-
2004; Urrutia et al. 2013; Vischetti et al. 2004). Generally, the nia, USA). Radiolabeled CFN [Ring-U-14C]-Carbofuran
performance of such variations in the composition of (14C-CFN; 2.89×109 Bq g−1; radiochemical purity 100 %;
biomixtures is evaluated only by determining the removal or chemical purity 99.59 %) was obtained from Izotop (Institute
half-life of one or more pesticides (Castillo et al. 2008), with- of Isotopes Co., Budapest, Hungary). Carbendazim-d3 (surro-
out considering the toxicological changes in the matrix during gate standard, 99.0 %) and carbofuran-d4 (internal standard,
the degradation process; the latter are essential to assay the 99.5 %) were purchased from Dr. Ehrenstorfer (Augsburg,
environmental impact of the use of BPSs. Similarly, the incor- Germany). Ultima Gold cocktail for liquid scintillation
poration of radiometric techniques to assess mineralization of counting was purchased from Perkin Elmer (Waltham, MA,
the pesticides to determine the extent of complete removal of USA). Solvents and extraction chemicals are listed in Ruiz-
potentially toxic transformation products has been limited in Hidalgo et al. (2014).
these matrices.
The volumetric composition of the originally proposed
biomixture was 50 % lignocellulosic substrate, 25 % humic Biomixture components and preparation
component, and 25 % soil (Torstensson and Castillo 1997).
This composition has been traditionally used to date, with Clay loam soil (sand 40 %, silt 27 %, clay 33 %; pH 5.9;
modifications related to the use of alternative materials. Sim- C, 2.71 %, N, 0.29 %) was collected from the upper soil
ilarly, Karanasios et al. (2012) optimized the use of BPS in layer (0–20 cm) of a beet/onion field with history of CFN
terms of the moisture content during wastewater applications. application, in Tierra Blanca, Cartago, Costa Rica (9° 55′
Given that biomixture composition strongly affects the perfor- 34.17″ N; 83° 53′ 11.76″ W). Soil was air-dried and
mance of biobeds (Karanasios et al. 2010b), optimization re- sieved through a 2-mm sieve. Garden compost (pH 7.3;
mains a necessary step in the design of improved BPS. C, 10.37 %, N, 0.76 %), employed as the humic compo-
Previous work has revealed that the biomixture made of nent, was collected from a composting station located at
coconut fiber (as the lignocellulosic substrate), garden com- Universidad de Costa Rica and sieved as described for the
post (as the humic component), and top primed soil has favor- soil after air drying. Coconut fiber (CF; lignin content,
able properties for the degradation of pesticides such as 56.1 %) acquired from a local market was used as the
carbofuran (CFN, 2,2-dimethyl-2, 3-dihydro-1-benzofuran- lignocellulosic substrate.
7-yl methylcarbamate) (Chin-Pampillo et al. 2015). This is a Biomixtures were prepared by mixing the lignocellu-
broad-spectrum insecticide from the family of carbamates losic substrate, the compost, and the pesticide-primed
which displays neurotoxic activity, and is primarily used in soil at the volumetric ratios shown in Table 1 in order
the control of nematodes. Due to its high toxicity and still wide to obtain a total of nine biomixtures of different com-
use in developing countries (despite being banned in the USA position, according to the design variables described in
and the EU), CFN was employed as the model pesticide in this the Experimental design, response surface methodology,
work. and statistical analysis section. The biomixtures were
This work aims to optimize the composition of a moistened to approximately 75 % of the maximum
biomixture for the degradation of CFN using a central com- water-holding capacity and aged at 25 °C during
posite design (CCD), employing a multifactorial approach that 1 month with weekly homogenization prior to use.
19186

Table 1 CCD design matrix and response values during the removal of CFN in FCS biomixtures

Run Actual factors Corresponding FCS Responses


biomixture composition

x1 x2 Soil Coconut Compost R1 CFN R2 residual toxicity after 8 days R3 mineralization


(%) fiber (%) (%) removal in
Soil Volumetric ratio 3 days (%) 24 h 48 ha k (day−1) Half-life
(%v/v) CF/compost (days)
(%/%) EC50 (%) CLb TU EC50 (%) CLb TU

