Removal of Myclobutanil and Detoxification in Modified Biomixtures: Fungal Bioaugmentation and Biochar Amendment

Abstract

Biopurification systems are designed for the treatment of pesticide-containing agricultural wastewater; their biologically active matrix, the biomixture, can be modified to enhance the pesticide removal capacity. Two approaches, fungal bioaugmentation with Trametes versicolor and amendment with biochar, were applied for the potential improvement of biomixtures’ capacity to remediate myclobutanil-contaminated wastewater. The conventional biomixture (B) and its modifications, either bioaugmented with Trametes versicolor (biomixture BT) or supplemented with pineapple biochar (5% v/v) (biomixture BB), were spiked with myclobutanil at a very high concentration (10,000 mg/kg) to simulate extreme on-farm events such as the disposal or leakage of commercial formulations. The dissipation followed a bi-phasic behavior in every case. Both modifications of the conventional biomixture increased the dissipation rates, resulting in estimated DT₅₀ values of 61.9 (BB) and >90 days (BT) compared to biomixture B (DT₅₀ = 474 days). The assessment of biomixtures’ detoxification was carried out with two different bioindicators: a seed germination test in Lactuca sativa and an algal growth inhibition test. Some degree of detoxification was achieved for all biomixtures in both indicators, with the exception of the biochar-containing biomixture, which, despite showing the fastest myclobutanil dissipation, was unable to maintain a steady detoxification trend towards the algae over the course of the treatment, probably due to biochar adverse effects. This approach seems promising for removing persistent myclobutanil from agricultural wastewater and demonstrates the dissipation capacity of biomixtures at extremely high pesticide concentrations likely to take place at an on-farm level.

1. Introduction

Both developed and developing countries are concerned about the serious problem of water contamination. The Sustainable Development Goal 6, launched by the 2030 Agenda for Sustainable Development (United Nations, Sustainable Development Goals), promotes the rational management of water and sanitation, emphasizing the significance of water quality. Water pollution control is recognized as one of the international objectives as a result of the UN 2030 Agenda, which is expected to have a significant impact on future policies and initiatives [1,2].

Pesticides are mainly utilized for the control of pests; however, less than 15% of sprayed pesticides really reach the intended sites, causing undesired chemical distribution and exposure in various environmental compartments [3], reaching natural water streams through a number of pathways, including runoff from agricultural activities, leaching, spray-drift, soil erosion, and deposition. Therefore, pesticides are considered significant environmental pollutants that contaminate soil, surface water, and groundwater. The European Commission recognized the high polluting potential of pesticides early on, as confirmed by the addition of several pesticide active ingredients on the list of water resources’ “priority substances” [4,5].

The broad-spectrum systemic fungicide myclobutanil ((RS)-2-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl) hexanenitrile) is a triazole fungicide used to manage various diseases in cereals, fruits, and vegetables, including summer patch, powdery mildew, dollar spot, and rusts. Its mode of action includes the inhibition of the sterol 14-demethylase enzyme, revealing a therapeutic, eradicating, and preventive capability [6]. With a half-life ranging from 35 d (field) to 365 d (lab) and a high chemical, biological, and photochemical stability, myclobutanil is persistent in soils and water, raising concerns about the potential negative impacts on the environment and human health [7,8]. In Australia, myclobutanil was one of the most frequently detected fungicides, with a maximum concentration of 2.9 μg/L in aquatic systems and up to 120 μg/kg in sediments, according to Wightwick et al. [9]. Similarly, Smalling et al. [10] demonstrated that myclobutanil was one of the most frequently determined fungicides in surface waters in a coastal lettuce-growing region located in California, USA.

Biopurification systems (BPSs) represent an efficient, economical, and ecologically beneficial on-farm biotechnological approach for the decontamination of agricultural wastewater with high concentrations of pesticide residues [11]. The level of pesticide concentrations to be disposed of with BPSs varies depending on the origin of the wastewater, being lower in the case of application remnants (diluted commercial formulations) and higher in situations such as the discharge of leftovers or expired commercial formulations.

The success of BPSs is highly reliant on the biomixture, the biologically active matrix which metabolizes and immobilizes pesticides through abiotic (adsorption and chemical degradation) and biotic (biodegradation) processes. A typical biomixture consists of topsoil, a lignocellulosic substrate, and a humic-rich component (peat or compost) [12]. Soil hosts the microorganisms responsible for pesticide degradation and increases the biomixture adsorption ability. The lignocellulosic substrate provides nutrients to microbial communities and promotes the colonization of ligninolytic fungi, whereas peat or compost can boost adsorption capacity, maintain aerobic conditions, and keep adequate moisture levels [13].

