*Article* **Di**ff**erential Expression of Maize and Teosinte microRNAs under Submergence, Drought, and Alternated Stress**

**Edgar Baldemar Sepúlveda-García 1, José Francisco Pulido-Barajas 2, Ariana Arlene Huerta-Heredia 3, Julián Mario Peña-Castro 1, Renyi Liu 4 and Blanca Estela Barrera-Figueroa 1,\***

Received: 15 August 2020; Accepted: 11 October 2020; Published: 15 October 2020

**Abstract:** Submergence and drought stresses are the main constraints to crop production worldwide. MicroRNAs (miRNAs) are known to play a major role in plant response to various stresses. In this study, we analyzed the expression of maize and teosinte miRNAs by high-throughput sequencing of small RNA libraries in maize and its ancestor teosinte (*Zea mays* ssp. *parviglumis*), under submergence, drought, and alternated stress. We found that the expression patterns of 67 miRNA sequences representing 23 miRNA families in maize and other plants were regulated by submergence or drought. miR159a, miR166b, miR167c, and miR169c were downregulated by submergence in both plants but more severely in maize. miR156k and miR164e were upregulated by drought in teosinte but downregulated in maize. Small RNA profiling of teosinte subject to alternate treatments with drought and submergence revealed that submergence as the first stress attenuated the response to drought, while drought being the first stress did not alter the response to submergence. The miRNAs identified herein, and their potential targets, indicate that control of development, growth, and response to oxidative stress could be crucial for adaptation and that there exists evolutionary divergence between these two subspecies in miRNA response to abiotic stresses.

**Keywords:** hypoxia; submergence; drought; alternated stress; maize; teosinte; microRNAs

### **1. Introduction**

As a consequence of global warming, hydrological fluctuation events such as excessive rainfall and droughts are common and projected to continue in the future, affecting economic activities [1]. Alterations in water availability in the field, either caused by water surplus or deficit, produce water stress in plants that negatively impacts the growth and productivity of crops worldwide [2].

Flooding affects the properties of soil and the composition of associated microbial communities [3] and reduces the availability of nutrients and flux of oxygen to the plant. This leads to hypoxic stress at the cellular level, especially when the column of water exceeds the length of the stem (i.e., submergence) [4,5]. In response to flooding stress, plants express genes known as hypoxia core genes (HCG) that promote anaerobic metabolism [6]. Additionally, some plants use the escape strategy by redirecting growth to stem elongation to overpass the column of water and maintain the oxygen

flux. Plants may also delay growth, flowering, and other processes that are energetically expensive, as part of a strategy known as quiescence to save resources useful to reestablish normal growth when the water level recedes [7]. On the contrary, when water availability is limited, as in the case of drought, plants undergo a similar series of changes to adapt to the adverse conditions, which include stomatal closure with limitations in CO2 uptake and reduction in photosynthetic activity, increase in root elongation rate, early or delayed flowering time, and other physiological and morphological changes [8].

Cellular dehydration caused by drought, and hypoxia driven by submergence, trigger a cascade of adaptive responses that are regulated at the molecular level in plants and are directed to maintain vital functions and protect structures from damage. Since cellular protection is a priority during stress, it has been shown that some responses to dehydration and excess water have common molecular effectors acting as nodes in the crosstalk between responses to drought and submergence [9–11]. This may have allowed wild plants to evolve tolerance mechanisms to cope with alternate drought and flooding events, which commonly occur consecutively in some regions of the world [12,13].

Maize (*Zea mays* ssp. *mays*) is one of the main dietary cereals in the world [14]. In the United States, the world's main maize producer, extreme drought and excessive rainfall are the first and second major causes of losses in maize production, respectively, with both affecting yields to a comparable extent [15]. According to archeological, botanical, and genetic evidence, maize was domesticated from its single ancestor teosinte (*Zea mays* ssp. *parviglumis*) around 10,000 years ago [16,17]. It is estimated that maize retained 83% of the nucleotide diversity from its ancestor [18]. During domestication and breeding, maize has been subject to selection for traits that are beneficial to improve feeding quality and yield, while other traits like tolerance to stress could have been lost. For this reason, teosinte is considered a living reservoir of genes and mechanisms that could be of importance to improve tolerance to stress in maize [19,20].

MicroRNAs (miRNAs) have been proposed as promising targets for developing plants with improved tolerance to multiple abiotic stresses [21]. miRNAs are small RNA molecules with pivotal roles in the response to environmental stresses in plants that usually act as negative regulators of gene expression via silencing target genes, which are recognized by sequence complementarity and subsequently targeted for degradation and/or translation inhibition [22]. With the development of new platforms for the high-throughput sequencing of small RNAs, several miRNAs involved in the response to drought or submergence have been described in plants such as *Arabidopsis thaliana* [23,24], *Brachypodium distachyon* [25,26], *Oryza sativa* [27], *Nelumbo nucifera*, [28] and *Zea mays* [29,30].

According to miRBase, 325 mature miRNAs from 174 miRNA precursors have been identified in maize [31], and some of them have been reported to be responsive to drought [32,33], submergence, [34] or waterlogging [35]. For example, miR159, miR164, miR167, miR393, miR408, and miR528 are upregulated by short-term waterlogging in roots of maize lines with high tolerance and suggest the involvement of hormonal control in the response mediated by miRNAs [30]. Another study of two maize lines, with contrasting tolerance to drought, observed the downregulation of miR164 and upregulation of miR159, miR390, and miR398 in the tolerant line compared with the sensitive line [29]. However, it is not clear how miRNAs are regulated in teosinte under submergence or drought stress and whether miRNA response to these stresses is conserved during maize domestication and breeding.

Therefore, this study aimed to identify microRNAs expressed in maize and teosinte under submergence and drought stress in order to analyze the conserved and differential mechanisms of response in these plants. For this, we used high-throughput small RNA sequencing to profile maize and teosinte under submergence, drought, and consecutive drought and submergence stresses. We found a group of miRNAs that were regulated by drought or submergence, including some miRNAs that were differentially regulated in the two species. Analyses of small RNA data from teosinte subject to alternate treatments of drought and submergence indicated that miRNA response to drought was attenuated when submergence was the first stress treatment, but miRNA response to submergence did not change when drought stress was applied first. The potential targets of these miRNAs indicate that control of development, growth, and response to oxidative stress could be crucial for understanding the conservation and divergence of stress tolerance between maize and teosinte.

#### **2. Results**

#### *2.1. Physiological Response of Maize and Teosinte to Submergence and Drought*

With the objective to determine a time period of treatment for RNA collection, maize seedlings with two vegetative leaves and 14 days after sowing (stage V2, 14 DAS) were exposed to submergence for 2, 4, and 6 days (Figure 1A). All treatments induced growth reduction in maize, but plants submerged for 4 or 6 days developed necrotic spots on the leaves, and plants treated for 6 days were not able to recover from the stress. Therefore, a 2 day submergence treatment was selected for subsequent assays. Similarly, drought assays were performed on maize seedlings that were deprived of water until they reached half their initial weight and were maintained under this level of water limitation for 2, 4, 6, and 8 days (Figure 1B).

**Figure 1.** Effects of submergence and drought on maize and teosinte plants. (**A**) Plants exposed to submergence for 2, 4, and 6 days, and (**B**) plants exposed to drought for 2, 4, 6, and 8 days. Response of teosinte and maize plants to (**C**) submergence for 48 h, expressed as submergence tolerance coefficient (STC), and (**D**) drought for 6 days and recovery 24 h after rehydration, expressed as relative water content (RWC) in the leaves. Data are means, with error bars representing ±SD of three biological replicates, each replicate consisting of six plants. Different letters above the error bars indicate statistically significant differences between samples (*p* < 0.05) in a Student's *t*-test.

Drought caused a reduction in the growth of maize plants when compared with control plants, but this reduction was stabilized, which may be explained by the fact that pots were maintained at a constant, intermediate water deficit condition, and not exposed to progressive drought stress. Based on these observations, maize and teosinte plants were exposed to submergence for 2 days and drought for 8 days, and the submergence tolerance coefficient (STC) and relative water content (RWC) were registered for the respective treatments. Submergence affected the growth of maize and teosinte seedlings to a similar extent, since STC was not significantly different (Figure 1C). There were also no

significant differences in RWC between maize and teosinte in control, drought, and 24 h of recovery after the stress (Figure 1D). These results suggest that both plants are able to sense and respond to stress by adjusting water status and growth to a similar extent at this stage. This is expected because both maize CML496 and teosinte grow in conditions prevalent in the tropics, where drought, high temperature, and extreme seasonal variations in monthly rainfall are commonly observed. Therefore, this constitutes a solid system to investigate the intra-specific conservation and divergence in the response to stress between maize and its direct ancestor that lead to adaptation in both plants.

#### *2.2. Overall Analysis of Small RNA Sequencing Data*

The alterations in growth, STC, and RWC observed in maize and teosinte seedlings in the assays suggest that the times and stress intensities can help to study molecular responses in both plants. Accordingly, maize and teosinte seedlings exposed to 2 days of submergence or 6 days of constant drought were selected for profiling the microRNA populations involved in the corresponding responses. In addition, teosinte seedlings exposed to alternated stress of drought-submergence and submergence-drought were also selected.

High-throughput sequencing of small RNA libraries prepared from teosinte and maize under submergence, drought, and alternated stress rendered from 16 to 46 million raw reads (Table 1), with PHRED score above 30 (Figure S1). After removing low-quality reads and adapters, the libraries were analyzed for size distribution of reads, revealing an enrichment of 24 nt sRNAs across the libraries (Figure S2). Reads less than 18 or longer than 35 nt were removed, resulting in a number of clean reads ranging from 14.8 to 41.9 million (Table 1).

**Table 1.** Summary of small RNA sequencing data from eight maize and teosinte libraries. Distribution of small RNAs in the corresponding non-coding RNA category.

Submerg: Submergence; mt: mitochondria; cp: chloroplast; mRNA: messenger RNA; rRNA: ribosomal RNA; tRNA: transfer RNA; snRNA: small nuclear RNA; snoRNA: small nucleolar RNA; lncRNA: long non-coding RNA.

