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INTRODUCTION
Macrophytes have a variety of roles in maintaining the physical and chemical functioning of freshwater ecosystems. They also provide habitats for other plants (particularly epiphytic algae) and animals, food for invertebrates and vertebrates (birds and fishes), and oviposition sites for invertebrates and fishes (Malby et al. 2010).
Therefore, the macrophytes are important in the agricultural landscape because they carry out a range of ecosystem functions that help to maintain the integrity of freshwater systems (Maltby et al. 2010).
In aquatic environments, pesticides might freely dissolve in the water or bind to suspended matter and sediments, and might be transferred to the organism’s tissues during bioaccumulation processes, resulting in adverse consequences to non-target species (Rodrigues et al. 2013), which can be observed and measured throughout biomarkers.
Biomarkers can be defined as measurable changes at the molecular, biochemical, cellular, physiological or behavioral levels in response to exposure to chemical contaminants and/or their effects (Gupta 2014). In this sense, the biomarkers can be used to understand the exposure of organisms to biologically available environmental pollutants (biomarker of exposure) and to quantify their effects (biomarker of effects) (Hugget et al. 1992).
These responses represent early warning systems of sublethal stress, which help to predict environmental risks and to define an efficient biological alert system (Hugget et al. 1992). In plants, several biomarkers, such as antioxidant enzymes (e.g. catalase and peroxidase activities), chlorophyll content, photosynthetic oxygen production, and chromosomal aberrations, are commonly used.
Among genetic biomarkers, cytogenetic biomarkers such as chromosomal aberrations in mitotic and meiotic cells and micronuclei are widely used and may be considered as highly sensitive biomarkers of early effects.
There are two types of possible mechanisms of the formation of chromosome aberrations in plant mitotic cells, which depend on the mode of action of the genotoxic agent. Aneugenic agents interfere with the normal formation of the mitotic spindle or interrupt the binding of the kinetochore to the tubulin fibers, while clastogenic agents induce breaks in DNA chains (Rank 2003).
Regarding physiological biomarkers, it has been shown that chlorophyll and carotenoid contents, both considered as biomarkers of early effects, are either equally or more sensitive stress indicators than biomass or relative growth rate (Brain and Cedergreen 2008).
In fact, if the contaminant disrupts photosynthesis or pigment biosynthesis, chlorophyll and carotenoid contents can be easy-to-measure and robust biomarkers, amenable to both laboratory- and field-based investigations (Brain and Cedergreen 2008).
In addition, chlorophyll a fluorescence (ChlaF) can be used to study the photosynthetic electron transport capacity of any plant. In fact, measurements of ChlaF have been widely used to study plant photosynthesis and stress response to “non-herbicidal” pesticides (Krugh and Miles 1996) and chromium (Nichols et al. 2000), among other pollutants. Indeed, both hydrophilic and lipophilic compounds have been shown to affect fluorescence parameters in aquatic plants and algae at earlier times (Brain and Cedergreen 2008).
The chemical destructive methods traditionally used for chlorophyll quantification required pigment extraction in a solvent and spectrophotometric determination of absorbance, which are relatively time-consuming. Thus, non-destructive optical methods based on the absorbance and fluorescence emission of light by the intact leaf were developed. Among them, Soil Plant Analysis Development (SPAD) is used to measure chlorophyll content, while ChlaF measures the potential yield of the photochemical reaction of photosystem II (PS II) (Krugh and Miles 1996, Richardson et al. 2001).
Bidens laevis L. (smooth beggartick) is a wetland perennial herbaceous plant widespread in North, Central and South America (Lahitte and Hurrell 1997). This species possesses particularly suitable cytological characteristics to perform genotoxicity tests, such as a high percentage of seed germination (> 70 %), almost null mortality of seedlings and young plants, large and clearly visible chromosomes, and a relatively low somatic chromosome number of 2n = 24 (Menone et al. 2015).
Previous works have shown that B. laevis shows high sensitivity to different toxicants (including pesticides and recognized mutagens), evaluated through chromosomal aberrations and enzymatic and molecular biomarkers (Pérez et al. 2008, 2011, 2014).
Strobilurin fungicides are considered a very valuable tool in agriculture, because of their apparent dual role in crop production: they provide plant protection from pathogenic fungi, and allegedly induce physiological and hormonal changes in plants, which may lead, in the long-run, to enhanced plant growth (Grossman and Retzlaff 1997, Balba 2007).