Biomixture formulation may vary depending on local needs and the availability of materials [14]. For instance, the adaptation of biomixtures in different latitudes has resulted in the assessment of alternative lignocellulosic substrates and other materials such as rice husks, sugarcane bagasse ash, and wheat straw biochar [15,16,17].

Additionally, the adoption of bioaugmentation techniques has attracted the attention of researchers in order to provide missing catabolic genotypes, speed up pesticide degradation activity, and prevent the creation of harmful metabolites in biomixtures [18]. Taking into account that ligninolytic fungi are well-known for their ability to break down diverse organic contaminants through the action of extracellular lignin-modifying enzymes and intracellular enzymatic complexes such the cytochrome P450 [19], and that the lignocellulosic substrate is commonly the major component of the biomixtures, the fungal bioaugmentation of these matrices represents a promising strategy to improve their removal performance [20]. In this respect, previous studies demonstrated an increase in the efficacy of the BPS to remove pesticides when the fungus Trametes versicolor was employed as a bioaugmentation agent [21,22,23]

Biochar, a solid substance rich in carbon that is produced by thermochemically converting biomass, has garnered significant interest in recent years [24]. Because of its low cost, constant characteristics, and unrestricted availability, it is considered an environmentally friendly option to promote pesticide removal. Previous studies have reported on biochar usage as a biomixture component [17,25,26], but there are still research gaps related to the fate of numerous pesticides in matrices containing biochar and the side effects of biochar on non-target organisms.

On the other hand, ecotoxicological evaluation is highly recommended, although not so widely applied, for the assessment of BPS performance [11,27], as it provides a more global panorama on the effects of potential transformation products and their interactions, not determined solely by pesticide quantification. Moreover, in the case of biochar-amended biomixtures, such ecotoxicological monitoring has not been reported in the scientific literature.

Taking into account the recognized positive effect of T. versicolor on pesticide removal and the need to assay the effect of biochar on biomixtures, this study aims to evaluate the impact of two modifications of biomixtures, based on a bioaugmentation with T. versicolor and the addition of a palm stubble biochar, on the removal of the fungicide myclobutanil. As most BPS studies use pesticide concentrations mimicking the disposal of diluted formulations, this study was designed to determine the dissipation considering extremely high pesticide concentrations, likely those obtained during leakages or the disposal of expired commercial formulations. In addition, phytotoxicity tests on the algae Raphidocelis subcapitata and the plant Lactuca sativa (lettuce) were assessed during the detoxification process in the matrix to determine the biomixture’s contribution to environmental safety. In this context, the present study provides the first integrated assessment of the simultaneous application of a T. versicolor bioaugmentation and a palm stubble biochar amendment in biomixtures for the treatment of myclobutanil-contaminated wastewater at extremely high pesticide concentrations. While previous research has focused separately on the role of ligninolytic fungi or biochar in enhancing pesticide dissipation, this work explores their combined effect on both removal efficiency and detoxification capacity too.

2. Materials and Methods

2.1. Reagents and Chemicals

The pesticide formulation of myclobutanil under the trade name Rally 40 WP (400 g active ingredient/kg) was purchased from a local supplier (San Jose, Costa Rica). Analytical standard myclobutanil was obtained from Chem Service Inc. (West Chester, PA, USA); carbendazim-d₄ (surrogate standard, 99.5%) and linuron-d₆ (internal standard, 98.5%) were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Solvents and extraction chemicals are listed elsewhere [15]. Physicochemical properties of myclobutanil are shown in

Table 1. Physicochemical properties and characteristics of myclobutanil [8].

Parameter	Value/Parameter
Molecular formula	C15H17ClN4
Substance group	Triazoles
Structural formula
Molecular weight (g/mol)	288.78
Water solubility at 20 °C (mg/L)	132
Octanol-water partition coefficient at 25 °C (LogKow)	2.89
Vapor pressure at 20 °C (mPa)	0.198
Adsorption coefficient Koc (L/Kg)	278.9
Henry’s Law constant at 25 °C (Pa m3/mol)	4.33 × 10−4

.