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On average, 99.7% of the clean reads were mapped to the maize genome. From the reads mapped to the maize genome, less than 3% represented plant miRNAs reported in the miRBase (Table 1). Interestingly, libraries from teosinte showed the lowest percentage of miRNAs (approximately 0.74% for teosinte and approximately 2.19% for maize), suggesting a potential underrepresentation effect caused by the absence of teosinte miRNAs in the miRBase. However, the percentage of teosinte reads that were mapped to the maize genome (approximately 99.75%) was the same as that for maize (approximately 99.77%), indicating that differences are likely due to lower levels of miRNA expression in teosinte. In addition, a reduced abundance of plant miRNAs under submergence stress was observed in both maize and teosinte, and in teosinte treated with drought-submergence stress compared with the corresponding controls (Table 1). After removing low frequency reads and normalization to tags per million (TPM), 178 miRNA sequences in total (118 mapping to maize and 60 to miRNAs of other plants) were expressed across the libraries (Table S1).

#### *2.3. Global Changes in Expression of miRNA Sequences in Response to Stress*

Further analysis revealed that 164 miRNA sequences (110 from maize and 54 annotated in other plants) were differentially expressed in maize and/or teosinte in response to at least one of the treatments (Table S1).

Regarding submergence stress, maize and teosinte showed common expression profiles of 10 upregulated and 72 downregulated miRNA sequences (Figure 2A). A total of 42 miRNA sequences (14 upregulated and 28 downregulated) were regulated exclusively in maize, while 27 miRNA sequences (4 upregulated and 23 downregulated) were exclusive of teosinte (Figure 2A). A similar trend in the number of miRNAs responsive to stress, with lesser miRNAs being regulated in teosinte than in maize, was observed for drought treatments (Figure 2B). The analysis revealed 70 miRNA sequences (21 upregulated, 49 downregulated) regulated exclusively in maize, while 27 were differentially expressed only in teosinte (17 upregulated, 10 downregulated).

The overlap between submergence and drought responses in maize and teosinte showed that in maize, most of the miRNAs (68 sequences) were downregulated in both submergence and drought (Figure 2C), whereas in teosinte, the number of sequences downregulated exclusively in submergence was the highest (69 sequences; Figure 2D).

In addition, small RNA libraries from teosinte exposed to alternated water stress were analyzed to assess the effects of consecutive treatments of drought and submergence on miRNA expression. Figure 2E shows that 18 miRNA sequences (5 upregulated and 13 downregulated) were differentially expressed in a similar pattern in response to submergence, drought, and alternated drought-submergence treatments. The number of miRNA sequences (69) overlapping between drought-submergence and submergence (representing 63% of total sequences in submergence) was higher compared with only 22 miRNA sequences between drought-submergence and drought (39% of total sequences in drought; Figure 2E), indicating that when submergence is the second stress, it has the strongest effect on miRNA expression.

When submergence is followed by drought, 21 out of 56 miRNA sequences regulated under drought overlapped between submergence-drought and drought treatments (37% of sequences in drought), indicating that when submergence is the first stress, it affects the subsequent response to drought in teosinte (Figure 2F).

**Figure 2.** Venn diagrams showing differentially expressed miRNA reads that overlap in maize and teosinte under (**A**) submergence and (**B**) drought treatments. Plant miRNAs overlapping in submergence and drought treatments in (**C**) maize, and (**D**) teosinte. Plant miRNAs overlapping in teosinte under single and alternated treatments of (**E**) drought followed by submergence, and (**F**) submergence followed by drought. Numbers are based on fold change (FC) (log2) values (treatment/control). Upward arrows represent upregulated miRNAs (FC ≥ 1) and downward arrows represent downregulated miRNAs (FC ≤ −1).

#### *2.4. Redundancy of miRNA Reads Annotated to miRBASE and Grouping of Representative Sequences*

The data showed a high level of redundancy of reads in the libraries annotated to miRBASE, mainly caused by positional variants of mature miRNAs and other sequences matching miRNA precursors. Regrouping of mature miRNAs, positional variants derived from the same mature sequence, and sequences that exactly match those from other plants but with a close similarity to

known maize miRNAs (i.e., differing by one or two nucleotides in the 5 or 3 ends), rendered 67 representative sequences belonging to 23 miRNA families (Table S2).

#### *2.5. Search for Novel miRNAs*

In order to identify new variants of known miRNAs in teosinte, a search was performed against the miRBASE, allowing up to two mismatches in the mature or precursor sequences. This rendered 102 polymorphic variants that showed the same expression profiles as those of their corresponding exact sequences (Table S3). However, these sequences failed to provide a perfect hit to the reference genome, which did not allow the analysis of a putative precursor sequence. We also searched for potential novel miRNAs in our datasets and generated several candidates, but after a detailed inspection, they did not comply with the criteria for annotation of novel miRNAs in terms of secondary structure and/or distribution of reads along the potential precursor [36].

#### *2.6. Analysis of miRNAs Responsive to Submergence and Drought in Maize and Teosinte*

In the group of 67 representative non-redundant miRNA sequences, 13 were identified as responsive to at least one of the stress conditions, with fold change values ranging from −4.39 for the most downregulated miRNA (miR166bd) to 2.17 for the most upregulated miRNA (miR319b) (Table 2). Besides these miRNAs, others showing differential expression such as miR1511, miR2916, miR482, and miR4995 (Table S2), were discarded from the analysis due to failure to hit the maize genome or originating from ribosomal RNA or transposons.

**Table 2.** Representative plant miRNAs responsive to submergence and drought in maize and teosinte.

1 Fold change (treatment/control) values ≥ 1 indicate upregulated miRNAs (orange), and values ≤ −1 indicate downregulated miRNAs (green).

Overall, the grouping of miRNAs presented a clear picture of the stress response. Changes in miRNA expression were more marked in maize than in teosinte for most miRNAs downregulated under submergence, suggesting that maize plants could be more sensitive or reactive to submergence than teosinte in terms of miRNA responses. miRNAs that were specifically downregulated in maize were miR159ab, miR164e, miR166bd, miR167cdeg, miR169cr, miR396cd, and miR398b (Table 2).

In maize, drought stress repressed the expression of five miRNAs (miR156k, miR164e, miR166bd, miR167cdeg, and miR528a). In teosinte, only miR166bd and miR408 were downregulated, while five

miRNAs were upregulated under drought (miR156k, miR159ab, miR164e, miR319b, and miR396cd) (Table 2).

#### *2.7. Analysis of miRNAs Responsive to Alternated Stress in Teosinte*

When plants were exposed to drought-submergence conditions, the miRNA expression profile resembled that of submergence, suggesting that at the end of the drought treatment, plants were not compromised to respond to a submergence event (Table 3). The changes between drought-submergence and submergence stress were limited only to the expression of miR408. This miRNA target gene encodes plastocyanin (PLC) and laccase (LAC) involved in copper homeostasis and the formation of lignin.

**Table 3.** Representative plant miRNAs responsive to single (submergence or drought) and alternated treatments (drought followed by submergence and submergence followed by drought) in teosinte plants.

1 Fold change (treatment/control) values ≥ 1 indicate upregulated miRNAs (orange), and values ≤ −1 indicate downregulated miRNAs (green).

When plants were exposed to submergence-drought conditions, most miRNAs were regulated in a way similar to that in plants under drought alone, but the response was attenuated. For example, miR159ab, miR319b, and miR396cd were upregulated in drought as a single stress, but when drought was preceded by submergence, regulation of these miRNAs was maintained within the same trend but at low levels (Table 3). These results suggest that plants are plastic in response to environmental cues and have the capacity to oscillate to adapt to different stresses.

### *2.8. Assessment and Validation of miRNA Expression by Quantitative RT-PCR*

Quantitative RT-PCR assays were performed using a modified SL-RTPCR method for seven miRNAs regulated by stress. Based on the fold change values of miRNAs expressed in the libraries, miR166c-5P was selected as a constitutive control for qRT-PCR assays, since it showed intermediate abundance levels and stable regulation in single and alternated stress conditions (Tables 2 and 3). SL-RTPCR assays showed changes in expression similar to those observed by analysis of sequencing data for miR156k, miR159ab, miR167cdeg, miR396cd, miR398ab, miR408b, and miR528ab (Figure 3A–G).

**Figure 3.** qRT-PCR validation of miRNAs response to submergence, drought, and alternated stress by SL-RTPCR for (**A**) miR156, (**B**) miR159, (**C**) miR167, (**D**) miR396, (**E**) miR398, (**F**) miR408, and (**G**) miR528. Relative fold change values were calculated as ΔΔCt for SL-RTPCR (blue bars) using miR166c as the constitutive control or as the treatment/control ratio from normalized reads for sRNA-SEQ data (black bars). Results are mean with error bars representing ±SD of three biological replicates, each replicate consisting of a pool of six plants and three technical replicates. Different letters above the error bars indicate statistically significant differences between samples (*p* < 0.05).

For example, based on sequencing data, miR167 had a fold-change value of 0.12 and 0.17 in maize under submergence and drought, respectively. The values obtained by SL-RTPCR were 0.39 (±0.03) and 0.40 (±0.06), respectively, which confirms that miR167 is downregulated by these treatments and thus the expression trends for upregulated or downregulated miRNAs were maintained in both methods.

#### *2.9. Other Small RNAs*

Mitochondria and chloroplasts are the main compartments for energy production that are affected by low water and oxygen availability and may be a source of signals to integrate the cellular response [37,38]. A search was performed in the libraries with the objective of identifying small RNAs potentially originating from maize mitochondria or plastids. For both mitochondria and plastids, the highest number of reads mapped to the subunit 2 of NADH dehydrogenase (Table S4). The reads were derived from a region spanning 53 bp of NADH dehydrogenase subunit 2 with an abundance of

up to 820 TPM, comparable to miR408 abundance (Table S2). The second highest number of reads mapped to the trnL-trnF intergenic spacer and tRNA-Phe (trnF) gene from the maize chloroplast. Interestingly, these reads derived from a 32 bp region of the gene did not show a clear expression trend in response to stress; however, a high accumulation of reads in teosinte compared with that in maize was evident, with up to 2273 TPM, comparable to miR398 abundance (Tables S2 and S5). The causes and implications of the existence of these small RNA signatures need to be investigated in the future.

