Growth Assessment of Marine-Derived Fungi in the Presence of Esfenvalerate and its Main Metabolites

The growth and biodegradation potential of marine-derived fungi were evaluated by measuring the radial growth of colonies. It was observed that Penicillium raistrickii CBMAI 931, Aspergillus sydowii CBMAI 935, Cladosporium sp. CBMAI 1237, Microsphaeropsis sp. Dr(A)6, Acremonium sp. Dr(F)1, Westerdykella sp. Dr(M2)4 and Cladosporium sp. Dr(M2)2 were able to grow and develop in the presence of the pyrethroid insecticide esfenvalerate (S,Sfenvalerate) and its main metabolites (3-phenoxybenzaldehyde, 3-phenoxybenzoic acid, 3-phenoxybenzyl alcohol and 2-(4-chlorophenyl)-3-methylbutyric acid), showing the possibility of esfenvalerate biodegradation by these strains. The presence of technical grade esfenvalerate and its metabolites caused significant growthinhibition, while fungal development was not affected by the presence of the commercial insecticide SUMIDAN 150 SC in the culture medium. This fact might show that the biodegradation of the esfenvalerate in the commercial insecticide is slower than that of the technical grade active ingredient, since slower biodegradation of esfenvalerate would reduce the concentration of phenolic compounds and thus the growth inhibition. Future studies will focus on the quantitative biodegradation analysis of technical grade esfenvalerate and active ingredient in the commercial insecticide.


Introduction
Synthetic pyrethroids have been developed to improve on the specificity and activity of pyrethrin, the natural insecticide produced by the flowers of pyrethrum species (Chrysanthemum cinerariaefolium and coccineum). Pyrethrin is known for its instability in light and air, which limits its effectiveness in crop protection. The synthetic pyrethroids were developed to increase the photostability while retaining the potent and rapid insecticidal activity and relatively low acute mammalian toxicity of pyrethrin. There are about 1000 different structures and some of them are very different from the original pyrethrin structure [1,2].
The structural diversity of synthetic pyrethroids was further enhanced by the discovery that the 2,2-dimethylcyclopropanecarboxylic acid moiety of the pyrethrins and most previous synthetic compounds could be replaced by an α-isopropyl phenylacetic acid moiety. This new series of compounds led to the discovery of the commercial insecticide fenvalerate [2].
Fenvalerate is a pesticidal active ingredient composed of four stereoisomers. Originally, a balanced mixture of all four isomers was marketed. However, since the S,S-isomer shows the highest insecticidal activity, the synthesis of fenvalerate has been modified to enrich the racemic mixture with the S,S-isomer, which is named esfenvalerate [3].
Pyrethroids such as esfenvalerate are esters, with an alcohol and an acid moiety, so that cleavage by esterases is the first step in the biodegradation pathway. Studies available in the literature show that 3-phenoxybenzaldehyde [4,5], 3-phenoxybenzoic acid [5,6], 3-phenoxybenzyl alcohol [7] and 2-(4-chlorophenyl)-3-methylbutyric acid are the main products of pyrethroid biodegradation such as fenvalerate ( Figure 1). adaptation to environmental change, since marine currents promote rapid temperature and pH alterations. Marine microorganisms may show efficient biodegradation because they possess a unique enzymatic system adapted to highly halogenated and oxygenated compounds, such as the esfenvalerate employed in this study [8]. Thus, marine-derived fungi might have great potential for bioremediation applications and deserve to be studied.
It is also noteworthy that esfenvalerate has been identified as having the potential to accumulate in aquatic sediments [9,10], making the study of its biodegradation in aquatic systems very important.
Marine-derived fungi have already been used in biodegradation processes. Examples are Aspergillus sclerotiorum CBMAI 849 and Mucor racemosus CBMAI 847, which were capable of metabolizing pyrene to the corresponding pyrenylsulfate and benzo[a]pyrene to benzo[a]pyrenylsulfate [11]. Marine-derived fungi have also been used in the bioremediation of raw textile mill effluents [12], molasses-based raw effluents [13] and the anthraquinone dye, Reactive Blue 4 [14].