1 25 2 25 50.0 25.0 49.2±7.0 NT – 0 NT – 0 0.0009±0.0001 815


2 25 2 25 50.0 25.0 70.8±7.6 NT – 0 NT – 0 0.0032±0.0004 214
3 25 2 25 50.0 25.0 62.5±7.4 NT – 0 NT – 0 0.0031±0.0004 221
4 25 2 25 50.0 25.0 50.4±7.0 NT – 0 NT – 0 0.0012±0.0001 598
5 25 2 25 50.0 25.0 72.9±7.7 86.7 85.0–100 1.2 86.4 85.0–100 1.2 0.0043±0.0004 161
6 40 3.4 40 46.4 13.6 95.5±8.6 NT – 0 NT – 0 0.0034±0.0002 204
7 40 1.4 40 35.0 25.0 65.1±10.4 NT – 0 NT – 0 0.0028±0.0002 251
8 10 1.4 10 52.5 37.5 82.1±13.2 NT – 0 NT – 0 0.0020±0.0001 350
9 10 3.4 10 69.5 20.5 87.2±9.1 47.9 25.0–50.0 2.1 47.9 25.0–50.0 2.1 0.0014±0.0001 495
10 25 4 25 60.0 15.0 98.4±8.7 37.4 25.0–50.0 2.7 37.4 25.0–50.0 2.7 0.00166±0.00006 418
11 46.2 2 46.2 35.9 17.9 92.4±9.2 38.3 25.0–50.0 2.6 32.2 25.0–50.0 3.1 0.00138±0.00006 502
12 25 0 25 0.0 75.0 76.8±19.3 27.3 25.0–50.0 3.7 50.0 25.0–75.0 2.0 0.0026±0.0001 268
13 3.8 2 3.8 64.1 32.1 93.5±8.6 NT – 0 NT – 0 0.0014±0.0001 485

NT non-toxic
a
Toxicity values at 48 h (expressed as TU) were employed in the CCD analysis
b
Confidence limits (for EC50 values)
Environ Sci Pollut Res (2015) 22:19184–19193
Environ Sci Pollut Res (2015) 22:19184–19193 19187

Experimental design, response surface methodology, residual toxicity after 8 days, and to maximize both CFN
and statistical analysis removal after 3 days and desirability.