#### **3. Discussion**

Drought and submergence represent complex conditions that can be dissected into several components such as hypoxia, nutrient, light, osmotic, temperature, and oxidative stress [39]. Understanding how the different components act to integrate the response to stress at all levels of regulation is fundamental to underpin the improvement of tolerance to drought and flooding stress in crops.

miRNAs are a class of small RNAs that act as regulators of gene expression at the transcriptional level. In this study, high-throughput sequencing and analysis of small RNA populations from maize and teosinte under submergence and drought revealed that submergence caused a global reduction in the representation of miRNA expression compared with control or drought conditions. This was observed in roots of wild tomato treated with hypoxia, where only 1.45% of total reads were expressed in hypoxia-treated roots against 2.45% expressed in control roots [40].

Further exploration of the expression of specific miRNA family members allowed the identification of 13 miRNAs with differential expression profiles in response to submergence and drought. The analysis of their expression patterns and functions of their predicted, or previously confirmed targets, allowed the recognition of two main components of the responses to submergence and drought in maize: transcriptional regulation and antioxidant activity.

#### *3.1. Transcriptional Regulation*

The growth and development processes are driven by hormonal signaling pathways that are fundamental in the response to submergence and drought in plants [41], where transcription factors and miRNAs participate. Several differentially expressed miRNAs identified in this work are known to act over target genes encoding transcription factors such as miR156 (squamosa promoter-binding protein like, SPL) [42–44], miR159 (giberellic acid-MYB transcription factor, GAMYB [28,29]), miR164 (NAM-ATAF-CUC domain transcription factor NAC, MYB [45]), miR166 (basic leucine zipper transcription factor, HD-ZIP III [46,47]), miR167 (auxin-responsive factor, ARF [30,35,48]), miR169 (nuclear transcription factor Y subunit alpha, NFYA [49–52]), miR319 (teosinte branched1/cycloidea/proliferating cell nuclear antigen factor, TCP [53]), and miR396 (growth-regulating factor, GRF [54,55]). Other target genes predicted in this study are listed in Table S6.

Most miRNAs in this category have been related to responses to abiotic stress. miR156 controls developmental transitions and flowering through the activity of their targets (SPL) [42–44] which is upregulated in *Arabidopsis* roots under hypoxia and in lotus under submergence [23,28]. Other miRNAs such as miR166 were upregulated in maize roots in response to waterlogging [34] and downregulated in *Arabidopsis* roots in response to hypoxia [23]. miR164 participates in the development of lateral roots [45] and the response to waterlogging in maize roots [30]. miR159 controls petiole elongation and flowering [56] and is upregulated in maize roots exposed to waterlogging [29]. miR167 is upregulated by waterlogging in maize roots [30,34], and downregulated in submerged lotus seedlings [28].

In this study, miRNAs in the category of transcriptional regulation overlapped between maize and teosinte in response to submergence and between submergence and drought in maize (Figure 4), indicating that the control of growth is of central importance for adaptation in maize and teosinte. Interestingly, all these miRNAs were downregulated in the overlaps. Downregulation of this set of miRNAs means that their targets may be released from post transcriptional control, which may activate hormone-responsive genes to initiate elongation (miR159), cell elongation and differentiation (miR167),

transition to flowering (miR156), and other responses. In addition, it is possible that changes observed in miRNA expression at the transcriptional level are not only directed to increase growth, but also to mediate feedback in hormonal pathways, or to prepare for reoxygenation or rehydration after stress. Interestingly, the response to drought in teosinte was regulated by five miRNAs that were upregulated (miR156, miR164, miR159, miR319, and miR396). The upregulation of these miRNAs suggests that growth processes may be restricted in teosinte during drought stress (Figure 4).

**Figure 4.** Hypothetical model of the regulation of miRNAs in the response to submergence and drought in maize and teosinte. The importance of antioxidant enzymatic activity, copper homeostasis, and control of growth and development as the main lines of response regulated by miRNAs in both plants is highlighted. Interrupted lines represent overlap between plants and/or treatments. Green, downregulated miRNAs; orange, upregulated miRNAs. GRF: growth-regulating factor; GAMYB: gibberellic acid-MYB transcription factor; ARF: auxin-responsive factor; NFYA: nuclear transcription factor Y subunit alpha; PLC: plastocyanin; LAC: laccase; SOD: superoxide dismutase; SBP1: selenium-binding protein1; bZIP: basic leucine zipper transcription factor; SPL: squamosa promoter-binding protein like; NAC: NAM-ATAF-CUC domain transcription factor; TCP: teosinte branched1/cycloidea/proliferating cell nuclear antigen factor; SBP1: selenium-binding protein 1.

#### *3.2. Antioxidant Activity*

Submergence and drought stress result in excessive production of reactive oxygen species (ROS) and efficient mechanisms to cope with oxidative injury are determinants of tolerance [57]. miR398, miR408, and miR528 are included in this category. miR398 target genes encode Cu/Zn superoxide dismutases (CSD1 and CSD2) that act in the defense against toxic ROS in *Arabidopsis* [58]. In *Phaseolus vulgaris,* miR398 is downregulated under drought and submergence [59] and is also downregulated in response to submergence in *Arabidopsis* [60]. We predicted other miR398 targets including a selenium-binding protein (SBP) (Table S6). An SBP was recently reported to be induced in *Arabidopsis* in response to submergence [60] and confirmed as a miR398 target by degradome analysis in maize [61]. In this study, miR398 was downregulated in maize and teosinte under submergence, thereby implicating a potential increase in antioxidant activity.

Other members, such as miR408 and miR528, target genes encoding the cupredoxins PLC and LAC [62–64], and other genes controlling circadian clock and flowering time [65,66]. PLC functions as an electron transporter and LAC participates in the formation of lignin by oxidation. miR408 and miR528 have been previously reported to respond to submergence in Lotus seedlings and maize roots [28,30]. In this study, miR408 and miR528 were downregulated in maize and teosinte under submergence, suggesting the maintenance of electron flux, oxidation homeostasis, and lignin synthesis (Figure 4).

#### *3.3. Comparison of miRNA Expression in Maize and Teosinte*

Previous work demonstrated a high level of conservation of miRNA mature sequences between maize and teosinte [67]. Thus, divergence in tolerance to stress is most likely the result of differences in gene expression between these plants. Overall, in this work maize and teosinte showed similar responses to submergence, with a clear trend toward downregulation of miRNAs involved in the control of growth, development, antioxidant response, and copper homeostasis. However, the response to submergence was more intense in maize than in teosinte for most of the miRNAs responsive to stress, especially for those miRNAs controlling growth. This suggests that maize is more reactive to submergence and responds by releasing the control on their target genes, which in turn may accelerate growth to escape from stress. In teosinte, these responses were less dramatic, suggesting that a finer tuning of expression could be a key for tolerance in this plant. A similar pattern was observed in a study comparing miRNA expression in inbred maize lines with different levels of tolerance to waterlogging, where the stress sensitive line reacted by downregulating most of the miRNAs and induced target genes to accelerate growth. Instead, the tolerant line responded with a moderate level of downregulation, or even upregulation of some miRNAs to repress growth under stress [30].

In the case of drought, maize responded in a similar way as that in submergence by downregulating miRNAs to induce growth, while teosinte upregulated miRNAs to restrict growth and probably save energy resources. Regarding defense against oxidative stress, oxidation homeostasis, and reinforcement of cell structure, both plants maintained the downregulation of miRNAs, suggesting the possibility that their target genes were actively protecting the plants from the oxidative damage caused by stress.

#### *3.4. E*ff*ects of Alternated Stress on Teosinte*

Alternated events of drought and submergence are common in some regions of the world where plants experience the succession of these events throughout their life cycle. In this study, teosinte plants treated with alternated stress revealed that drought as the first stress had no effect on the capacity to respond to a subsequent event of submergence (Figure 5). In fact, plants treated with alternated stress, and plants treated only with submergence had resembling replicates for most of the miRNAs. This suggests that teosinte was able to rapidly reverse the effects of drought and redirect the response towards submergence, even though drought had induced clear symptoms of stress in plants. The only miRNA that did not respond to submergence as the second stress was miR408, but a functional overlap with miR528 may have supplied the activity of LAC and PLC.

**Figure 5.** Hypothetical model of the response to alternated stress in teosinte. The influence of the first stress and the consequences in miRNA responsiveness to the second stress are indicated. Green, downregulated miRNAs; orange, upregulated miRNAs.

In the opposite order, submergence followed by drought produced an attenuated response to drought. The effects of submergence as the first stress had a long-term influence, considering that between the end of submergence and the beginning of drought, there was a gap of 8 days. This effect also suggests the plastic nature of plants to adapt to adverse factors and oscillate between different stresses.

Research has shown that recovery from submergence represents an additional stress by itself. Upon de-submergence, plants suffer reoxygenation stress conducive to the accumulation of ROS, reillumination stress, dehydration, and senescence [68]. During these processes, hormonal signals, ROS, protective proteins, and other molecular effectors could be induced in plants over a period of time that could be useful to survive any other event of stress such as drought, establishing an overlap between submergence and drought tolerance, as described in rice harboring the SUB1A gene [9].

Several aspects in the involvement of miRNAs in the responses to stress still need to be addressed for constructing a solid system that improves stress tolerance in plants. These include a detailed study of expression dynamics between miRNA and target genes during exposure to combined or alternated stress and during recovery in lines with contrasting tolerance, implications of other factors in the response such as intensity and quality of light during stress, circadian cycle on miRNA expression, plant age, the function of other sRNAs involved, and additional layers of regulation of gene expression. The present study constitutes a base for information that could be useful in further efforts to broaden the knowledge on the roles of miRNAs in stress response and explore miRNAs as tools for improving tolerance to multiple stresses.