Some research on the biodegradation of pesticides by marinederived fungi has been carried out in this laboratory. The organochlorine insecticide dichlorodiphenyltrichloroethane was biotransformed by Trichoderma sp. [15] and the organophosphate insecticide profenofos [16] by the strains Aspergillus sydowii CBMAI 935 and Penicilium raistrickii CBMAI 931.
The culture medium was sterilized in an autoclave (AV-50, Phoenix, Brazil) at 121°C for 20 minutes and manipulations involving marine fungi were carried out in a laminar flow cabinet (FUV-18, Veco, Brazil). Since the microorganisms used in this study were isolated on  various culture media, 3% malt was used as a nutrient source because it is a rich and appropriate medium for marine microorganisms [17,18].
It is noteworthy that, except for the commercial insecticide, all the xenobiotic compounds were predissolved in 100 µL of DMSO per plate to enhance the dissipation of xenobiotic in the culture medium.

Results and Discussion
Radial growth experiments of marine-derived fungi, not previously used in any study of pyrethroid biodegradation, were performed in the presence of xenobiotics. These experiments were carried out to assess the growth of these strains in the presence of esfenvalerate and its main metabolites, which are known to be toxic and recalcitrant.
In the experiment with the strain Microsphaeropsis sp. Dr(A)6 (Table 1, S.I. 1), it was observed during 7 and 14 days of incubation that the presence of the EsfCom had no significant effect on colony growth (Exp. B, 102% and 104%, respectively), relative to the growth in 3% malt in the absence of the insecticide (Exp. A).
However, when xylene (Exp. D), EsfTec (Exp. E) or xylene plus EsfTec (Exp. F) was added to the medium, a smaller colony, with a 75% diameter of the reference colony diameter (Exp. C) was seen after 7 days of incubation. After 14 days, the growth inhibition was still apparent, but the colony diameter differed from the reference diameter less than after 7 days. Xylene (Exp. D) and EsfTec (Exp. E) led to smaller colonies of approximately 60% of the reference colony size (Exp. C), throughout the assessed period. It was also observed that in the presence of xylene plus EsfTec (Exp. F), the inhibitory effect was additive, inducing the least-developed colony observed, reaching only 33% of the reference colony diamenter (Exp. C). The presence of ClAc (Exp. G) did not cause any difference from the reference M3+DMSO (Exp. C).
It is important to note that PBAc (I) stimulated the colony growth from 21 days of incubation, resulting in a colony diameter of 124% of the reference M3+DMSO (C).
In the experiment with the strain Acremonium sp. Dr(F)1 (Table However, when xylene (Exp. D), EsfTec (Exp. E) or xylene plus EsfTec (Exp. F) was added to the medium, after 7 days a smaller colony was observed, approximately 70% of the size of the reference colony (Exp. C) in the presence of xylene (Exp. D) or EsfTec (Exp. E), and approximately 60% of the reference size in the presence of xylene plus EsfTec (Exp. F). After 14 days of incubation, the growth inhibition was still present, but the colony diameter reduction relative to the M3+DMSO reference was smaller, approximately 90%.
In the presence of the possible metabolites of biodegradation [PBAc (Exp. I), PBAlc (Exp. H) and ClAc (Exp. G)], growth inhibition was not observed, a colony of approximately 100% of the reference diameter (Exp. C) being measured after 7 and 14 days of incubation. After 21 days of incubation, growth stimulation was observed, resulting in a colony diameter of 110% of the reference (Exp. C) diameter.   Data are means of triplicate ± standard deviation.  The presence of xylene (Exp. D) and EsfTec (Exp. E) did not produce a significant difference from the reference experiment M3-DMSO (Exp. C), since approximately the same diameter was observed. However, in the presence of xylene plus EsfTec (Exp. F), some growth inhibition occurred, since the colony diameter was about 90% of the reference size (Exp. C) after 7 days, and approximately 80% after 14, 21 and 28 days of incubation. It is important to note that the strain Cladosporium sp. Dr(M2)2 stopped growing after 14 days of development, even on the reference plates. This strain did not grow well or developed properly on the selected culture medium.