A CCD methodology with two factors (k=2) was applied to Experimental procedures
optimize the composition of a biomixture composed of CF,
compost, and soil, to remove CFN. The design variables or Degradation experiments
factors were the volumetric content of soil (%) (x1) and the
volumetric ratio CF/compost (x2). The effect of these factors The degradation of CFN and the formation of its transforma-
was observed on the following response variables: the per- tion products, 3-hydroxycarbofuran and 3-ketocarbofuran,
centage of CFN removal after 3 days (R1), the residual acute were assayed by weighing 5 g of each biomixture into
toxicity of the matrix after 8 days (R2), as evaluated in an 50-mL polypropylene tubes. Each sample was spiked with
immobilization Daphnia magna test, and the rate of 14C- commercial CFN (30 mg kg−1), manually homogenized, and
CFN mineralization over a period of 35 days (R3). CCD em- incubated in the dark at (25±1)°C during 3 or 8 days. The
ploys 2k factorial points representing all combinations of the remaining pesticide concentration was determined by sacrific-
codified values (±1), 2k axial points at a distance ±α from the ing duplicate systems (quintuplicates for the central point of
origin, and at least three central points in the origin (encoded the experimental design) at times 0 h and 3 days, as described
as 0,0). The factor levels were normalized and coded in the in the Analytical procedures section. Additional unitary sys-
range (−α, +α). The α value corresponds to 1.414 (α=F1/4, tems (10 g each) obtained after 8 days of treatment (eight per
where F=2k). biomixture) were combined as a composite sample to perform
Nine combinations of the design variables were evaluated; the acute toxicity test with D. magna.
to determine the experimental uncertainty, the central point
was performed by quintuplicate. This resulted in an experi- Mineralization studies
mental design that included 13 runs. The experimental design
matrix is shown in Table 1 and includes actual values for the The mineralization of 14C-CFN was determined through
14
different combinations of factors x1 and x2. The CCD was CO2 production in biometric systems containing 14CO2
centered in the point where x1 =25 % and x2 =2, which corre- traps with 10 mL KOH (0.1 M) (Ruiz-Hidalgo et al. 2014).
sponds to the most commonly described volumetric composi- For each biomixture, 50 g was weighed into the biometric
tion of biomixtures employed in BPS, that is, lignocellulosic flask and spiked with commercial CFN (30 mg kg−1) and
substrate/compost/soil at a ratio of 50:25:25 (Castillo et al. 14
C-CFN (50 Bq g−1). The systems were incubated in the dark
2008). The volumetric content of soil, x1, ranged from 3.8 % at (25±1)°C during 35 days. The KOH in the flasks was
to 46.2 %, considering that this is the main source of withdrawn at selected times (two times weekly) and replaced
degrading microbiota. The ratio CF/compost, x2, was chosen with the same amount of fresh KOH. Activity of 14C from the
14
in order to estimate the role of lignin degrading microbiota on CO2 mineralized was analyzed in the KOH samples as de-
CFN removal and ranged from 0 (which represents absence of scribed in the Analytical procedures section.
lignocellulosic substrate) to 4.
Each response variable can be fitted to a second-order poly- Analytical procedures
nomial model (k=2), according to the following equation:
Extraction and analysis of CFN and transformation products
y ¼ b0 þ b1 x1 þ b2 x2 þ b12 x1 x2 þ b11 x21 þ b22 x22 ð1Þ
Unitary samples (5 g) obtained from degradation experiments
The estimation of the model parameters (bi) and the statis- were extracted using 10 mL of water, 15 mL of acidified
tical analysis were performed using the software Design Ex- acetonitrile (formic acid 1 %v/v), 1.5 g of anhydrous sodium
pert 9.0 (Stat-Ease Inc., Minneapolis, USA). The quality of acetate, 6 g of anhydrous magnesium sulfate, and 1 g of sodi-
the fit polynomial model was determined by the Fisher’s F- um chloride. Samples were shaken manually during 2 min and
test, model terms were evaluated by the P value with 95 % then centrifuged at 4500 rpm during 7 min at 10 °C.
confidence level, and results were completely analyzed by Carbendazim-d4 was added as a surrogate standard. An ali-
analysis of variance (ANOVA) employing the same software. quot (3 mL) of supernatant was cleaned adding 900 mg of
In addition, a variable called Bdesirability^ (which ranges anhydrous magnesium sulfate, 150 mg of Bondesil-PSA,
from 0 to 1) that encompasses the simultaneous effects of and 75 mg of silica-C18 and then shaken during 1 min and
the response variables was also determined. Optimization of centrifuged at 4500 rpm during 7 min at 10 °C. A 1.5-mL
the biomixture composition was conducted with response aliquot of supernatant was evaporated until dryness and
surface methodology by the analysis of contour plots and then carbofuran-d 3 was added as internal standard and
numerical solutions by the software, in order to minimize reconstituted to a final volume of 1.5 mL using acidified water
19188 Environ Sci Pollut Res (2015) 22:19184–19193