#### **4. Materials and Methods**

#### *4.1. Plant Materials*

Maize seeds (*Zea mays ssp. mays*) of tropical homozygous line CML496, and teosinte (*Zea mays ssp. parviglumis*) were obtained from Centro de Investigación y de Estudios Avanzados (CINVESTAV) and Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP) seed collections, respectively. The seeds were germinated directly in pots with Sunshine Mix 3 (N: 36 ppm from

NO3/9 ppm from NH4; P: 7 ppm; K: 49 ppm; Ca: 45 ppm; Mg: 30 ppm; S: 39 ppm; B: 0.046 ppm; Cu: 0.01 ppm; Fe: 0.478 ppm; Mn: 0.251 ppm; Mo: 0.035 ppm; Na: 10 ppm; Cl: 8 ppm; Al: 0.48 ppm) (Sun Gro Horticulture Distribution Inc., Agawam, MA, USA), three seeds per pot. In the case of teosinte, the seeds were mechanically scarified to remove the coat before sowing.

#### *4.2. Growth Conditions and Treatments*

Maize and teosinte plants were grown in a plant growth room at 23 ◦C and 16 h/8 h light/dark cycle in order to lengthen the vegetative growth phase. The drought stress applied in this study was set to maintain plants at a constant intermediate intensity of drought rather than a progressive drought stress. For drought stress, pots with plants in vegetative stage V2, 12 days after sowing (12 DAS) were water-deprived for 6 days by interrupting water supply until half of their initial weight (water deficit) was reached. Then, water was added in small amounts on a daily basis to constantly maintain the plants in a water deficit condition at half their weight for 6 days. Submergence stress was applied on plants at 26 DAS, with pots in empty 32 cm deep submergence tanks that were filled with filtered tap water to increase the water column by 6 cm every 2 h for 10 h, until the maximum capacity was reached, and plants were fully submerged. The plants were maintained in this condition for 48 h after the first addition of water. For all the treatments, growth conditions were maintained at 23 ◦C and 16 h/8 h light/dark cycle.

For drought followed by submergence (drought-submergence), teosinte plants (14 DAS) were water-deprived for 6 days followed by 6 days under constant drought stress and then submerged for 48 h as previously described. For submergence followed by drought (submergence-drought), teosinte plants (12 DAS) were submerged for 48 h and removed from the water tank followed by depriving them of water until the plants reached half their initial weight (8 days). Then, small amounts of water were added to maintain a constant water deficit condition for 6 days. The single and alternated stress experiments with maize and teosinte were designed to end simultaneously at 28 DAS. At the end of the experiments, the shoots were immediately collected, frozen in liquid nitrogen, and stored at −80 ◦C for further processing. All the assays, including control and treatments, were performed with three biological replicates, with each replicate containing six plants.

#### *4.3. Assessment of Physiological and Morphological E*ff*ects of Stress in Maize and Teosinte*

For drought stress, the relative water content (RWC) was measured in the second leaf of maize plants under drought stress and control conditions, as previously described [69]. Briefly, square leaf sections (2 × 2 cm) were excised from plants and weighed to obtain the fresh weight, then submerged in deionized water for 24 h to obtain the turgid weight, and finally dried at 70 ◦C until constant dry weight was achieved. Then, RWC was calculated as [(fresh weight-dry weight)/(turgid weight-dry weight) × 100].

For submergence stress, the submergence tolerance coefficient (STC) was adapted from the coefficient applied for waterlogging [70]. Briefly, the length of shoots and roots of plants exposed to submergence or control conditions were noted at the end of experiments. The plants were then dried at 70 ◦C until a constant dry weight was reached. The sums of length and dry weight of plants exposed to the stress and control conditions were used to calculate the STC as [(sum of treated plants)/(sum of control plants)]. RWC and STC measurements were performed in three biological replicates, each consisting of six plants per treatment.

#### *4.4. Total RNA Extraction and Construction of Small RNA Libraries*

Total RNA was extracted from treated and control samples (three biological replicates, each consisting of a pool of six plants) with TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Total RNA was quantified using a Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and visualized by 1% agarose gel electrophoresis under denaturing conditions. Total RNA from triplicates of the same treatment was

pooled (4 μg from each biological replicate in a total of 12 μg per treatment). The small RNA fraction (20–30 nt) was purified from total RNA in a denaturing 15% acrylamide gel. Next, a pre-adenylated adapter (linker1, Integrated DNA Technologies, San Diego, CA, USA) was ligated to the 3' end of small RNAs with T4 RNA ligase II (New England Biolabs, Ipswich, MA, USA) in the absence of ATP. Then, the RNA adapter Illumina RA5 was ligated to the 5´ end of small RNAs with T4 RNA ligase 1 (Ambion, Austin, TX, USA). The ligated products were used as templates in a cDNA synthesis reaction with the primer RT Bridge and the retrotranscriptase MMuLV (Thermo Fisher Scientific Inc., Waltham, MA, USA), followed by a 15-cycle PCR reaction with Phusion High-Fidelity DNA polymerase (New England Biolabs). The PCR products were purified, quantified, and sequenced in the Illumina HiSEQ2500 at the Genomics Core of the University of California-Riverside (Riverside, CA, USA). Adapters and primers are listed in Table S7.

#### *4.5. Bioinformatics Analysis of Small RNA Libraries*

Raw sequencing datasets from small RNA libraries were processed with the CLC Genomics Workbench 12 (QIAGEN, Hilden, Germany) to remove adapters, low-quality reads, and reads shorter than 18 nt and longer than 35 nt. Clean reads were mapped to the B73 RefGen\_v5 maize genome [71] and classified into the different non-coding RNA categories (rRNA, tRNA, snRNA, snoRNA, lncRNA, and repeats). In addition, clean reads were mapped to the maize chloroplast and mitochondria DNA (B73 RefGen\_v4). The clean reads were searched against the plant miRNAs deposited in the miRBASE (Release 22.1) [31], allowing (1) zero mismatches, or (2) up to two mismatches to detect polymorphisms. miRNA sequence counts were normalized as TPM relative to the total clean read counts in the corresponding library. Individual miRNA sequences were then analyzed for differential expression. Fold change values were calculated as log2 (normalized counts in treatment/normalized counts in control) (Table S1). Only miRNA sequences with raw counts ≥ 50 in at least one of the libraries were considered for differential expression analysis, and fold change values ≥1 or ≤−1 were considered as upregulated or downregulated, respectively.

miRNA sequences with a high level of similarity were then grouped. All sequences that differed from the mature sequence by one or two nucleotides in the 5 or 3 ends were considered positional variants and were then grouped with the mature sequence to constitute a representative miRNA group. The sum of reads and fold change values were recalculated for grouped miRNA sequences (Table S2).

Small RNA sequencing data were deposited into the NCBI/GEO database with accession number GSE155050.

#### *4.6. Assessment of miRNA Di*ff*erential Expression by Quantitative RT-PCR (qRT-PCR)*

A modified stem-loop method based on that previously described [72,73] was used to assess miRNA expression. Briefly, total RNA from treated and control plants were treated with DNase I (Thermo Fisher Scientific, Inc, Waltham, MA, USA.). Subsequently, 0.1 μg of treated RNA was used in 10 μL-cDNA synthesis reactions using the TaqMan miRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) and miRNA specific stem-loop primers. For quantitative PCR, 20 μL-PCR reactions were prepared using Maxima Probe qPCR Master Mix (Thermo Fisher Scientific Inc, Waltham, MA, USA) with a miRNA-specific direct primer, PCR stem-loop reverse primer, and a universal fluorescein amidite (FAM)-labeled probe, in a PikoReal real-time thermal cycler (Thermo Fisher Scientific Inc.). Differential expression values were calculated using the ΔΔCt method [74] with the use of miR166c as an internal control to normalize the relative abundance of each miRNA. All primers and probes used are listed in Table S7.

#### *4.7. Prediction of Target Genes*

The psRNATarget analysis server (http://plantgrn.noble.org/psRNATarget/) was used for identification of target genes of miRNAs responsive to submergence and drought [75]. DPMIND [76] was used to search for miRNA targets in nine maize degradomes.

*Plants* **2020**, *9*, 1367

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2223-7747/9/10/1367/s1, Figure S1: PHRED score distribution of small RNA reads in maize and teosinte libraries; Figure S2: Size distribution of small RNA reads in maize and teosinte libraries; Table S1: Known miRNA sequences expressed in maize and teosinte libraries; Table S2: Grouped sequences of known microRNAs expressed in maize and teosinte libraries; Table S3: Sequence variants of microRNAs expressed in maize and teosinte libraries; Table S4: The most abundant reads that mapped to NADH dehydrogenase subunit 2 from the *Zea mays* organelle DNA; Table S5: The most abundant reads that mapped to the trnL-trnF intergenic spacer and tRNA-Phe (trnF) gene from the maize chloroplast; Table S6: List of predicted targets of submergence and drought-responsive miRNAs; Table S7: List of adapters and primers used in this study.

**Author Contributions:** Conceptualization, R.L. and B.E.B.-F.; formal analysis, E.B.S.-G., J.F.P.-B. and B.E.B.-F.; funding acquisition, J.M.P.-C., R.L. and B.E.B.-F.; investigation, E.B.S.-G., J.M.P.-C., R.L. and B.E.B.-F.; methodology, E.B.S.-G., J.F.P.-B. and A.A.H.-H.; resources, J.M.P.-C. and B.E.B.-F.; supervision, B.E.B.-F.; writing—original draft, B.E.B.-F.; writing—review & editing, E.B.S.-G., A.A.H.-H., J.M.P.-C. and R.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This material is based upon work supported by a grant from the University of California Institute for Mexico and the United States (UC MEXUS) and the Consejo Nacional de Ciencia y Tecnología de México (CONACYT) to B.E.B.-F. and R.L., grants from Consejo Nacional de Ciencia y Tecnología CONACYT—México (Ciencia Básica 287137) to J.M.P.-C., and (Ciencia Básica 169619) to B.E.B.-F., CONACYT postdoctoral fellowship to E.B.S.-G. and A.A.H.-H., and CONACYT postgraduate fellowship to J.F.P.-B.