The mode of action of strobilurin fungicides involves the inhibition of mitochondrial respiration in pathogenic fungi by their binding to the Qo site of the cytochrome-bc1 complex located in the inner mitochondrial membrane (Bartlett et al. 2002), which then leads to a decrease in ATP synthesis.
Although these fungicides were originally designed to control fungal pathogens, their mode of action is not specific to fungi, so they can be potentially toxic to a wide range of non-target organisms (Rodrigues et al. 2013).
Garanzini and Menone (2015) have recently reported that the fungicide azoxystrobin (AZX) causes phytotoxic and genotoxic effects in the aquatic submerged macrophyte Myriophyllum quitense, by inducing changes in the antioxidant system and DNA fragmentation. In that study, the active ingredient of AZX was in contact with the whole body plant, being able to be uptaken by the roots, stem and leaves. However, information concerning other potential effects on plants is scarce.
Particularly in wetland plants, whether AZX can be uptaken from roots and translocated to aboveground tissues is not known. If AZX translocates to leaves, possible negative effects on the chloroplast electron transport chain could be expected, and this potential effect could be evidenced as damage in the photosynthetic apparatus.
In a study performed by Battaglin et al. (2011) in the USA, AZX was the fungicide most frequently detected (45 % of 103 samples) in 17 sites of 11 states, reaching maximum concentrations of 1.13 µg/L, whereas in a study by Berenzen et al. (2008) in streams in Braunschweig (northern Germany), AZX levels were as high as 29.7 µg/L.
In Argentina, although AZX is extensively applied in several crops, as wheat, corn, barley, potato and rape (Pérez et al. 2017), little is known about AZX levels in aquatic ecosystems. In this sense, the risk of AZX to reach aquatic ecosystems and to induce adverse effects on wetland organisms creates a high concern.
For this reason, the objectives of the present work were to assess whether the fungicide AZX induces chromosomal damage in roots of the wetland macrophyte B. laevis and/or photosynthetic system damage in leaves, shortly after a brief (1 or 2 days) exposure.
MATERIALS AND METHODS
Chemicals
Azoxystrobin [(methyl (E)-2-2-(6-(2-cyanophenoxy) pyrimidine-4-yloxy) phenyl-3-methoxyacrylate)] (CAS: 131860-33-8) and methyl methanesulfonate (MMS) (CAS: 66-27-3) reference standard (purity > 95 %) were purchased from Sigma-Aldrich. These compounds were used as received from the company without any additional purification. Dimethyl sulfoxide (DMSO, Mallinckrodt) and N,N-dimethylformamide (N,N-DMFA, ACS Reagents), which were used as solvents, were purchased from a local seller.
Plant material
Seeds of B. laevis were collected from La Brava Lake (37º 53´ S, 57º 59´ W), Argentina, in May 2013, and then stored in paper bags in darkness and without humidity until use. Seeds were sterilized in a 30 % solution of commercial bleach (DEM Argentina, 5.5 g/L) for 5 min, rinsed several times in distilled water, and placed in Petri dishes with damp filter paper for germination at 20 ºC.
Rooted seedlings were transferred to soil-containing pots and maintained for two months until full expansion of the first emergent leaf and appearance of the third pair of leaves. Prior to the start of the exposure, two-month-old plants were removed from the pots, and carefully rinsed in tap water to remove the soil.
Experimental solutions and design
Stock solutions of pure AZX were prepared in DMSO in a concentration of 2000 mg/L. AZX treatments were prepared by diluting the corresponding volume of stock solution in Hoagland medium to a final volume of 350 mL. Thereby, the five final exposure solutions of AZX contained 0.1, 1, 10, 50 and 100 µg/L of active ingredient and 0.004 % of DMSO.
In addition, the following control exposure solutions were used: one negative control (Co-1), with Hoagland Solution; another negative control (Co-2) in which DMSO was added to the Hoagland solution at 0.004 % (similar to the concentration of DMSO used for the AZX exposures); and one positive mutagenic control (Co+) consisting of 10 mg/L of MMS in Hoagland solution, used only in the cytogenetic analysis.
A total of 90 plants in the same physiological stage were selected to perform the experiments. One set of 42 plants (n = 6 per each treatment) for experiment 1 (photosynthetic biomarkers) and one set of 48 plants for experiment 2 (cytogenetic biomarkers) were randomly placed into individual 350-mL glass flasks containing a known exposure solution.
A piece of aluminum foil was used to cover the flasks to provide support to the stems and darkness to the roots. In this sense, the flasks only served as the container for root exposure.