(formic acid 1 %v/v). Sample extract was filtered through a Beckman LS6000SC counter. The total cumulative 14CO2 ac-
0.45-μm syringe Teflon filter (Millipore, Billerica, MA), be- tivity evolved (mineralized) and the initially added 14C-CFN
fore liquid chromatography–mass spectrometry (LC-MS) were used to calculate the percentage of 14C-pesticide miner-
analysis. Recovery of extraction methodology in the matrix alized. Pesticide mineralization was modeled according to a
was 91 %. first-order model to determine mineralization rate constants.
CFN and its transformation products were analyzed by LC-
MS/MS using ultra high performance liquid chromatography Toxicological analysis
(UPLC 1290 Infinity LC, Agilent Technologies, CA, USA)
coupled to a triple quadrupole mass spectrometer (6460, An acute test based on D. magna immobilization was
Agilent Technologies, CA, USA). Chromatographic separa- employed to estimate the residual toxicity in the biomixtures.
tion was done at 40 °C by injecting 6-μL samples (2-μL The test was conducted following the US EPA guidelines
loop) in a Poroshell 120 EC-C18 column (100×2.1 mm i.d., (EPA 2002) and some modifications as described by Ruiz-
particle size 2.7 μm; Agilent Technologies, CA, USA), and Hidalgo et al. (2014), using elutriates from the biomixtures
using acidified water (formic acid 0.1 %v/v, A) and acidified at time 0 h (after CFN spiking) and after 8 days of treatment,
methanol (formic acid 0.1 %v/v, B) as mobile phases. The as analytical matrix. Elutriates were obtained according to the
mobile phase flow was 0.3 mL min−1 at the following condi- protocol EPA-823-B-01-002 (EPA 2001); briefly, 40 mL dis-
tions: 30 % B for 3 min, followed by a 15-min linear gradient tilled water were added to 10-g samples, and the mixture was
to 100 % B, 4 min at 100 % B, and 0.1-min gradient back to mechanically shaken for 1 h and centrifuged (3500 rpm); the
30 % B, followed by 5 min at initial conditions. Selected supernatant was used as elutriate. Tests were performed in
transitions for the analytes are shown in Table 2. The mass triplicates in glass vials containing five daphnid neonates (less
spectrometer employed a jet stream (electrospray) ionization than 24 h) each, and 10-mL elutriate dilutions. EC50, the con-
source operating at the following conditions: gas temperature centration producing 50 % of immobilization in the daphnids
300 °C, gas flow 7 L min−1, nebulizer 45 psi, sheath gas (bioassay endpoint, determined at 24 and 48 h), was
temperature 250 °C, sheath gas flow 11 L min−1, capillary determined using the binomial probability test on the
voltage 3500 V (for positive and negative), nozzle voltage TOXCALC—Toxicity Data Analysis Software from Tidepool
500 V (for positive and negative), and heater MS1 and MS2 Scientific Software. Toxicity results were expressed as
100 °C. Recovery of the method was 91 % for CFN, 98 % for toxicity units (TU), calculated according to the expression
3-hydroxycarbofuran, and 95 % for 3-ketocarbofuran. Limit TU=(EC50)−1 ·100.
of detection (LOD) and limit of quantification (LOQ) were 13
and 26 μg kg−1 for CFN and 3-ketocarbofuran, and 16 and
32 μg kg−1 for 3-hydroxycarbofuran. Results and discussion

Determination of 14CO2 from mineralization assays Removal of CFN from the biomixtures: optimization

Scintillant liquid (10 mL) was added to 2-mL aliquots from A previous screening of several lignocellulosic substrates, hu-
the removed KOH solution samples, and the 14C activity from mic components (compost and peat), and one CFN-primed
the trapped 14CO2 was measured by liquid scintillation using a soil employed in the elaboration of biomixtures (at a

Table 2 Selected transitions and


other parameters in the detection Compound Retention time Transition Fragmentor Collision energy Type of
of CFN and its transformation (min) (V) (V) transitiona
products using the dynamic
MRM method 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
Carbofuran-d3 7.92 225→165 86 9 Q
225→123 21 q
a
Q, quantification transition; q, qualifier transition
Environ Sci Pollut Res (2015) 22:19184–19193 19189