**Acknowledgments:** We wish to thank Axel Thiessen-Favier (CINVESTAV-Irapuato; *in memoriam*) for providing maize seeds; Flavio Aragon Cuevas (INIFAP Oaxaca Valles Centrales) for providing teosinte seeds; Mikael Roose (UCR) for academic advice and administrative assistance; Glenn Hicks and John Robert Weger (Genomics Core, UCR) for technical advice and performing sRNA sequencing; Fabiola Hernández, L.E. María de Jesús Hernández, L.C. Yenny Reyes Roque and Francisco Campos (Universidad del Papaloapan) for administrative assistance.

**Conflicts of Interest:** The authors declare no conflict of interest. The sponsors had no role in the design, execution, interpretation, or writing of the study.


*Article* **Study of the Enhancements of Porous Structures of Activated Carbons Produced from Durian Husk Wastes**

**Tongsai Jamnongkan 1,\*, Nitchanan Intaramongkol 1, Nattharika Kanjanaphong 1, Kemmika Ponjaroen 1, Wasana Sriwiset 1, Rattanaphol Mongkholrattanasit 2, Piyada Wongwachirakorn 3, Kun-Yi Andrew Lin 4,\* and Chih-Feng Huang 5,\***

**Abstract:** The idea of generating high-value practical materials, such as activated carbons, from agricultural wastes as a raw material has been a quite important trend recently due to its positive contributions to the environment and resource savings from biomass. In this paper, activated carbons prepared from durian husk waste by the KOH chemical activation method are studied. We focus on the effects of stages of the activating temperature on their properties. The optimum conditions for activation were a KOH/char ratio of 1:2 at the first and second activation process at the temperatures of 400 and 800 ◦C, respectively. The characterization results of Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and Brunauer–Emmett–Teller (BET) surface area showed that the obtained activated carbons have a high surface area and small pore size. The adsorption/desorption isotherms of the obtained activated carbons showed type I and type II isotherms. The chemical structure of obtained activated carbons did not show any variation in the surface functional groups. A feasible method to produce the activated carbons with a high surface area and high adsorption capability from durian husk waste was eventually demonstrated.

**Keywords:** activated carbons; durian husk; activation; waste utilizations; adsorption

#### **1. Introduction**

Durian is a one of most famous fruits consumed locally and exported in the Southeast Asia region, especially in Malaysia and Thailand. A large quantity of durian fruit is produced per year for commercialization [1]. However, it is well known that not the entire durian fruit is edible. The outer part of the durian skin normally weighs more than half of the total of this fruit, and this cannot be eaten and is thrown away. Large quantities of durian husk waste are being produced, which is becoming an environmental problem. Therefore, it is worthwhile to find a solution to this problem. Generally, durian husk waste is usually discarded at a landfill site and ends up in open burning, which can release harmful pollution into the air, increasing the air pollution to the environment [2,3]. In the past decade, the idea of utilizing biomass from agricultural wastes as a raw material for the production of valuable material has attracted the interest of global researchers.

**Citation:** Jamnongkan, T.; Intaramongkol, N.; Kanjanaphong, N.; Ponjaroen, K.; Sriwiset, W.; Mongkholrattanasit, R.; Wongwachirakorn, P.; Lin, K.-Y.A.; Huang, C.-F. Study of the Enhancements of Porous Structures of Activated Carbons Produced from Durian Husk Wastes. *Sustainability* **2022**, *14*, 5896. https://doi.org/ 10.3390/su14105896

Academic Editors: Hafeezullah Memon, Xiaoke Jin, Tian Wei and Chengyan Zhu

Received: 7 April 2022 Accepted: 9 May 2022 Published: 12 May 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Activated carbons, carbonaceous material with a large surface area and high porosity, are one of the most commonly obtained materials produced from agricultural wastes. In recent times, this material has received much attention from the scientific community as it is renewable, low-cost, and environmentally friendly. The surface area and pore size distribution are important factors in the determination of activated carbon's performance, such as its adsorption and mechanical properties [4].

Recently, activated carbons with high surface area and pore volumes have been produced from various sources of agricultural wastes such as coal [5–7], coconut shell [8,9], sawdust [10,11], sugarcane bagasse [12], rice straw, and husk [13] plant materials [14–18], including agricultural industrial by-products such as coffee bean husk [19,20]. Among these precursors, durian husk waste is also a main source within agricultural industrial practice in the production of potential activated carbon due to its effectiveness and inexpensive cost as a raw material. In general, activated carbon is used in a wide range of environmental applications. In particular, it is a common material used as an adsorbent for adsorption or treatment in water purification, such as heavy metal removal in groundwater [21–23], wastewater treatment [24,25], and for the removal of volatile organic compounds (VOCs) [26], carbon dioxide [27], pigments, and odors [28]. It is well known that the potential efficiency of activated carbons depends on their surface area and active surfaces. Thus, it is very important to prepare activated carbon with a high surface area and high porosity in order to enhance its adsorption capacity due to these characteristics, which are particularly suitable for specific applications [29–31]. Currently, activated carbon is manufactured via physical or chemical stimulation processes, such as the physical activation of pyrolysis to produce char followed by steam gasification [32,33], and chemical activation, such as using solutions of zinc chloride [34], phosphoric acid [35], potassium hydroxide [36], sodium hydroxide [37], and so on. Besides this, the nature of raw materials has also commonly affected the pore structure and pore size distribution of activated carbon. A functional activated carbon will be obtained through a two-step process: (i) hydrothermal carbonization of the raw materials in an atmosphere without oxygen followed by the activation of the resulting char. In this stage, most of the noncarbon elements, such as hydrogen and oxygen, are removed in a gaseous form by pyrolytic decomposition, resulting in carbon with a fixed mass and a rudimentary pore structure; (ii) secondly, the activation step can be performed as follows: an appropriate amount of obtained char is mixed with an activating agent and then initiated by heating the samples in the furnace from room temperature until the desired temperature is reached. This step is employed to enlarge the diameter of fine pores and also to create new pores so that the adsorptive power of the carbonization product is enhanced [38–40]. It is well known that the activated carbon is not only used as an adsorbent for the removal of polluting molecules in the environment but is also widely used for supercapacitors' electrode materials, catalyst carriers, and reinforcement materials [41–44]. Recently, several studies have reported about the method of activated carbon production, but there have been no studies into the effect of the activating temperature on the surface area and porosity structures of the obtained activated carbon. Therefore, it is worthwhile to further examine this parameter and how it affects the properties of activated carbon.

In this research, carbonized durian husk was used as a precursor for the production of activated carbon by chemical activation with potassium hydroxide; due to its cost effectiveness, it is used as an activating agent for producing activated carbon. The effects of the stages of the activating temperature on the properties of activated carbon were investigated. Then, a comparison of the iodine absorption capacity of activated carbon was performed to study the specific surface area and the amount of porosity in adsorption. In addition, the results of Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and Brunauer–Emmett–Teller (BET) surface area are investigated and discussed.

#### **2. Materials and Methods**

*2.1. Materials*

Durian husk (*Monthong varieties*) was collected from a local durian market in the Chonburi province of Thailand. The characteristics of durian husk waste are given in Table 1. Hydrochloric acid with a purity of 85% was purchased from Sigma–Aldrich (Germany). Potassium hydroxide and sodium hydroxide were purchased from Ajex chemical (Germany) and sodium thiosulfate of AR-grade was purchased from Loba Chemie (India). Other chemical agents were analytical grade purity and used as received without further purification.

**Table 1.** The characteristics of durian husk waste.

#### *2.2. Activated Carbon Preparations*

As a first step, the durian husk waste was cut to 3–5 cm. Then, it was repeatedly washed with distilled water to remove dust and other inorganic impurities. After that, the clean durian husk was dried at a temperature of 100 ◦C for 24 h in the hot-air oven to reduce its moisture contents, and it was stored in a desiccator. In the carbonization process, the char was produced by carbonizing the samples of dried durian husk in a fixed-bed batch reactor in an atmosphere without oxygen. The reactor was filled with samples and was placed in a furnace (Model 30400, Barnstead Thermolyne, USA). The furnace was heated from room temperature to 600 ◦C, kept at that temperature for 3 h, and then cooled down to room temperature overnight. The obtained char was crushed in a crusher (Model ZM200, Restch, Germany) to obtain particles with sizes between 800 to 1000 μm and then stored in desiccators for further use. After the carbonization process, the mass yield of the char was calculated, and we found that the yield average was 73.58% of the total mass. The activation process of char was carried out as follows: the char was impregnated by soaking appropriate amounts of the char with different ratios of potassium hydroxide at room temperature for 24 h, as shown in Table 1. After the impregnation process was completed, we used into two processes for activations: (i) in Process I, denoted as DH1–DH3, the impregnated precursors were poured onto a porcelain disc and placed in the furnace for their first activation at a temperature of 400 ◦C for 1 h. Then, the activation process was carried out by adjustment to the second activation at a temperature of 600 ◦C for 3 h, and then the material was cooled down to room temperature. (ii) In Process II, defined as DH4–DH6 code samples, these activated carbons were heated to the second activation temperature of 800 ◦C for 3 h under the same conditions as in the Process I preparation. Details of the process conditions for preparing activated carbon are shown in Table 2.

**Table 2.** Sample identification, activation process conditions, and yield for activated carbon preparation.

All activated carbons were washed sequentially with a 0.5 N HCl solution in order to eliminate excess KOH. Consecutively, activated carbon powders were repeatedly washed with distilled water until reaching a neutral pH. After that, these powders were dried in an oven at 110 ◦C for 24 h and stored in a desiccator before further use.

#### *2.3. FTIR Spectroscopy*

The chemical structure of the obtained activated carbon was verified by using a FTIR spectrometer (Invenio, Bruker, USA) in ATR mode equipped with a diamond crystal, which was used for the tests. All the spectra were recorded in transmittance mode with 4 cm−1 resolution in the wavenumber range of 4000–400 cm−1 under ambient conditions.