2.2. Biomixture Materials, Experimental Setup, and Sampling

Controlled-experiment biomixture batches were prepared to determine the effect of T. versicolor and the addition of palm stubble biochar on myclobutanil dissipation. Topsoil (0–20 cm), coconut fiber, garden compost, and palm stubble biochar were used as biomixture materials. Topsoil was collected from a watermelon production field with a history of myclobutanil application, located in Guanacaste, Costa Rica. Garden compost and coconut fiber were acquired from a local supplier and sieved (2 mm) before biomixture preparation. Palm stubble biochar, corresponding to the entire pineapple plant after cutting off the edible part, was air-dried, chopped, and then pyrolyzed (at 600 °C for 1 h). The resulting biochar (pH 7.07) was ground to small particles (2 mm) [28].

T. versicolor (ATCC 42530), employed as a bioaugmentation agent, 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 as described in Madrigal-Zúñiga et al. [29].

Three different biomixtures (treatments) were evaluated using the aforementioned materials. The conventional biomixture (“B”) contained coconut fiber, garden compost, and topsoil at a volumetric composition of 50:25:25. The second biomixture (“BB”) consisted of coconut fiber, garden compost, palm stubble biochar, and topsoil at a volumetric ratio of 50:20:5:25, as recommended in other studies using biochar [17,25,26]. The last biomixture (“BT”) consisted of a fungally bioaugmented version of “B”, in which the coconut fiber was pre-colonized with T. versicolor through the inoculation of blended mycelial suspension as described in Murillo-Zamora et al. [30] before biomixture preparation. Briefly, flasks containing the proper amount of humidified coconut fiber (ratio 1:2, dry material/water, w/v) were sterilized at 121 °C for 15 min prior to the inoculation of blended T. versicolor mycelial suspension (0.5 mL/g of dry coconut fiber); after fungal colonization for 10 d at 25 °C, biomixtures were prepared by adding soil and compost at the above-mentioned volumetric ratio. An aqueous solution of myclobutanil was applied to biomixtures B, BB, and BT to reach a nominal concentration of 10,000 mg/kg d.w.; such concentration reflects extreme conditions of BPS use, i.e., the disposal of expired commercial formulations or leakages from concentrated pesticide solutions (during the preparation of spraying solutions).

Experiments were carried out in 50 mL falcon tubes containing 10 g of each biomixture, incubated at 25 °C; replicate tubes for each sampling time point per treatment were sacrificed to determine myclobutanil residual concentration. The sampling time points were set at 0, 21, 60, and 90 days after pesticide spiking. Also, analogous biomixture systems were prepared to perform ecotoxicological assays (seed germination test in L. sativa and algal growth inhibition test), and were sampled at 0, 60, and 90 days after myclobutanil spiking. Throughout the experiment, ultra-pure water was added to keep the moisture at 50% of the biomixtures’ water-holding capacity (WHC).

2.3. Extraction and Quantification of Myclobutanil

Extraction of myclobutanil was performed as described in Chin-Pampillo et al. [15]. Analyses were performed in a gas chromatograph (7890B, Agilent Technologies, Santa Clara, CA, USA) coupled to a mass spectrometer (7000 C, Agilent Technologies). Chromatographic separation was carried out with a column HP-5MS UI (30 m × 250 µm × 0.25 µm, Agilent Technologies), employing the following oven temperature program: 60 °C, hold 1 min, rate 40 °C/min to 170 °C (hold 0 min), then 10 °C/min to 310 °C (hold 3 min), for a total run time of 20.75 min and 5 min post-run; a post-column (Inert Fused Silica, 2.5 m × 180 µm × 0 µm, Agilent Technologies) was used. A volume of 2 µL was injected using the solvent vent mode in the Multimode Inlet (MMI), starting at 60 °C (hold 0.35 min) and then 900 °C/min to 300 °C (hold 0 min). Conditions in the MS/MS detector were as follows: ionization mode EI (−70 eV); at 300 °C; quadrupole temperature 150 °C; filament off 4 min. The transference line was set at 280 °C; He (2.25 mL/min) and N₂ (1.5 mL/min) were the quench and collision gas, respectively. The retention time was 11.885 min. The acquisition mode was MRM, from which three transitions were determined for myclobutanil: 179 → 90 (CE = 35 V); 179 → 125 (CE = 20 V); 179 → 151.8 (CE = 7 V) (dwell time 10 ms).