volumetric proportion of 50:25:25) revealed the fastest CFN Table 4 Regression results from the CCD experiments for the
modeling of response variables R1 (CFN removal after 3 days) and R2
removal by the biomixture composed of CF-compost-soil
(residual toxicity after 8 days) according to the selected quadratic model
(FCS) (Chin-Pampillo et al. 2015). This biomixture showed
an estimated CFN half-life of around 2.5 days, and 79.6 % Model Model Coefficient Standard F-value P value
elimination after 4 days, a better performance than the other term (coded factors) error
matrices. For this reason, and considering the lack of known CFN removal Intercept 63.7 4.15
reports on formal biomixture optimization, the FCS after 3 days x1 −1.28 3.32 0.15 0.7099
biomixture was selected for composition optimization.
x2 9.59 2.82 11.54 0.0094
Taking into account that the half-life of CFN was 2.5 days
x12 14.87 3.65 16.61 0.0036
in the FCS biomixture (50.25:25), its removal after 3 days was
x22 6.27 1.87 11.19 0.0102
selected as the response variable to compare the performance
Residual Intercept 0.42 0.33
of biomixtures of different composition. The removal capacity toxicity x1 0.13 0.36 0.13 0.7274
of such biomixtures after 3 days of treatment is shown in after 8 days
x2 0.48 0.30 2.63 0.1437
Table 1 and ranged from 49.2 % (% soil=25, CF/compost=
x1x2 −0.78 0.48 2.69 0.1399
2) to 98.4 % (% soil=25, CF/compost=4).
x22 0.46 0.19 5.76 0.0432
Response surface methodology was employed to analyze
the correlation between the variables (% soil and CF/compost)
and the CFN removal after 3 days (R1). The data was used to
fit a second-order polynomial model, according to Eq. (2)
which represents the regression in terms of the actual factors: on the degradation extent in 3 days; this means that higher
CF/compost ratios improve CFN removal. On the other hand,
R1 ¼ 120:21−3:39x1 −20:49x2 þ 0:066x21 þ 6:27x22 ð2Þ the effect of % soil (x1) is negative; however, this latter effect
is much lower than that exerted by the ratio CF/compost (co-
The model was statistically analyzed to determine the ac- efficients 9.59 vs −1.28). This finding suggests that, despite
curacy of the fit (Table 3). ANOVA demonstrated that the essential, the amount of soil used in the biomixtures plays a
model was significant, as revealed by the low probability val- negligible role on the removal of CFN; this is in agreement
ue (Pmodel >F=0.0084), and the lack of fit (LOF, not signifi- with Sniegowski et al. (2012), who reported that concentra-
cant, P=0.7804), which implies a significant relationship be- tions as low as 0.5 % pesticide-primed soil may be efficient
tween design and response variables within the model. More- enough to provide the degrading population for proper biobed
over, the determination coefficient (R2 =0.7881) indicates an performance. The coefficients for the quadratic factors x1 and
acceptable goodness in the fit, and the adequate precision x2 showed a positive and significant effect on the removal;
(5.985, desired over 4) suggests an adequate signal over noise other interactions were not significant and, therefore, they
in the model. were not included in the model.
Table 4 shows the coefficient values of the fitted model as The response surface and the contour plots are the graphi-
coded factors. These coded factor coefficients permit to esti- cal representations of the regression equation (Fig. 1). As it
mate the relative impact of the factor on the response variable. can be observed on the figure, two regions in the contour
The positive value of the coefficient related to the ratio CF/ maximize the removal of CFN, those corresponding to high
compost (x2) indicates that this variable has a positive effect CF/compost ratios (x2, around 3.4) and either low (10 %) or
high (40 %) amounts of soil (x1). Optimization under the cri-
terion of maximizing R1 in the Expert Design software was
performed. According to the model, the maximum removal of
Table 3 Analysis of variance (ANOVA) and other statistical CFN in 3 days takes place at conditions x1 =41.9 % and
parameters for the quadratic model employed to fit the removal of CFN x2 =3.52, that is, in a biomixture containing CF/compost/soil
after 3 days in the FCS biomixtures (F/C/S) at a ratio of 45:13:42. Interestingly, lower R1 values
Source of Sum of df Mean F-value Probability were obtained near the central point, which corresponds to the
variations squares square P (>F) typical ratio of 50:25:25, widely employed to prepare
biomixtures since the appearance of biobeds in the 1990s.
Model 2622.94 4 655.73 7.44 0.0084 Two typical transformation products from CFN metabo-
Residual 705.21 8 88.15
lism, 3-hydroxycarbofuran and 3-ketocarbofuran, were ana-
Lack of fit 213.56 4 53.39 0.43 0.780
Pure error 491.640 4 122.910
lyzed in all the biomixtures. Only 3-ketocarbofuran was de-
Total 3328.14 12
tected at the time of spiking at a concentration of 13.2 μg kg−1;
however, none of the products was detected after 3 days in any
R2 =0.7881; adjusted R2 =0.6822; adequate precision=5.985 of the biomixtures. This finding suggests that regardless of the
19190 Environ Sci Pollut Res (2015) 22:19184–19193

A 98.43 B
49.2 Removal after 3 d (%)
3.8
100 100
90
90
140
80
120
Removal after 3 d (%)

2.975 70
100

80

CF/compost
60
2.15
40 ×

3.8 1.325

2.975
80
42
2.15 0.5
35.2
CF/compost 28.4 8 14.8 21.6 28.4 35.2 42
1.325 21.6
14.8 % soil
0.5 8 % soil