#### *2.4. Iodine Number Analysis*

The iodine number is a technique commonly employed to determine the adsorption capacity of activated carbons. In this paper, the iodine number was determined according to the standard test method ASTM D 4607–94 [45]. The procedure of the experiment can be briefly explained as follows: 1.0 g of dried activated carbon samples were placed in a 250 mL flask, and 10 mL of 5%wt. hydrochloric acid was added. The flask was swirled until the activated carbon became wet; then, the flask was boiled for 1 minute to degas the sulfur dioxide on the surface samples and cooled down to room temperature. Then, 100 mL of stock iodine solution (12.7 g of Iodine (Merck) and 19.1 g of potassium iodide (Merck) in 1 L of de-ionized water) was added into the sample flask, and the mixture was shaken for 1 minute in the magnetic stirrer (Model C-MAG HS7, IKA, Germany) and the samples were filtered. Then, 50 mL of filtrate was titrated with 0.1 N sodium thiosulphate until the solution become pale yellow. Then, approximately 1 mL of starch solution (1 wt.%), used as an indicator, was added, and titration slowly continued with sodium thiosulphate until the solution changed from blue color to colorless. The iodine number is the amount of adsorbed iodine per gram of samples (X/M), the value of which was calculated by Equations (1) and (2):

$$\text{V}(\text{X/M}) = \frac{\left\{ (\text{N}\_1 \times 126.93 \times V\_1) - \left[ (\text{V}\_1 + \text{V}\_{\text{HCl}}) / \text{V}\_F \right] \times \left( \text{N}\_{\text{Ni}\_2\text{S}\_2\text{O}\_3} \times 126.93 \right) \times \text{V}\_{\text{Ni}\_2\text{S}\_2\text{O}\_3} \right\}}{\text{M}} \tag{1}$$

$$\mathbf{C} = \left(\mathbf{N}\_{\text{Na}\_2\text{S}\_2\text{O}\_3} \times \mathbf{V}\_{\text{Na}\_2\text{S}\_2\text{O}\_3}\right) \tag{2}$$

where N1 and NNa2S2O3 are the concentration of iodine solution and sodium thiosulfate solution (N), respectively. V1, VHCl, VF, and VNa2S2O3 refer to the volume of iodine solution, hydrochloric acid, filtrate used in titration, and the consumed volume of sodium thiosulfate solution (mL), respectively. M is the mass of activated carbon (g).

#### *2.5. Morphological Measurement*

The morphological surfaces of the obtained activated carbon were examined via SEM (JSM-6610LV, JEOL, Japan) at an accelerating voltage of 10 kV. Each sample was attached to the stubs with double-sided carbon tape. Then, the samples were sputtered with gold under vacuum before observation. The average pore sizes for each activated carbon were investigated by using the image analysis software Image J (ver. 1.45, USA).

#### *2.6. Specific Surface Area*

The specific surface area, total pore volume (VT), micropore volume (V ), mesopore volume (Vm), and average pore size (Dp) of the obtained activated carbon were scrutinized via nitrogen adsorption Brunauer–Emmett–Teller (BET) analysis on a Micrometrics 3Flex Version 5.02 apparatus. The cell containing the sample was weighed before degassing. Approximately 0.2 g of sample powders were placed in a test tube in order to removes contaminants such as water vapor and adsorbed gases from the samples. The samples were degassed at 200 ◦C for 16 h before the measurements.

#### **3. Results and Discussion**

#### *3.1. FTIR Spectroscopy*

FTIR spectra were collected for the characterization of the surface functional groups of prepared activated carbon. Figure 1 depicts the FTIR spectrum of all activated carbons obtained after the chemical treatment with KOH followed by the different conditions, as shown in Table 1. The IR spectrum of the char sample was used to compare the spectra of the obtained activated carbons. We found that the weaker broad band around 3380 cm−1 revealed the stretching vibrations of the O-H bonds of the mixtures that were adsorbed onto the surface of activated carbon. A peak at around 2100 cm−1 shows the presence of a triple bond between carbon–carbon atoms (alkynes group) with stretching vibration [46]. Compared to the char spectrum, it was found that some changes could be noted in the position and intensity of some peaks in the activated carbon spectrum. A new strong peak appeared around 1030–1100 cm−1, which was because of the C-O stretching vibrations of carboxyl or carbonyl groups. This probably indicates the formation of an oxidation reaction during treatment with KOH in the activation process. Furthermore, we also found that the absorption peak in the regions between 1500 and 1600 cm−1 could be ascribed to C = C aromatic ring stretching vibration, which is generally found in carbonaceous material [47].

**Figure 1.** FT-IR analysis of the obtained char and activated carbons.

#### *3.2. Iodine Number Analysis*

It is well known that the indirect value of the iodine number also indicates the porosity of activated carbon, which is correlated with the micropore volume. A higher iodine number meant a higher porosity on the surface determined by iodine absorption. Therefore, iodine number analysis was used to analyze the porosity of DH1–DH5, and char was also used for comparison. The iodine adsorption capacity of all the activated carbon is illustrated in Figure 2. It is confirmed that the process parameters affected the porosity of the obtained activated carbon, which was found to be higher than char. Besides this, we found that the impregnation ratio between KOH and char has a profound effect on the adsorption properties of activated carbon. At the given activation temperatures, the iodine numbers of the activated carbons increased with an impregnation ratio increased from 1:4 to 1:2 and decreased with the KOH:char ratio increased to 1:1. From our experimental results, the highest iodine number was achieved at the impregnated ratio of 1:2, as shown in Figure 2. The iodine numbers of char, DH1, DH2, and DH3 are 717, 959, 971, and 933 mg/g, respectively. In addition, the effect of the activation process on the adsorption capacity was studied. It was found that the iodine numbers increased with increases in the temperature of the second activation process, which can be compared with DH2 and DH5 in Figure 2. At the given impregnation ratio, DH5 showed the highest values, at 1037 mg/g, of its iodine number compared with the other activated carbon. This is probably due to the effect of pore structure formation within the char structure during the activation process. As mentioned above, the iodine number is commonly used in industries as a measure of adsorption capacity, reflecting the number of micropores in the carbon. This value indicates the approximate internal surface area of activated carbon. The relationship between the iodine number and the internal surface area of activated carbon has been well-established in the literature for various precursors [48–50]. It is well known that the iodine number has a direct proportional relationship with the specific surface area. Comparing recent studies, the iodine number of activated carbon from agricultural wastes has been reported to be up to 1000 mg/g, which is in accordance with the previous studies by Saka [51], Toledo et al. [52], and Anderson et al. [53]. Thus, the results of this study can suggest that the biochar from durian husk can be used as a candidate as a precursor in the manufacture of activated carbon with a highly porous structure for specialized industrial sorbents.

**Figure 2.** Effect of various preparation conditions on iodine number of activated carbon.

#### *3.3. Morphological Measurement*

An SEM instrument was used to observe the surface morphology of the materials obtained. Based on the iodine numbers, DH2 and DH5 were selected as desired activated carbons due to their high exhibited iodine number values. Therefore, we focused on investigating their surface morphologies, and the char was also compared in this study. Figure 3 shows the SEM micrographs of char, DH2, and DH5, respectively. From the image, it is clear that the durian husk can produce char and activated carbon composed of porous structures, with the appearance of roughly similar structures from all samples. However, the images suggest that the average pore diameters of the samples are quite different. As mentioned above, the pore sizes of activated carbon would be directly affected in terms of several of their properties, such as adsorption and mechanical properties. Therefore, it is worthwhile to further examine the pore sizes of the obtained activated carbon. Figure 4 shows the SEM images and the surface porous diameter distributions for the char and activated carbon materials. DH5 shows that the surface porosity is smaller than DH2 and char, respectively. In Figure 4, it can be seen that the average surface porous diameters of char, DH2, and DH5 are 6150 ± 2.86, 5060 ± 2.01, and 3790 ± 2.37 nm, respectively. The averages diameter of the pore structure decreased with an increasing activation temperature. This investigation qualitatively confirms in the same trends the results of the iodine number discussed earlier. Thus, we suggest that the surface structures of obtained activated carbon depend on the temperature and method of the activation process.

**Figure 3.** SEM images of the surface morphologies of char (upper), DH2 (middle), and DH5 (lower) with different magnifications.

**Figure 4.** SEM images and the surface porous diameter distributions of char, DH2, and DH5.

#### *3.4. Specific Surface Area*

In this part, DH2 and DH5 were selected as desired activated carbons due to their high iodine number values. In addition, char was also investigated for comparison. Figure 5 illustrates the nitrogen adsorption/desorption isotherms of the char, DH2, and DH5. The adsorbed volume of DH5 is higher than DH2 and char, respectively. However, it was found that the isotherm curves for the char sample showed that N2 adsorption/desorption exhibited a pattern of a type II isotherm, according to IUPAC classification of isotherms [54]. This type of isotherm is a general characteristic of porous carbons that have large-sized pores. On the other hand, the N2 adsorption/desorption isotherms of DH2 and DH5 were exhibited as types I and II, indicating that there were many micropores in the materials. The rapid rise of adsorption/desorption isotherms in the low relative pressure region also indicated that there were microporous or small mesoporous structures. The textural characteristics of char and activated carbon prepared under different activation conditions

are listed in Table 2. The SBET and SLangmuir increased significantly compared to char. Additionally, it can be observed from the data that the BET and Langmuir surface area increased with increases in the temperature of the second activation process when comparing DH2 and DH5. On the one hand, the pore size of DH5 was found to be the smallest of the three materials, which corresponded with similar trends in the results for the surface area. This is probably due to the increase in the activation temperature indicating the increase in extent of the reaction between KOH and char. Thus, the increased extent of the reaction aids in the enhancement and generation of new pores in the char, resulting in a higher surface area and pore volume, as presented in Table 3. It is well known that the activation process can contribute to the carbon structure, allowing the process to produce a more ordered structural skeleton. Typically, the pore creation process would occur in four stages: (i) the opening of previously accessible pores, (ii) creation of new pores, (iii) widening of the existing pores, and (iv) combination of the existing pores due to pore wall breakage [55].