2.4. Estimation of the Dissipation Rate of Myclobutanil

The estimation of the dissipation rate of myclobutanil was calculated using the measured concentrations during the experiments and the appropriate kinetic model. The experimental data were fitted to the single first-order (FO) and Hockey-stick (HS) kinetic models [31]. The agreement between the measured values and the values predicted by the two models was visually evaluated, considering the correlation coefficient (r). The FO model is described by the following equations:

$$C=C_0{e}^{-kt}$$

(1)

$${DT}_{50}={\displaystyle \frac{ln2}{k}}$$

(2)

where C is the pesticide concentration at time t (day); C₀ is the initial concentration at t = 0; k (1/day) is the removal rate constant; and DT₅₀ (day) is the pesticide half-life.

The HS model is a bi-phasic kinetic model in which the pesticide degrades in two phases: a rapid initial decline in pesticide concentration followed by a slower decline. According to this model, pesticide concentration initially decreases following first-order kinetics with a rate constant, k₁. At a certain point in time (defined as the breakpoint), the removal rate constant changes to a different value, k₂. For typical bi-phasic patterns, the rate constant, k₁, is usually greater than the removal rate constant, k₂. The HS model is described by the following equations:

$$C=C_0{e}^{-k_{1}t}\text{ for }t\le t_b$$

(3)

$$C=C_0{e}^{-k_{1}t}{e}^{-k_2(t-t_b)}\text{ for }t>t_b$$

(4)

$${DT}_{50}={\displaystyle \frac{ln2}{k_1}}\text{ if }{DT}_{50}\le t_b$$

(5)

$${DT}_{50}=t_b+{\displaystyle \frac{ln2-k_1{t}_b}{k_2}}\text{ if }{DT}_{50}>t_b$$

(6)

where k₁ (1/day) and k₂ (1/day) are removal rate constants; DT_x (day) is the time required for the pesticide concentration to decrease by x percent; and t_b (day) is the breakpoint. Data were analyzed using the software CAKE 3.7 (Syngenta).

2.5. Ecotoxicological Assays

Elutriates from biomixture samples were prepared according to the protocol EPA-823-B-01-002 [32]; briefly, a 1:4 (w/v) ratio of biomixture to corresponding media (distilled water for lettuce assay and Woods Hole MBL medium for algae assay) was mechanically shaken (2500 rpm, 1 h) and centrifuged (10 min at 3500 rpm); supernatants were finally filtered (Whatman GF/C filter, 1.2 m porosity, 47 mm diameter) and used as elutriates for ecotoxicological assays.

2.5.1. Seed Germination Tests in L. sativa

Seed germination tests with lettuce (L. sativa var. Georgia) were carried out to keep track of the residual phytotoxicity in the matrix, according to Lizano-Fallas et al. [33]. Triplicates of 20 seeds were exposed to the elutriates (5 mL) from biomixture samples from each treatment; assays were carried out in glass Petri dishes and incubated for 6 days in the dark at room temperature (20–25 °C). Relative seed germination (SG) and relative root elongation (RE) were determined after the incubation period, considering the germination in controls obtained by exposure to distilled water; these parameters were calculated as described elsewhere [34] and were employed to calculate the germination index (GI).

2.5.2. Algal Growth Inhibition Test

Prior to the assay, R. subcapitata was cultured in Bold’s modified basal freshwater nutrient solution under white light, with constant aeration and a controlled temperature (23 ± 2 °C). The growth inhibition on the algae R. subcapitata was evaluated in accordance with OECD and US EPA criteria, with modifications for usage in microplates [34,35]. Algae were exposed for 72 h to dilutions of each elutriate sample (diluted in MBL at 0%, 12.5%, 25%, 50%, 75%, and 100%). Each well contained 300 μL final test volume per well and 50 μL inoculum at an initial cell density of 10⁴ cells mL⁻¹; five replicates were employed for each elutriate dilution. MBL medium was utilized as the control. Microplates were incubated in an orbital shaker at 25 °C with a 24 h^L:0 h^D photoperiod [36]. Absorbance was recorded at 750 nm throughout the assay using a microplate spectrophotometer (MultiscanSky, ThermoScientific, Waltham, MA, USA); growth rates were estimated according to the following equation:

$${\mu }_{i-j}={\displaystyle \frac{{lnAbs}_j-{lnAbs}_i}{\Delta t}}$$

(7)

where Abs_i is the absorbance (750 nm) corresponding to the cell density used to start the test (10⁴ cells/mL), Abs_j is the absorbance (750 nm) at the end of the test, and Δt is the time interval (days).