Fig. 1 Surface response for the removal of CFN after 3 days (R1) in coconut fiber/compost/soil biomixtures of different composition. Three-dimensional
response surface curve (a); contour plot (b). Standard composition (50:25:25) is indicated with “×”

composition, the FCS biomixtures are efficient at eliminating is based not only on soil content but also on a balance between
these transformation products. In other words, such metabo- its components.
lites are removed at higher rates than they are produced in the On the other hand, the lower mineralization rates correlated
biomixture, thus limiting their detection with the analytical the lowest removal values after 3 days; nonetheless, for higher
approach employed. These results remark the suitability of removal values, there was no correlation with mineralization
FCS biomixtures with respect to other biomixtures that show rates. This finding could be ascribed to the fact that CFN can
some accumulation of transformation products from CFN be used as a C source by microbiota in the biomixtures, and
(Chin-Pampillo et al. 2015). therefore, an important fraction of the 14C was assimilated into
microbial biomass and not trapped in the KOH as 14CO2;
Mineralization of 14C-CFN similarly, part of the 14C contained in the transformation prod-
ucts was probably retained through the adsorption of these
The mineralization rate of 14C-CFN was determined in all the molecules to organic matter in the matrix. Moreover, the much
runs for a period of 35 days; the rate constants and estimated longer mineralization half-lives (compared to the degradation
half-lives are shown in Table 1. Rate constants ranged from half-lives) suggest that the removal of CFN metabolites is the
0.0009 to 0.0043 days−1; however, those extreme values were limiting step in the mineralization, and not the initial transfor-
obtained from replicates of the central point of the CCD. A mation of the pesticide. Despite that removal rates are
correlation between removal (Removal of CFN from the much faster in the biomixtures compared to other systems,
biomixtures: optimization section) and mineralization was
not found in the biomixtures; this can be explained given that
mineralization evaluates not only the transformation of paren-
tal CFN but also the complete oxidation of the molecule to
CO2 and water; particularly, with the radiolabeled CFN
employed (labeled in the ring moiety), mineralization is deter-
mining the breakdown of the ring, while removal considers
only the initial transformation (dissipation) of CFN. The re-
sults from mineralization assays (R3) could not be modeled in
the Expert Design software; however, some interesting trends
were observed. First, leaving out the central point, the higher
mineralization rates were achieved in the biomixtures contain-
ing 40 % soil, regardless of the ratio CF/compost (1.4 vs 3.4)
(Fig. 2). It is suggested that larger initial microbial populations
contained in pesticide-primed soil (not necessarily CFN de-
graders) might accelerate the degradation of the metabolites
Fig. 2 Mineralization of 14C-CFN represented as the percentage of
released from CFN transformation (Sniegowski et al. 2011); 14
CO2 evolved from several biomixtures composed of coconut fiber/
nonetheless, run 11 (46.2 % soil, the highest) showed a low compost/soil at different proportions. Biomixtures: run 6 (●); run 8 (○);
mineralization rate, pointing out that biomixture performance run 11 (▼); run 12 (Δ)
Environ Sci Pollut Res (2015) 22:19184–19193 19191