In the experiments with Cladosporium sp. CBMAI 1237 ( Table  6, S.I. 6), it was observed that the commercial insecticide generated growth inhibition, since the observed colony diameters in the presence of EsfCom (Exp. B) were 80% of that of the reference culture on M3 agar (Exp. A).
The presence of xylene (Exp. D), EsfTec (Exp. E) or xylene plus EsfTec (Exp. F) did not affect the growth of the fungus, since approximately the same colony diameter was observed on the reference plate (Exp. C).  Data are means of triplicate ± standard deviation.  Data are means of triplicate ± standard deviation. The presence of PBAc (Exp. I) and ClAc (Exp. G) caused growth inhibition; the colony diameter observed being approximately 85% of the reference diameter after 7 days and approximately 90% after 14 days of growth. PBAlc (Exp. H) also inhibited the growth of this strain, the colony diameter being about 60, 70, 80 and 90% of the reference M3+DMSO (Exp. C) after 7, 14, 21 and 28 day of incubation, respectively.
In the experiments with the strain Aspergillus sydowii CBMAI 935 (S.I. 7), it was not possible to measure the colony diameter, since secondary colonies spread over the plate as the spores dispersed. However, it was possible to note that, in the presence of the possible esfenvalerate metabolites [ACl (Exp. G), FBAlc (Exp. H), FBAc (Exp. I), and FBAld(Exp. J)], even though the fungus had spread all over the plate, the colony was visibly less dense than the reference on M3-DMSO (Exp. C).
Several particular characteristics were observed in the fungal growth in the presence of the xenobiotic compounds under study. Generally, it was observed that the possible metabolite ClAc, showed weaker inhibition effects than the other possible metabolites (PBAc, PBAld and PBAlc), while some strains, such as Westerdykella sp. Dr(M2)4 and Acremonium sp. Dr(F)1 did not exhibit any inhibitory effects at all for this possible metabolite, with colonies of approximately 100% of the reference diameter. However, no growth effects were observed for the strain Acremonium sp. Dr(F)1 and even growth stimulation was observed in the Westerdykella sp. Dr(M2)4 culture, possibly because of the use of this compound as a carbon source or enzyme effector.
The inhibition caused by ClAc, FBAld, FBAlc and FBAc may be due to the formation of phenolic compounds, which are known for their disinfectant action [19,20], these have been described as metabolites of bacterial biodegradation [6,21] and thus might be produced by fungi too.
Except by Cladosporium sp. CBMAI 1237, all the tested strains were significantly inhibited in the presence of esfenvalerate, xylene and esfenvalerate plus xylene. Growth inhibition by esfenvalerate and xylene can be attributed to the formation of phenolic metabolites by biodegradation, which would produce toxic effects. It was observed that the commercial insecticide SUMIDAN 150 SC did not cause growth inhibition effects, suggesting that the biodegradation of esfenvalerate in the commercial insecticide is slower than that of the technical grade compound. A more efficient biodegradation would generate a higher concentration of phenolic compounds, which are the probable cause of growth inhibition.
Another possibility is that the polysaccharides present in the commercial insecticide stimulate the fungal growth and compensate the growth inhibition generated by phenolic metabolites. However, this is not very likely, since a rich medium (malt 3%) was used in these experiments.

Conclusions
The marine fungal strains Penicillium raistrickii CBMAI 931, Aspergillus sydowii CBMAI 935, Cladosporium sp. CBMAI 1237, Microsphaeropsis sp. Dr(A)6, Acremonium sp. Dr(F)1, Westerdykella sp. Dr(M2)4 and Cladosporium sp. Dr(M2)2 were grown in the presence of esfenvalerate and its main metabolites, showing the potencial of esfenvalerate biodegradation by these strains. It was observed that technical grade esfenvalerate and its metabolites had inhibitory effects on growth, reducing the size of the colony.
However, the fungal development was not affected by the presence of the commercial insecticide SUMIDAN 150 SC in the culture medium, possibly showing that the biodegradation of esfenvalerate in the commercial insecticide is slower than that in the technical grade compound, since a slower biodegradation of esfenvalerate reduces the concentration of phenolic compounds and thus the growth inhibition. Future studies will focus on the quantitative biodegradation analysis of technical grade esfenvalerate and active ingredient in the commercial insecticide.