Τhe percentage of growth inhibition for each treatment was calculated according to the following equation:

$$\%I_r={\displaystyle \frac{{\mu }_c-{\mu }_r}{{\mu }_c}}$$

(8)

where %I_r is the percentage of the growth inhibition, µ_c is the growth rate (µ) for the control, and µ_r is the growth rate for each treatment. EC₅₀, the concentration of the elutriate that causes a 50% reduction in algal growth, was calculated after 72 h using R version 4.3.2 and the DCR package [37].

3. Results and Discussion

3.1. Dissipation Rate of Myclobutanil

The necessity to assess the removal of myclobutanil in BPSs is underscored by the considerable environmental risk associated with the poor handling of pesticide-contaminated wastewater at an on-farm level, as well as the detection frequency of the fungicide residues in natural water compartments. Moreover, most myclobutanil biodegradation studies have been conducted considering diffused-pollution-derived concentrations and not point-source pollution sites. Interestingly, although potentially myclobutanil-degrading bacterial strains have been identified [38], the formal assessment of fungicide transformation or methods aimed at its biodegradation have not been developed. Moreover, even though myclobutanil is regarded as a persistent pesticide in soil, with a half-life ranging from 35 days to over 500 d [7,8], degradation studies exploring either the parent compound or its potential metabolites in BPSs remain notably unreported. This study evaluates the capacity for myclobutanil removal of a biomixture locally used in Costa Rica for pesticide dissipation and employs two alternative approaches to potentially improve the elimination of the fungicide, either by the use of palm stubble biochar to partially replace the garden compost (as a humic component in the biomixture) or through a bioaugmentation with the fungal biomass of T. versicolor.

FO and HS kinetic models [31] were used to describe the dissipation rate of myclobutanil in the different biomixture treatments (Figure 1), and the corresponding kinetic parameters are presented in

Table 2. Kinetic model parameters for myclobutanil dissipation and correlation coefficients (r) between measured and estimated myclobutanil concentrations by first-order and Hockey-stick models.

Parameters	Biomixture B	Biomixture BB	Biomixture BT
First-order model (FO)
r	0.9549	0.9283	0.8529
k (1/d)	0.00193	0.01001	0.006455
DT50 (d)	360	69.3	107
Hockey-stick model (HS)
r	0.9967	0.9945	0.9975
tb (d)	20.98	20.09	29.28
k1 (1/d)	0.00431	0.0233	0.01868
k2 (1/d)	0.00133	0.005382	1.24 × 10−12
DT50, k1 (d)	161	29.8	37.1
DT50, k2 (d)	521	129	ND
DT50, overall (d)	474	61.9	ND

t_b = breakpoint of the Hockey-stick model.

(experimental points can be seen in Figure S1, Supplementary Material). Regardless of the treatment, the dissipation rate shows a time dependence; more specifically, the removal is faster during the first 21 days, but then the rate decreases until the end of the experiment (90 days). The correlation coefficient (r) values according to the FO and HS kinetic models for biomixture B were 0.9549 and 0.9967, respectively, which indicate that myclobutanil dissipation is better fitted by the latter. Such a trend was also observed for BB and BT biomixtures and correlates with the bi-phasic behavior described above, which is better described by the HS model.

Overall, the unconventional biomixtures containing biochar (BB) and T. versicolor (BT) exhibited a faster dissipation of myclobutanil compared to the conventional biomixture B. According to the HS model, higher initial dissipation rates (k₁) were determined for BB and BT, translating into an estimated DT₅₀ (based on k₁) of less than 40 d in both cases, while biomixture B showed a significantly longer estimated DT₅₀ value of 161 d (based on k₁) and 474 d (overall), which makes it the least suitable for the fungicide removal. Similarly, a higher k₂ value was determined in BB with respect to B; nonetheless, as the dissipation reached a plateau in the case of BT, no k₂ value (or global estimated DT₅₀) could be determined by the model. Consequently, with a global DT₅₀ of 61.9 d (considering both k₁ and k₂), biomixture BB represents the most promising matrix for myclobutanil removal. The same pattern is observed comparing the estimated DT₅₀ values according to the less accurate fit of the FO model: 360 d (B) > 107 d (BT) > 69.3 d (BB). Despite the better dissipation performance in the alternative biomixtures, the fungicide was not totally removed by the end of the treatment (90 d), and between 40.5 and 56.2% of the initial concentration was still detected in the biomixtures, compared to 82.8% in the B biomixture.