mineralization half-lives resemble those obtained in soils pre- response surface, it seems not to determine the extent of tox-
viously unexposed to CFN (Ou et al. 1982). Nonetheless, icity elimination. The use of higher amounts of soil might
most of the mineralization rates were similar or higher than favor the toxicity removal, perhaps due to a largest initial
those obtained for CFN in other biomixtures containing peat degrading population that results in a shorter lag phase in the
or compost, and lignocellulosic substrates such as rice husk, combined removal of toxic metabolites. On the other hand, the
cane bagasse, and woodchips (Chin-Pampillo et al. 2015). combined effect of x1 and x2 markedly exerts a desirable and
Similarly, the lack of correlation between the mineralization large effect on the biomixture, as it results in the largest extent
rates and the residual toxicity also indicates that mineraliza- of toxicity reduction. From the other terms in the equation,
tion is not indispensable to achieve complete removal of tox- only the quadratic effect of x2 was significant with a positive
icity from the biomixtures, which is in the end the goal of the effect, which indicates that more residual toxicity remains in
system. the biomixtures at higher values of x22, in agreement with the
linear effect of x2.
The response surface and the corresponding contour plot
Toxicity reduction during CFN removal: optimization
are shown in Fig. 3. The standard 50:25:25 biomixture
succeeded at minimizing residual toxicity and therefore ful-
In order to estimate differences among biomixtures of differ-
filled desired behavior, contrary to optimization results for R1.
ent compositions, the end point to determine toxicity was set
The contour plot confirms that most of the surface results in
in 8 days because previous screening of biomixtures prepared
very low residual toxicity values, as desired. Therefore, one
with several lignocellulosic residues produced almost com-
single optimal combination of design variables was not select-
plete toxicity removal after 60 days (including a CF-based
ed to minimize residual toxicity. However, the area that com-
biomixture) (Chin-Pampillo et al. 2015).
bines % soil values from 8 % to 12 % and CF/compost ratios
The FCS biomixtures showed a marked ability to reduce
above 3 results in higher residual toxicity values; therefore,
the toxicity in the matrix during CFN removal. Initial toxicity
such combinations should be avoided in the design of
values of around 200 TU obtained from the immobilization
biomixtures for CFN removal.
test with D. magna were sharply decreased after 8 days of
treatment to residual values below 4 TU. Moreover, no toxic-
Global optimization of the biomixture composition
ity was obtained in 8 out of the 13 runs. Besides the degrada-
tion and mineralization processes, the overall toxicity decrease
Simultaneous optimization was performed using the variable
achieved in the matrices could have been also favored by
Bdesirability,^ which considers the models explaining the
some extent of adsorption of residual CFN and its transforma-
behavior of both response variables, R1 and R2. The criteria
tion products to the organic matter in the biomixtures on the
for optimization were: maximizing the CFN removal after
course of the degradation process or Baging^ of the pesticide
3 days and minimizing the residual toxicity after 8 days. The
in the matrix (Alexander 2000).
response surface and the contour plots for desirability are
The correlation between the design variables and the resid-
presented in Fig. 4. From the numerical solutions yielded
ual toxicity after 8 days (R2) was analyzed through response
by the software, the one with the highest desirability
surface methodology. Toxicity values were so low that fitting
(0.889) was obtained for the values: % soil= 42.0 and
a model was a difficult task; a second-order polynomial model
CF/compost=3.45, which correspond to a volumetric pro-
partially fitted the data; the mathematical regression model for
portion of 45:13:42 (F/C/S). The optimized composition is
residual toxicity in the actual factors is given as follows in
almost identical to that selected only taking into account the
Eq. (3):
response R1 (Removal of CFN from the biomixtures: optimi-
R2 ¼ −1:44 þ 0:13x1 −0:43x2 −0:05x1 x2 þ 0:46x22 ð3Þ zation section), and permitted to distinguish between the two
optimum regions for R1: the region containing low values for
According to ANOVA employed to estimate the accuracy x1 and high for x2 does not result in optimum desirability as it
of the fit, this is not a significant model (Pmodel >F=0.1289); produces relatively higher residual toxicity values; in the
however, the adequate precision (4.697; desired values over 4) meantime, the area that comprises high values for both x1
indicates that the model can be used to navigate the data and, and x2 produces almost negligible residual toxicity. This latter
therefore, it is useful to see the trend. area coincidently contains the optimum combination of x1 and
The coefficient values (as coded factors) of the fitted model x2 to maximize both desirability (as described above), and R1.
are shown in Table 4. Analysis of coefficients indicates that The optimized composition markedly differs from the
individual linear factor x2 exerts a positive relative impact on standard 50:25:25 biomixture and basically suggests that a
the residual toxicity, that is, higher x2 values tend to leave larger amount of soil should be employed in biomixture
some low residual toxicity in the matrix. Similarly, the relative preparation, by slightly reducing the amount of the lignocel-
effect of soil content (x1) is quite low, and as observed in the lulosic substrate and half-reducing the amount of compost to
19192 Environ Sci Pollut Res (2015) 22:19184–19193

3.11
A B
0 Residual toxicity after 8d (TU)
3.4

4
1
Residual toxicity after 8d (TU)

3
2.85
2

CF/compost
1

0 2.3

-1
0

1.75
3.4
1
2.85 42
35.2
2.3 28.4
CF/compost 21.6 1.2
1.75
14.8 8 14.8 21.6 28.4 35.2 42
1.2 8
% soil
% soil

Fig. 3 Surface response of the residual toxicity of the matrix after 8 days (R2) during the removal of CFN in coconut fiber/compost/soil biomixtures of
different composition. Three-dimensional response surface curve (a); contour plot (b). Standard composition (50:25:25) is indicated with “×”