**Figure 5.** Nitrogen adsorption–desorption isotherms for char, DH2, and DH5, respectively.

**Table 3.** Microstructural characteristics of char and activated carbons.

#### **4. Conclusions**

We successfully produced activated carbons with a high porosity and high surface area to volume structures from durian husk waste via a chemical activation method. The condition of the activation process directly affected the physical properties of the obtained activated carbons. The porosity and surface areas of activated carbons increased with the increases in temperatures during the second activation process. The average iodine number was exhibited to be up to 1320 mg/g. FTIR spectra also indicated only characteristic peaks of activated carbons without any new sign of functional groups. SEM images also revealed that all the activated carbons with various pore sizes and porosities were related to the temperatures in the second activation process. The specific surface area, pore volume, and pore size were further confirmed by BET investigations. It was found that the DH5 exhibited the highest surface area and pore volume of the three samples, which agreed with the results of iodine numbers and SEM investigations. The adsorption/desorption isotherms of activated carbons produced within the range of temperatures and impregnation ratios were a mixture of type I and II isotherms. In summary, we demonstrated the advantages of the utilization of such an inexpensive material through a simple preparation method. This work opens up a new approach to producing activated carbons for applications in the environment and industries, such as for adsorbents and battery electrodes.

**Author Contributions:** Conceptualization, T.J. and C.-F.H.; methodology and investigation, N.I., N.K., K.P. and W.S.; formal analysis, T.J.; resources, P.W.; writing—original draft preparation, T.J.; writing—review and editing, R.M.; K.-Y.A.L. and C.-F.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was financially supported by the Two-Institution Co-Research Scholarship provided by Kasetsart University and National Chung Hsing University.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We also acknowledge the Faculty of Science at Sriracha, Kasetsart University for providing funding and convenient laboratories.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


*Edited by Ivan X. Zhu*

Wastewater treatment and sludge disposal are important for protecting receiving rivers, lakes, and other water bodies, and vital for human health. Since excessive discharge may cause eutrophication and deterioration of aquatic systems, the US EPA and other national agencies have set guidelines for wastewater discharge standards. Conventional technologies are well developed and widely applied worldwide for wastewater treatment; however, new ideas and new technologies are gaining additional interest for the sake of water and energy reuse. While water is essential in arid regions, wastewater reuse and recycling have been playing an important role in human life. Although there are no universal standards for industrial and agriculture reuse, balancing wastewater treatment and public health protection presents challenges and opportunities.

ISBN 978-1-78923-476-3

Sewage

Published in London, UK © 2018 IntechOpen © Kayla Gibson / unsplash

**SEWAGE**


*<IP_ADDRESS>* Lactobacillus reuteri *strain*

*L. reuteri* is the probiotic strain of the probiotics *L. reuteri*, is a well-known probiotic. *L. reuteri* ATCC 55730 colonizes the stomach effectively and can reduce the occurrence of watery diarrhea associated with rotavirus. In addition, it recently showed effectiveness in the treatment of acute diarrhea with oral rehydration in

#### **Table 5.** *Probiotic microorganisms [82].*

#### *Probiotics in Processed Dairy Products and Their Role in Gut Microbiota Health DOI: http://dx.doi.org/10.5772/intechopen.104482*

children from 6 to 36 months of age [84]. A Indrio [85], research shows that *L. reuteri* DSM 17938 treatment has lowered stomach distension, expedited gastric emptying, and reduced regurgitation in children with normal gastroesophageal reflux. A recent study shows that this strain also has a favorable effect on the medication of baby colic if employed in several clinical studies or as a therapy for prophylaxis.

#### *2.8.3* Saccharomyces *spp. with probiotic properties*

*Saccharomyces* genus contains several yeasts like: *Saccharomyces cerevisiae* that is used for the preparation of wine, bread, beer, *Saccharomyces bayanus* is used to produce wines, and *Saccharomyces boulardii* utilized in medicine as a probiotic [86].

*S. boulardii* is frequently advertised as a lyophilized probiotics to treat diarrhea and retains an excellent safety reputation [87]. Most reports show that *S. Boulardii's* clinical advantages are reducing diarrhea duration regardless of causation and thus reducing the social and economic benefits associated with hospitalization. *S. boulardii* dispensation has had a positive effect on the prevention and treatment of retroactive inflammatory bowel disease and moderate symptoms of ulcerative colitis [88] patients with irritable bowel syndrome. Current *Clostridium difficile* pseudomembranous colitis infection can also be drastically reduced through the administration of *S. boulardii* regular dosages together with standard antibiotics. No malformations were reported in the previously referred examination of probiotic safety during pregnancy [89]. It should be borne in mind, however, that *S. boulardii* can lead to fungal diseases or localized infections in immunocompromised people or in other patients.

### *<IP_ADDRESS> Escherichia coli strains with probiotic properties*

Although it is known mainly for its highly virulent serotype (e.g., *E. coli* O157:H7), *E. coli* is a very common lower gut inhabitant and even a probiotic strain is known to be *E. coli* Nissle 1917; *E. coli* genus belongs in a Gram-negative family called Enterobacteriaceae (EcN). As previously mentioned, it has been proven that constipation [90] and inflammatory bowel disease were treated with other probiotics in 1917 [91]. This strain could also alleviate gastrointestinal disorder, ulcerative colitis, Crohn's disease and even colon cancer.


**1. Introduction**

196 Pesticides in the Modern World - Risks and Benefits

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#### **1.1 Levels of organisation in biological systems and their relationships**

Scales in nature can be difficult to define and understand because several ecological factors can interact. The study of different biological scales contributes to information that varies in its quality and significance for humans. Observations at the ecosystem scale are of great ecological significance but can be of low quality or provide little information about causes; at the other extreme, molecular studies that provide exact determinations of causes can have very little relevance to effects at a larger scale (Figure 1). In the middle of these extremes are observations that provide more or less significant and relevant information. Increasing the level of biological complexity in our observations can lead to an unexpected increase in the number of variables to be considered, requiring the consideration of n-adimensional conditions.

Fig. 1. Relationship between spatial and temporal scales with the quality information obtained

Each level of study is influenced by the level below it, and each level affects the level above it, which is mediated by interspecific relationships that influence ecosystem structure, and this can vary according to the heterogeneity of an ecosystem. When biological complexity

Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World 199

different formulations available. In addition, the creation of new compounds, the genetic modification of plants to withstand pesticides, and the improved effectiveness of the methods of pesticide application have had substantial economic support for research and development. Unfortunately, studies of the damage caused by these chemicals have not had

Fig. 2. Land use in South America indicating the surface of Orinoco, Amazon and La Plata basins, approximately. A) urban development and level (A) farm and industrial activities (B). Scale indicates differences in the intensities of the activities (modifies of Collins et al., in

Thus, the man invades and uses natural systems, intensively modifying them, and the fauna suffers extreme stress. Aquatic systems, although generally not targeted by direct application of pesticides, are impacted as a result of runoff after rain. This water, with all the elements that may be associated with it, goes into rivers or depressed areas. Groundwater may also be contaminated by percolation after rainfall. This mobility of elements occurs

Moreover, the higher populations in cities have caused an increase in the urban area required to accommodate people and their families. This, together with increased biocide use and the increased area of impermeable surfaces in cities has meant that household chemicals and waste products are transported rapidly to aquatic systems during rains (José

Watersheds are continually being impacted, and care must be taken to ensure their quality control because these watersheds provide people with water to live. In South America, there are three major basins with water flows ranging from 18.000 m-3s-1 to approximately 220.000 m-3s-1 (Bonetto & Waiss, 1995; Lewis et al., 1995). These basins are the Amazon, Orinoco and La Plata. Of these three, the most densely populated watershed, with the greatest number of agricultural enterprises and the largest number of factories, is La Plata Basin (Figure 2)

more intensively when the fields have no vegetative cover.

similar financial support.

press).

de Paggi et al., 2008).

(Collins et al., in press).

increases, it is important to consider that temporal and spatial dimensions are interconnected, e.g., molecular reactions occur in spaces smaller than one-hundredth of a millimetre and at reaction times of less than one second. At the same time, the effects of the predation of one species on the populations of other species may play out in spaces at the scale of kilometres and at timescales that can exceed a year. According to the heterogeneity of a system, its component species and the processes involved, variations in the time and space involved in a given process can be very important (Figure 1).

## **1.2 Fauna in aquatic systems**

Year after year, the quality of aquatic environments is recognised as a priority for humanity, with particular emphasis on the quantity and quality of freshwater.

Among the faunal components of aquatic environments, decapods, an order of crustaceans, are an interesting group that possesses biological characteristics useful in assessing the quality of inland aquatic systems. In addition, some species of decapods may be used as food by humans and are part of the food chain of other species used by humans as food, mainly fish and birds.

Five decapod families occur in southern South America and east of the Andes. Some of these decapods are endemic at the family level, others at the genus level and still others at the species level. These families include prawns and shrimp (Palaemonidae and Sergestidae), crabs (Trichodactylidae), pseudocrabs (Aeglidae) and crayfish (Parastacidae) (Collins et al., 2007).

Some of these families live in burrows constructed of fine sediment (some Trichodactylidae and Parastacidae). Others live in the background using clasts, rocks or tree trunks for hide under this cover (Aeglidae). Some decapod families live among aquatic vegetation (some Trichodactylidae and Palaemonidae), while others live all or part of their lives in the water column (some Palaemonidae and Sergestidae). Thus, the habitats used by this group are very diverse, and different taxa have different relationships to the land environment. The densities of decapods can be very high at certain times of year and may exceed 500 animals per square meter (e.g., Palaemonidae). Their diets are varied and may include plant matter (e.g., aquatic plants and phytoplankton debris), microinvertebrates (e.g., protozoa, cladocerans, rotifers, and copepods), macroinvertebrates (Palaemonidae insect larvae, oligochaetes, molluscs) and vertebrates (fish). The trophic resource used by decapods is mainly composed of live animals, but dead animals are also commonly fed upon. Consumption intensities are very high, transferring energy and material from various bottom levels (e.g., oligochaetes, chironomid larvae, zooplankton, and vegetal remains) to the top trophic levels (e.g., fish, mammals, reptiles, birds) (Collins & Paggi, 1998; Collins 1999; Williner & Collins, 2002; Collins 2005; Collins et al., 2006).