The observed bi-phasic removal pattern observed in all biomixtures may be caused by pesticide sorption to the biomixture, which increases as the matrix ages; the sorption process translates into a decrease in the bioavailable fraction of the pesticide with time, consequently lowering the removal rate. Such an effect is known to slow down the removal of several organic pollutants [39] and may take place in biomixtures [40]. Another possible reason behind the delayed dissipation of myclobutanil over this period could be attributed to the appearance of its metabolites, which in some cases are known to exhibit a high toxicity towards soil organisms [8]. In a similar study, Parlakidis et al. [14] conducted a small-scale experiment to evaluate the removal performance of local biomixtures (containing topsoil, sunflower crop residues, and peat) in Greece under various treatments and observed an analogous delay in the dissipation rate of myclobutanil, which lasted from 7 days to 120 days of the experiment.

The removal of myclobutanil determined in this work provides a promising remediation strategy, considering the high concentration treated and the very persistent character of this fungicide, whose DT₅₀ values in soil range from 35 to 560 d [8]. Factors such as the toxicity of this compound (or its transformation products) would become more critical at extreme concentrations, thus potentially hindering its transformation, even if part of the dissipation might be due to abiotic factors, as described below. Most studies on the dissipation of triazoles and other fungicides in biomixtures are performed at concentrations that mimic the disposal of ready-to-apply pesticides containing wastewater (diluted formulations) at starting levels ranging from 0.5 to 50 mg/kg [14,30,41]. Consequently, this work presents promising results on the performance of biomixtures in the worst-case scenarios of the treatment of very high pesticide concentrations.

3.2. Impact of Biochar on Myclobutanil Dissipation

The elevated organic carbon content, water-holding capacity, and nutrient availability of biochar offer ideal conditions to enhance biodiversity richness, and are known to cause variations in the composition of soil microbial communities [42]. Such features may boost microbial activity or stimulate certain microbial groups. As a result, the breakdown of various pesticides can be accelerated, as observed in the case of atrazine, whose removal rate was enhanced by the amendment of soil with biochar [43,44]. The enhancement in habitat conditions produced by biochar for soil microorganisms and consequently for certain microbial populations could occur as well in biomixtures, and might explain the higher removal rate observed for myclobutanil in the biomixture BB compared to biomixture B.

The extent of the microbial degradation of pesticides when biochar is applied is known to depend on the pyrolysis process conditions and sorption capacity of biochar. First, the low temperature biochar formation procedure is more favorable for microbial development than the high temperature processes [45]. This effect was observed by Ren et al. [46] during the degradation of carbaryl in soil treated with biochar made from rice and corn, which improved in the soil treated with biochar produced at 350 °C but decreased with the use of biochar produced at 700 °C. In our case, the used biochar was pyrolyzed at 600 °C, and, contrary to what was observed, the dissipation rate of myclobutanil in the biomixture BB was expected to be partially limited. Second, several authors support that biochar addition adversely affects pesticide biodegradation by promoting pesticide adsorption and thus decreasing pesticide bioavailability [45].

The physicochemical characteristics of the palm stubble biochar used in this study indicate a moderately porous and carbon-rich material with alkaline properties, consistent with previous studies on pyrolyzed agrowastes. According to Chin-Pampillo et al. [28], the sorption and persistence of herbicides in tropical soils amended with charred materials depend strongly on the type of feedstock and the degree of pyrolysis. They showed that torrefied materials produced at 300 °C from palm oil fiber and pineapple stubble increased the sorption of bromacil and diuron up to sixfold compared with unamended soils (K_oc = 18–1309 L/kg), whereas biochars produced at 600 °C exhibited a weaker sorption. This effect was attributed to the abundance of oxygenated functional groups, mainly hydroxyl and carbonyl moieties that promote hydrogen bonding and π–π interactions with pesticide molecules, rather than to the total organic carbon content or surface area [46,47,48,49,50,51]. The reduced sorption capacity of biochars obtained at higher temperatures was associated with the loss of such polar sites and the development of more condensed aromatic structures, leading to a greater hydrophobicity [52,53,54,55,56].

Despite the potential limitations due to the temperature of biochar production used in this case and the expected decreased pesticide bioavailability, our data suggest that the overall effect of biochar amendment was positive for myclobutanil removal in biomixtures. Similar enhanced pesticide dissipation effects have been described by Mukherjee et al. [57] during the treatment of pyrimethanil and boscalid in biomixtures amended with 5% biochar; this effect is partly ascribed to the sequestration of the pesticides in the biochar-containing matrix and could also explain the improved dissipation observed for myclobutanil in our study.