13 % as humic component. Most studies that employ com- populations will probably translate into shorter lag phases
post as a substitute for peat use the same concentration of for the processes of toxicity decrease and pesticide removal.
25 % (Fogg et al. 2003; Karanasios et al. 2010a, b); none-
theless, Coppola et al. (2011) demonstrated higher removal
of isoproturon and bentazone in biomixtures containing Conclusions
50 % compost (compared to 87.5 %), which supports the
finding that lower amounts of compost results in better per- Optimization of a FCS biomixture intended for the removal of
formance of biomixtures. On the contrary, a biomixture CFN resulted in a volumetric composition of 45:13:42 (F/C/S).
made of soil and compost (75:25v/v) was more efficient to This composition differs from the standard 50:25:25 biomixture
degrade chlorpyrifos than a straw/compost/soil biomixture that has been mostly employed since biobeds appeared. As it
(25:25:50) (Kravvariti et al. 2010). These contrasting results happened for the treatment of CFN, the optimal removal of other
highlight the importance of optimizing biomixtures accord- pesticides in BPS might take place at different biomixture com-
ing to their intended application. positions; therefore, conditions should be evaluated in each case.
Differences among the standard and the optimized compo- Moreover, the optimization of biomixtures for the simultaneous
sitions for carbofuran degradation relies in the fact that a larger removal of several pesticides is highly recommended, particular-
amount of soil might contribute with larger populations at ly taking into account the diverse pesticides employed in the
the moment of biomixture preparation; the effect of such application program for a specific crop. In this work the

A 1 B
Desirability
3.6
0
0.8

1
3
0.8
0.6
0.6
Desirability

0.6
CF/compost

0.4
0.2 2.4

0 0.8

3.6 1.8

3 42

2.4 33.5
1.2
25
CF/compost 8 16.5 25 33.5 42
1.8
16.5 % soil
% soil
1.2 8

Fig. 4 Surface response for the desirability (which combines the effect of composition. Three-dimensional response surface curve (a); contour plot
the CFN removal and residual toxicity as response variables) during the (b). Standard composition (50:25:25) is indicated with “×”
removal of CFN in coconut fiber/compost/soil biomixtures of different
Environ Sci Pollut Res (2015) 22:19184–19193 19193

optimization included not only the removal of CFN but also the EPA (2002) Methods for measuring the acute toxicity of effluents and
receiving waters to freshwater and marine organisms (EPA/821/R-
elimination of toxicity within the biomixture during the treatment
02/012). Office of Water (4303T), Washington DC
of the pesticide, thus providing a global assess of the environ- Fogg P, Boxall ABA, Walker A, Jukes A (2003) Pesticide degradation in
mental suitability of the matrix to be employed in BPS. To obtain a Bbiobed^ composting substrate. Pest Manag Sci 59:527–537
more insights into potential residual toxicity of the optimized Fogg P, Boxall ABA, Walker A, Jukes A (2004) Leaching of pesticides
biomixture, the use of toxicity tests employing organisms from from biobeds: effect of biobed depth and water loading. J Agric
Food Chem 52:6217–6227
different levels of the trophic chain is highly recommended,
Karanasios E, Tsiropoulos NG, Karpouzas DG, Ehaliotis C (2010a)
particularly if the biomixture is intended for the simultaneous Degradation and adsorption of pesticides in compost-based
treatment of several pesticides. biomixtures as potential substrates for biobeds in southern Europe.
J Agric Food Chem 58:9147–9156
Acknowledgments This work was supported by Vicerrectoría de Karanasios E, Tsiropoulos NG, Karpouzas DG, Menkissoglu-Spiroudi U
Investigación, Universidad de Costa Rica (projects 802-B2-046 and (2010b) Novel biomixtures based on local Mediterranean lignocel-
802-B4-503), the Costa Rican Ministry of Science, Technology and Tele- lulosic materials: evaluation for the use in biobed systems.
communications, MICITT (project FI-093-13/802-B4-503), and the Joint Chemosphere 80:914–921
FAO/IAEA project TC COS5/029. Karanasios E, Papadi-Psyllou A, Karpouzas DG, Tsiropoulos NG (2012)
Optimization of biomixture composition and water management for
maximum pesticide dissipation in peat-free biobeds. J Environ Qual
41:1787–1795
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