For photosynthetic biomarkers, a 24-h exposure was performed, whereas for cytogenetic biomarkers, a 48-h exposure followed by a recovery period of 24 h in Hoagland solution was used to allow the completion of the cell cycle of the exposed cells (Grant and Owens 2002).
Both experiments were carried out in a controlled-environment chamber (12 h light/12 h darkness at 22 ºC). The photosynthetic and cytogenetic biomarkers were measured at the end of the exposures.
Experiment 1: Photosynthetic biomarkers
Chlorophyll a fluorescence
A portable chlorophyll fluorescence meter (Z990 Fluorpen Handheld Chlorophyll Fluorometer, System Inc., Canada) was used to measure ChlaF through the Fv/Fm ratio (where Fv is the maximum variable fluorescence emission and Fm is the maximum total fluorescence).
Each measurement was done in triplicate on the youngest fully expanded leaf. Data were expressed as the average value of the Fv/Fm ratio per plant and in terms of the average ± standard error (SE) for each treatment.
Chlorophyll content (SPAD measurement)
The total chlorophyll content was measured as SPAD values (Minolta SPAD-502 chlorophyll meter) in triplicate on the youngest fully expanded leaf. Data were expressed as the average of SPAD values per plant and in terms of the average ± SE for each treatment.
Chlorophyll content (chemical extraction)
Sample preparation and spectrophotometric measurement: Immediately after fluorescence and SPAD determination, leaf discs (0.2 g) of the youngest fully expanded leaf were placed into 10-mL vials with 2 mL of N,N-DMFA for 72 h at 4 ºC in darkness. An aliquot of 1 mL was placed into the spectrophotometric vial to assess chlorophyll a (Chla) at 664.5 nm and chlorophyll b (Chlb) at 647 nm.
A spectrophotometer Bausch & Lomb model Spectronic 20 was used to measure the chlorophyll pigments. The formulas used to calculate the concentrations of Chla, Chlb and total chlorophyll (TCh) were the following:
where the coefficients represent the molar extinction coefficients of Chla and Chlb in N,N-DMFA (Inskeep and Bloom 1985).
In addition, the ratio between Chla and Chlb (Chla/b) was calculated. Data were expressed as mg of Chla, Chlb and TCh per g of fresh tissue in each plant, and in terms of the average ± SE for each treatment.
Experiment 2: Cytogenetic biomarkers
Root tips (1 cm long) were fixed in ethanol:glacial acetic acid (3:1, v/v) for 24 h and maintained in 70 % ethanol in a refrigerator until analysis. Then, they were macerated in 1 M HCl at 60 ºC for 10 min, stained with Feulgen reagent for 2 h in darkness, squashed in a solution of 1-2 % carmine in 45 % acetic acid, and observed in an optical microscope Olympus CX31.
For all cytogenetic biomarkers, one or more slides per plant were prepared to observe the number of cells required at each stage of mitosis. In this sense, the mitotic index (MI) was calculated as the number of cells at any stage of mitosis per 1000 observed cells per plant. This parameter was measured to confirm that the number of mitotic cells was high enough to carry out the analysis of chromosome aberrations (Pérez et al. 2014).
The MI was expressed as a percentage of cells in mitosis for each plant and in terms of the average ± SE for each treatment.
Chromosomal aberrations in metaphase
One hundred cells in metaphase per plant were observed to score chromosomal aberrations in metaphase (CAM), which included chromosomes not congregated at the metaphase equator. Data were expressed as a percentage of CAM/100 metaphases for each plant and in terms of the average ± SE for each treatment.
Chromosomal aberrations in anaphase-telophase
Two hundred cells in anaphase-telophase per plant were observed, and the different chromosomal aberrations in anaphase-telophase (CAAT), including bridges, fragments, vagrants and laggards, were scored. Data were expressed as a percentage of each type of CAAT/200 anaphase-telophases per plant and in terms of the average ± SE for each treatment.
Statistical analyses
Photosynthetic biomarkers
As the values of all variables were continuous, normality and homogeneity of residuals were first verified by Shapiro-Wilk and Levene tests, respectively, and then, parametric tests were applied to analyze differences in these biomarkers. In this regard, ANOVA was applied and, a posteriori, differences between treatments were tested by Tukey’s test.
Cytogenetic biomarkers
As the values of all variables were discrete, data were first analyzed as a percentage and later transformed using the arcsine function to apply a parametric statistical test. In this regard, ANOVA was applied and, a posteriori, differences between treatments were tested by Tukey’s test.