3.3. Impact of T. versicolor on Myclobutanil Dissipation

The addition of fungal biomass showed a positive impact on the removal of myclobutanil, reaching slightly lower (initial) dissipation rates compared to the biochar containing biomixture, but markedly higher than the dissipation estimated for the conventional biomixture B. Nonetheless, the dissipation became steady after 21 d (at 43.8%), and the bi-phasic behavior described by the HS model indicated no additional dissipation during the second phase of the curve. The ability of T. versicolor to transform several organic pollutants including pesticides has been described for compounds such as chlorpyrifos, dicofol, cypermethrin, imiprothrin and carbofuran [58]. In particular, a few works have reported the effective bioaugmentation of biomixtures with this fungus, which boosted the removal and detoxification of pesticides (e.g., carbofuran), in matrices containing peat instead of compost [29], in contrast to some that report no enhancement in pesticide dissipation, mostly in compost-based biomixtures [20,30]. Accordingly, it is suggested that fungal bioaugmentation is preferable in situations with lower pH and greater C/N ratios, both contributed by peat as opposed to compost [21,29,30]. Data from this study reveal that compost-based biomixtures can be at least successfully bioaugmented in conditions of more neutral pH and lower C/N ratio, thus highlighting the versatility of compost as the humic-rich component used in Mediterranean and tropical biomixtures.

The addition of an external fungus in bioaugmentation approaches may fail at improving the removal of pollutants, as changes in bacterial degrading communities might take place as a result of disturbances triggered by the inoculation of large amounts of fungal biomass and the production of competitor substances [59], or when the autochthonous microbiota or physicochemical characteristics of the biomixture may prevent the fungus from effective colonization and activation, thus reducing its degrading ability [29,30,33,60]. Some of these situations, coupled to the eventual inactivation of the fungus by myclobutanil or its metabolites, might have resulted in the steady dissipation observed during the second stage of our removal curve. Instead, the desired panorama of removal enhancement takes place in situations in which some synergistic effects between bacterial and fungal communities potentiate pesticide transformation, such as the case described for aldicarb, atrazine, and alachlor in mixed cultures of bacteria and T. versicolor [61]. Considering the enhancement on the initial removal of myclobutanil in the biomixture containing T. versicolor, transformation assays using pure cultures of the fungus should be performed to better describe the extent of its removal capacity and the potential mechanisms underlying the degradation of the fungicide [29,30,33]. Also, the search for strategies to promote fungal activity for longer periods is recommended to cover normal treatment time in biomixtures.

3.4. Ecotoxicological Changes During the Treatment of Myclobutanil

The ecotoxicological evaluation of the biomixtures during myclobutanil treatment showed some extent of detoxification, which did not match the removal patterns. A detoxification of the matrices was determined in every case (with respect to initial toxicity) (

Table 3. Ecotoxicological parameters determined during the removal of myclobutanil in biomixtures, according to germination tests in L. sativa (germination index, GI) and growth inhibition of R. subcapitata (EC₅₀).

Time (d)	Conventional Biomixture (B)		Biochar-Containing Biomixture (BB)		Fungal Bioaugmented Biomixture (BT)
	GI (L. sativa) (%)	EC50 (Algae) (%)	GI (L. sativa) (%)	EC50 (Algae) (%)	GI (L. sativa) (%)	EC50 (Algae) (%)
0	37.6	0.00355	28.5	0.0124	20.6	0.0124
60	35.6	8.41	30.4	4.95	21.7	9.97
90	42.0	9.20	33.1	2.23	40.9	9.21

GI values are given as a percentage compared to the control seeds. EC₅₀ values correspond to a percentage of the elutriate, where 100% represents the undiluted elutriate.