Since the industrial revolution, the human population has been growing rapidly and has therefore required more intensive management of natural environments. This need for intensive management has included the use of more land for growing food, causing the conversion of forests, jungles, and grasslands, among other ecosystems, into farmland. Subsequently, different poisons (e.g., herbicides, insecticides, fungicides) have been employed with the aim of eliminating those plants and animals that could use the crop resources (cereal or other crops), which humans call "pests". The use of these chemicals grew during the last century in an unprecedented manner in both volume of use and in the

increases, it is important to consider that temporal and spatial dimensions are interconnected, e.g., molecular reactions occur in spaces smaller than one-hundredth of a millimetre and at reaction times of less than one second. At the same time, the effects of the predation of one species on the populations of other species may play out in spaces at the scale of kilometres and at timescales that can exceed a year. According to the heterogeneity of a system, its component species and the processes involved, variations in the time and

Year after year, the quality of aquatic environments is recognised as a priority for humanity,

Among the faunal components of aquatic environments, decapods, an order of crustaceans, are an interesting group that possesses biological characteristics useful in assessing the quality of inland aquatic systems. In addition, some species of decapods may be used as food by humans and are part of the food chain of other species used by humans as food,

Five decapod families occur in southern South America and east of the Andes. Some of these decapods are endemic at the family level, others at the genus level and still others at the species level. These families include prawns and shrimp (Palaemonidae and Sergestidae), crabs (Trichodactylidae), pseudocrabs (Aeglidae) and crayfish (Parastacidae) (Collins et al.,

Some of these families live in burrows constructed of fine sediment (some Trichodactylidae and Parastacidae). Others live in the background using clasts, rocks or tree trunks for hide under this cover (Aeglidae). Some decapod families live among aquatic vegetation (some Trichodactylidae and Palaemonidae), while others live all or part of their lives in the water column (some Palaemonidae and Sergestidae). Thus, the habitats used by this group are very diverse, and different taxa have different relationships to the land environment. The densities of decapods can be very high at certain times of year and may exceed 500 animals per square meter (e.g., Palaemonidae). Their diets are varied and may include plant matter (e.g., aquatic plants and phytoplankton debris), microinvertebrates (e.g., protozoa, cladocerans, rotifers, and copepods), macroinvertebrates (Palaemonidae insect larvae, oligochaetes, molluscs) and vertebrates (fish). The trophic resource used by decapods is mainly composed of live animals, but dead animals are also commonly fed upon. Consumption intensities are very high, transferring energy and material from various bottom levels (e.g., oligochaetes, chironomid larvae, zooplankton, and vegetal remains) to the top trophic levels (e.g., fish, mammals, reptiles, birds) (Collins & Paggi, 1998; Collins

Since the industrial revolution, the human population has been growing rapidly and has therefore required more intensive management of natural environments. This need for intensive management has included the use of more land for growing food, causing the conversion of forests, jungles, and grasslands, among other ecosystems, into farmland. Subsequently, different poisons (e.g., herbicides, insecticides, fungicides) have been employed with the aim of eliminating those plants and animals that could use the crop resources (cereal or other crops), which humans call "pests". The use of these chemicals grew during the last century in an unprecedented manner in both volume of use and in the

space involved in a given process can be very important (Figure 1).

with particular emphasis on the quantity and quality of freshwater.

1999; Williner & Collins, 2002; Collins 2005; Collins et al., 2006).

**1.2 Fauna in aquatic systems**

mainly fish and birds.

2007).

different formulations available. In addition, the creation of new compounds, the genetic modification of plants to withstand pesticides, and the improved effectiveness of the methods of pesticide application have had substantial economic support for research and development. Unfortunately, studies of the damage caused by these chemicals have not had similar financial support.

Fig. 2. Land use in South America indicating the surface of Orinoco, Amazon and La Plata basins, approximately. A) urban development and level (A) farm and industrial activities (B). Scale indicates differences in the intensities of the activities (modifies of Collins et al., in press).

Thus, the man invades and uses natural systems, intensively modifying them, and the fauna suffers extreme stress. Aquatic systems, although generally not targeted by direct application of pesticides, are impacted as a result of runoff after rain. This water, with all the elements that may be associated with it, goes into rivers or depressed areas. Groundwater may also be contaminated by percolation after rainfall. This mobility of elements occurs more intensively when the fields have no vegetative cover.

Moreover, the higher populations in cities have caused an increase in the urban area required to accommodate people and their families. This, together with increased biocide use and the increased area of impermeable surfaces in cities has meant that household chemicals and waste products are transported rapidly to aquatic systems during rains (José de Paggi et al., 2008).

Watersheds are continually being impacted, and care must be taken to ensure their quality control because these watersheds provide people with water to live. In South America, there are three major basins with water flows ranging from 18.000 m-3s-1 to approximately 220.000 m-3s-1 (Bonetto & Waiss, 1995; Lewis et al., 1995). These basins are the Amazon, Orinoco and La Plata. Of these three, the most densely populated watershed, with the greatest number of agricultural enterprises and the largest number of factories, is La Plata Basin (Figure 2) (Collins et al., in press).

Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World 201

on the chemical nature of the pesticide involved. Matsumura (1977) summarised the specific properties that influence uptake into aquatic organisms: lipid and water solubility, chemical stability against degradative action by biological systems (biotransformation), and the molecular weight of the chemical. These physicochemical properties determine the affinities of toxic compounds for the materials comprising the arthropod cuticle and plasma

Because lipids constitute a substantial part of the plasma membrane, lipid solubility is a very significant factor determining the rate of penetration of many toxic compounds (such as organochlorine pesticides) by passive diffusion through the non-polar portion of the membranes. Lipid solubility is usually characterised by the octanol/water partition coefficient (Kow). In other cases, both facilitated diffusion and active transport are required for the passage of toxic into the cell through channel proteins and via their association with carrier proteins, respectively (Newman & Unger, 2003). The passage through a protein channel occurs down a concentration gradient that may be subject to saturation kinetics, and it is influenced by the size of the molecule, which determines a lower permeability of the membrane with increasing molecular size (Zitko, 1980). Moreover, the uptake of several pesticide compounds requires an active process with an expenditure of metabolic energy in living tissue. Through these pathways, toxicants enter cells and cause alterations in the physicochemical properties of the cytoplasm and the pH of the medium, destruction of the membranes of the organelles, disruption of the normal functioning of the cell proteins, and

Because in multicellular organisms the distribution of toxicants occurs in more than one compartment, within the crustacean body, haemolymph circulation may be involved in the transport of these chemicals to their sites of action and even more so if it is an open system that flows around the organs. In other arthropods, such as insects, Brooks (1974) reported that phosphoric acid penetrates the cuticle more rapidly than organochlorine insecticides, and having passed this barrier, the toxicant enters the haemolymph and may be transported to all parts of the organism in solution, if water soluble, or bound to proteins or dissolved in lipid particles, if lipophilic. The relatively hydrophilic molecules are much more likely to remain in this circulatory fluid than small, hydrophobic molecules, which are rapidly distributed in several organs and stored in lipid tissue (Hartley & Graham-Bryce, 1980).

The adverse effects of toxic products on crustaceans depend on its concentration and affinity, activity (intrinsic toxicity, which is function of molecular structure) and chemical biotransformations (James, 1987) and the acclimation responses of the individual (Klerks, 1999). For biocides, such as organophosphates and carbamate anticholinesterases (anti-ChEs), intrinsic toxicity can be judged by measuring the inhibition of cholinesterase and

While some organic compounds are sufficiently water-soluble (hydrophilic) for excretion and can be eliminated rapidly, many lipophilic components cannot be directly excreted and would accumulate if not processed to more polar derivatives. Because the unaltered toxicant and any of its transformation products (metabolites) may be excreted, excretion represents a possible protective mechanism against the toxicant (Newman & Unger, 2003). Usually, organic pesticides are subject to modifications through enzyme-catalysed biotransformations leading to *detoxification* or *activation* (Figure 4). Chemical

propagation of action potentials on synaptic transmission (see biomarkers section).

inhibition of the actions of the enzymes (Sohna et al., 2004; Collins, 2010).

membrane of the cell (Hartley & Graham-Bryce, 1980).

**2.2 Toxicity and biotransformation**

#### **1.3 Biocides**

The variety of active ingredients used as biocides and their commercial formulations, solvents and coadjuvants or related chemicals is immense. All of them are used by application with agricultural aircraft, sprayers, hand-held units, or trucks that carry the spraying equipment, according to the extension land, application protocols, crop types and soil characteristics. Studies on native fauna are scarce, and only for very few taxa have the biological effects of biocides been studied. Studies on the interrelationships among the fauna components in relation to pesticide use have also been scarce. The actions of each biocide cause different biological responses, e.g., cypermethrin provokes an increase in metabolic activity and glyphosate a decrease (Collins et al., in press). The action of each pesticide is different, and the scarce information in their effects makes it very difficult to recognise the magnitude of the harm caused by these biocides on non-target species and on aquatic environments. The studies that have been conducted have focused on assays involving the active ingredient; however, it is not only the active ingredients that cause damage to the environment but also those compounds that are in the formulation and are considered inert. These compounds can increase the toxicity of the active ingredient, facilitating its ingression in biological systems, or may be toxic by themselves. It is therefore necessary for studies not to ignore commercial formulations, because they may include several compounds that can affect aquatic systems.

Fig. 3. Structure of typical area more affect by biocides through of sprayer with airplane, runoff after rain or groundwater potentially contaminated