Statistical analyses were carried out using the R software package (v. 3.2.2.), with 0.05 and 0.01 % significance levels.
RESULTS
Experiment 1: Photosynthetic biomarkers
Non-destructive methods
The ChlaF and chlorophyll content measured with SPAD showed no statistically significant changes at any of the AZX concentrations tested with respect to the negative controls (p > 0.05) (Table I).
TABLE I PHOTOSYNTHETIC BIOMARKERS IN LEAVES OF Bidens laevis EXPOSED TO DIFFERENT AZOXYSTROBIN TREATMENTS. AVERAGE OF SIX REPLICATES ± STANDARD ERROR
| Treatment | Non-destructive methods | Destructive methods | |||||
| ChlaF | SPAD | TChl | Chla | Chlb | Chla/b Ratio | ||
| Co-1 | 0.66 ± 0.01 | 29.64 ± 0.93 | 567.73 ± 43.80 | 416.10 ± 37.39 | 151.77 ± 9.92 | 2.75 ± 0.23 | |
| Co-2 | 0.67 ± 0.01 | 33.56 ± 1.22 | 608.43 ± 20.28 | 446.53 ± 10.74 | 162.06 ± 7.92 | 2.79 ± 0.21 | |
| 0.1 µg/L | 0.68 ± 0.01 | 33.28 ± 1.21 | 614.66 ± 29.36 | 435.95 ± 22.81 | 178.88 ± 6.74 | 2.43 ± 0.04 | |
| 1 µg/L | 0.69 ± 0.00 | 30.72 ± 0.64 | 575.21 ± 28.82 | 430.50 ± 17.86 | 144.86 ± 11.56 | 3.02 ± 0.17 | |
| 10 µg/L | 0.68 ± 0.01 | 32.03 ± 1.18 | 596.71 ± 21.49 | 444.00 ± 17.41 | 152.87 ± 9.32 | 2.90 ± 0.04 | |
| 50 µg/L | 0.63 ± 0.02 | 31.16 ± 0.86 | 519.63 ± 33.44 | 411.28 ± 18.59 | 108.58 ± 15.46 | 3.97 ± 0.33* | |
| 100 µg/L | 0.67 ± 0.00 | 32.06 ± 0.97 | 567.66 ± 36.37 | 467.50 ± 28.79 | 100.28 ± 7.68* | 4.68 ± 0.08* | |
| ANOVA p value | 0.148 | 0.126 | 0.385 | 0.674 | < 0.0001 | < 0.0001 | |
Co-1: Hoagland solution, Co-2: Hoagland solution + 0.004 % of dimethyl sulfoxide, ChlaF: chlorophyll a fluorescence measured by Fv/Fm, SPAD: total chlorophyll measured by SPAD units, TChl: total chlorophyll measured by chemical extraction with N,N dimethylformamide, Chla: chlorophyll a, Chlb: chlorophyll b, Chla/b ratio: chlorophyll a divided chlorophyll b.
*Statistically significant to negative control at 0.05 level of probability (Tukey’s test).
Destructive methods
The TCh and Chla showed no statistically significant changes at any of the AZX concentrations tested with respect to the negative controls (p > 0.05) (Table I). However, Chlb showed a statistically significant decrease at 100 µg/L AZX respect to the negative controls (p < 0.05). Also, the Chla/b ratio showed a statistically significant increase at 50 and 100 µg/L AZX with respect to the negative controls (p < 0.05) (Table I).
Experiment 2: Cytogenetic biomarkers
The MI varied from 2.6 to 8.4 in Co-1 plants, from 2.5 to 7.2 in Co-2 plants, from 2.9 to 8.1 in Co+ plants, and from 5.3 to 11.9 in AZX-treated plants. In all cases, it was high enough to score the required number of cells for the cytogenetic analysis (Table II).
TABLE II MITOTIC INDEX (MI) IN ROOT CELLS OF Bidens laevis EXPOSED TO DIFFERENT AZOXYSTROBIN TREATMENTS. AVERAGE OF SIX REPLICATES ± STANDARD ERROR
| Controls | Azoxystrobin (µg/L) | ||||||||
| Co-1 | Co-2 | Co+ | 0.1 | 1 | 10 | 50 | 100 | ||
| MI | 6.11 ± 1.20 | 5.41 ± 0.72 | 5.86 ± 0.77 | 6.91 ± 0.58 | 8.41 ± 0.54 | 7.94 ± 0.48 | 7.35 ± 0.43 | 9.20 ± 0.86 | |
Co-1: Hoagland solution, Co-2: Hoagland solution + 0.004 % of dimethyl sulfoxide, Co+: methyl methanesulfonate at 10 mg/L
The CAM that included chromosomes not congregated at the metaphase equator showed a statistically significant increase in plants exposed to 0.1-100 µg/L AZX, with respect to plants exposed to negative controls (p = 0.0001) (Table III).