), most notably in the tests using algae, and more discreetly in the germination index in L. sativa; however, such detoxification took place at a larger extent in the conventional (B) and bioaugmented (BT) biomixtures, even though the estimated half-life for myclobutanil was significantly longer in the former. In this respect, detoxification could also be related to pesticide sequestration through adsorption/aging processes within the matrix which result in a decreased pesticide bioavailability, and not only due to biological activity. The biochar-containing biomixture, the one that exhibited the fastest removal of the fungicide, was not capable of keeping a consistent detoxification trend along the treatment and, for algae final toxicity after 90 d, was slightly higher than that at 60 d, even after an initial detoxification (compared to the initial EC₅₀ value of 0.0124%); moreover, the BB biomixture globally showed the highest final toxicity values for both benchmark organisms. The presence of biochar might be promoting the accumulation of more toxic myclobutanil metabolites after 60 d, in case this component is causing the selection of a particular degrading pathway linked to a specific degrading microbial population; however, detailed studies of the produced metabolites and their role on studied non-target organisms would be required to confirm this hypothesis. Pesticide sequestration and the consequently reduced bioavailability and toxicity were mostly expected in the biochar-containing biomixture; on the contrary, the content of harmful substances in biochar, which is mostly dependent on the feedstock employed in its production [62], could also favor an increased toxicity in the biomixture, although this would likely be observed as initially higher toxicity values compared to the other matrices.

The content of such stressors in the biochar composition could promote undesired synergistic interactions with myclobutanil or some of its metabolites, thus translating in the final increase in the toxicity towards algae observed in the biomixture. Beyond the intrinsic sorptive properties, biochar can act as a dynamic source of chemical and physical stressors, influencing microbial function and detoxification efficiency. Variations in feedstock origin and pyrolysis conditions can lead to distinct redox-active surfaces, affecting the bioavailability of nutrients and the formation of reactive oxygen species (ROS) within the biomixture. Such processes may suppress the activity of ligninolytic enzymes or shift microbial community composition toward stress-tolerant taxa, thereby modifying pesticide degradation pathways. In particular, low-temperature biochars often contain volatile organic residues and partially carbonized lignocellulosic fractions that can interfere with fungal colonization and enzymatic secretion, whereas high-temperature biochars may introduce oxidative stress [63,64]. These combined effects may explain the residual toxicity patterns observed despite a substantial pesticide dissipation, highlighting that biochar-induced stress modulation should be considered a key factor in designing optimized remediation formulations.

Even though the ecotoxicological evaluation has not been commonly applied in the assessment of biomixture performance, most available reports refer to Daphnia magna (not employed as an indicator this time due to its relatively mild sensitivity towards myclobutanil), for which detoxification has been achieved in several cases for other pesticides [20]. Similarly, detoxification towards L. sativa has been described in biomixtures used for the treatment of individual pesticides or their mixtures, usually containing herbicides [2,65]. Nonetheless undesirable toxicological outcomes have also been reported [33]. The use of algae for the evaluation of biomixtures was not found in the scientific literature; however, the detoxification patterns towards this indicator described in this work not only support the applicability of the proposed matrices, but also suggest this could be a useful benchmark organism for the assessment of the performance of biomixtures.

4. Conclusions

This work describes the treatment of myclobutanil-contaminated wastewater in biomixtures at extremely high concentrations, simulating scenarios such as the disposal of expired commercial formulations during on-farm pesticide residue management. The results show that the persistent fungicide myclobutanil can be partially removed from biomixtures at such levels, albeit at relatively slow rates, while achieving a measurable degree of detoxification. The application of alternative formulations, namely biochar addition and fungal bioaugmentation, enhanced the removal efficiency compared to the conventional matrix.

According to the Hockey-stick kinetic model, the overall DT₅₀ was 474 days for the conventional biomixture (B) and 61.9 days for the biochar-amended system (BB), while the fungal biomixture (BT) exhibited a faster initial dissipation (DT₅₀ = 37.1 days, k₁ phase) but reached a plateau thereafter. Despite these improvements, a complete removal was not achieved within 90 days, and 40–56% of the initial concentration remained in the alternative matrices. The ecotoxicological evaluation confirmed a partial detoxification of the treated effluents; however, the detoxification trends did not always mirror the dissipation kinetics. Detoxification occurred in all cases compared with the initial toxicity, particularly in the algal bioassay and, to a lesser extent, in the Lactuca sativa germination tests. The effect was more consistent in the conventional and bioaugmented biomixtures, even though the estimated half-life was considerably longer in the former.

Overall, despite the improvements achieved in myclobutanil removal, further optimization of biomixtures is required prior to on-farm application, particularly to maximize pesticide removal while ensuring detoxification. The present findings emphasize the versatility of biomixtures to treat pesticide residues not only at low concentrations corresponding to diluted effluents from field applications, but also under worst-case scenarios involving extremely high pesticide loads. Future work should focus on optimizing biochar–fungus combinations, refining pyrolysis parameters (300–600 °C), and assessing long-term stability, regeneration potential, and microbial community evolution under repeated pesticide exposure to ensure sustainable and effective large-scale implementation.