TABLE III CHROMOSOME ABERRATION FREQUENCIES (%) IN MITOTIC ROOT CELLS OF Bidens laevis EXPOSED TO DIFFERENT AZOXYSTROBIN TREATMENTS. AVERAGE OF SIX REPLICATES ± STANDARD ERROR
| Treatment | Metaphase (CAM) | Anaphase-telophase (CAAT) | Others (OCA) | Total CA | ||||||
| Cnc | Bridges | Fragments | Laggards | Vagrants | Cm | Cc | ||||
| Co-1 | 0.66 ± 0.66 | 0.16 ± 0.16 | 0.00 ± 0.00 | 0.66 ± 0.16 | 0.33 ± 0.16 | 0.00 ± 0.00 | 0.00 ± 0.00 | 1.11 ± 0.22 | ||
| Co-2 | 0.16 ± 0.16 | 0.08 ± 0.08 | 0.00 ± 0.00 | 0.33 ± 0.10 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.33 ± 0.14 | ||
| Co+ | 6.16 ± 1.8 | 0.66 ± 0.27 | 0.08 ± 0.08 | 2.58 ± 0.50 | 0.16 ± 0.16 | 0.08 ± 0.08 | 0.00 ± 0.00 | 4.44 ± 0.97* | ||
| 0.1 µg/L | 8.16 ± 1.66** | 0.58 ± 0.08 | 0.08 ± 0.08 | 3.41 ± 0.68* | 0.50 ± 0.18 | 0.66 ± 0.30 | 0.33 ± 0.27 | 6.44 ± 0.89** | ||
| 1 µg/L | 8.00 ± 1.39** | 0.83 ± 0.16 | 0.00 ± 0.00 | 4.91 ± 0.80** | 1.00 ± 0.48 | 0.33 ± 0.24 | 0.66 ± 0.35 | 7.83 ± 1.47** | ||
| 10 µg/L | 10.6 ± 1.43** | 0.50 ± 0.27 | 0.00 ± 0.00 | 5.90 ± 0.87** | 1.10 ± 0.43 | 0.70 ± 0.48 | 0.30 ± 0.30 | 9.20 ± 1.07** | ||
| 50 µg/L | 7.83 ± 1.53** | 0.91 ± 0.23 | 0.08 ± 0.08 | 5.91 ± 0.90** | 1.58 ± 0.59* | 1.50 ± 0.17 | 0.00 ± 0.00 | 8.44 ± 1.40** | ||
| 100 µg/L | 7.25 ± 1.31* | 1.00 ± 0.61 | 0.00 ± 0.00 | 4.62 ± 0.92** | 0.37 ± 0.23 | 0.62 ± 0.31 | 0.00 ± 0.00 | 6.83 ± 1.09** | ||
| ANOVA p value | 0.0001 | 0.1647 | 0.7875 | < 0.0001 | 0.0482 | 0.2289 | 0.1720 | < 0.0001 | ||
Co-1: Hoagland solution, Co-2: Hoagland solution + 0.004 % of dimethyl sulfoxide, Co+: methyl methanesulfonate at 10 mg/L, CAM: chromosome aberrations in metaphase, CAAT: chromosome aberrations in anaphase-telophase, OCA: other chromosomal aberrations, Cnc: chromosomes not congregated in metaphase, Cm: colchicine mitosis, Cc: chromosome clumping, Total CA: total chromosomal aberrations
*Statistically significant to negative control at 0.05 level of probability (Tukey’s test)
**Statistically significant to negative control at 0.01 level of probability (Tukey’s test)
Among CAAT, only laggard chromosomes showed statistically significant changes between negative control plants and AZX-treated plants (Table III). This type of chromosomal aberration increased in plants exposed to 0.1-100 µg/L AZX (p < 0.0001) (Table III).
Other chromosomal aberrations (OCA) observed, as c-mitosis and chromosome clumping, were found only in plants exposed to Co+ and in some AZX-treated plants. However, their frequencies were not statistically different from the negative controls (p > 0.05) (Table III).
Regarding total CA, no differences between Co-1 and Co-2 plants were observed. On the other hand, a significant increase in total CA was found in Co+ plants with respect to both negative controls (Table III). In addition, statistically significant increases were found in all AZX treatments compared to the negative controls (p < 0.0001) (Table III). The chromosomal aberrations in mitotic root cells of B. laevis exposed to AZX are shown in figure 1.
DISCUSSION
Fungicides are extensively used in agriculture because they constitute the most effective method to control pathogenic fungi in crops. However, there is a growing concern about the environmental pollution generated by their use and their toxicity to non-target organisms.
In this sense, the impact of the exposure to strobilurin fungicides on non-target aquatic and wetland macrophytes that inhabit close to agroecosystems has recently become a topic of research because the results obtained are useful to predict negative effects on wild plants. The present work addressed the potential use of Bidens laevis as a biomonitor plant to assess the potential physiological and cytogenetic effects of the widespread strobilurin fungicide AZX.
Photosynthetic biomarkers
The AZX is a systemic fungicide that affects respiration in fungi by inhibiting electron transport in mitochondria, leading to cellular oxidative stress and disruption of fungal metabolism and growth (Bartlett et al. 2002). Recent studies have indicated that AZX also disrupts mitochondrial respiration and induces DNA damage in fishes (Olsvik et al. 2010, Han et al. 2016, respectively).
In addition, Garanzini and Menone (2015) reported that AZX changes the activity of the antioxidant enzymes in the aquatic macrophyte Myriophyllum quitense, indicating that this fungicide induces oxidative stress in this non-target plant. This raised the possibility that AZX may interfere with photosynthetic activity. In fact, several fungicides are known to interfere with the metabolic pathways of plants in the photosynthetic process (Petit et al. 2012).
Nason et al. (2007) tested different strobilurin fungicides, including AZX, in soy, wheat and barley plants, and observed that fungicides had similar effects on plant physiology but differed in their persistence and potency. These authors also found that the application of fungicides to whole plants by using a spray gun-led to a decrease in the net rate of photosynthesis, compared to the leaves of control plants.
Although the mechanism of the photosynthetic effects is unknown, these authors hypothesized that strobilurin fungicides act directly on ATP production in guard cell mitochondria or by stomata responding to strobilurin-induced changes in mesophyll photosynthesis.
Nason et al. (2007) also observed that, shortly after exposure to AZX, plants were stressed, as revealed by a decrease in ChlaF, possibly due to a blockade in the electron transport between PSII and PSI, by binding to the Qi site of the chloroplast cytochrome bf complex. Although these findings were described after a short-term exposure to AZX (after 1, 3 and 7 days), AZX was applied directly over the leaves, whereas, in the present work, the plants were in contact with AZX through the roots. As B. laevis is a wetland species, the experiments were designed to simulate a realistic condition in the field, in which an acute pulse of AZX could take place through runoff by root uptake.
The effects of AZX on the photosynthetic apparatus were assessed through SPAD and ChlaF. These measurements are common in plant ecophysiological studies, but are rare in ecotoxicological research. The analysis of these non-destructive optical measures did not allow us to observe AZX effects on B. laevis leaves.
The results showed only a decrease in Chlb at the highest AZX concentration tested through destructive chemical methods. This result is in agreement with those of Brain et al. (2004), who established that Chlb is generally more sensitive than Chla to be degraded under xenobiotic stress.
However, no changes were found in total chlorophyll content under any AZX treatment or in the other photosynthetic biomarkers tested (SPAD, ChlaF) (Table I). This lack of response in the photosynthetic biomarkers may be attributed to different possibilities. First, strobilurin fungicides have been shown to induce cytokinin synthesis (Grossmann and Retzlaff 1997).
It is well known that cytokinins increase the activity of RuBisCo, the main photosynthetic enzyme (Boonman et al. 2007) and, consequently, leaf photosynthetic capacity (Song et al. 2013). Cytokinins also promote tetrapyrrole biosynthesis (Yaronskaya et al. 2006) and often increase chlorophyll concentration. It is therefore possible that any negative impact of AZX on the photosynthetic electron transport could be counterbalanced by a cytokinin-mediated effect.
Second, in the present work root tissues were directly exposed to the fungicide, and the exposure time might not have been enough to allow the translocation of AZX from roots to leaves. In that case, no effects on leaves could be expected, mostly taking into account that AZX is an organic compound with low lipophilicity (log Kow = 2.5) (US-EPA 2010).
Future studies should be carried out to determine root uptake and distribution rate in leaves, stems and flowers, as it has been done with the insecticide endosulfan in B. laevis (Pérez et al. 2013).
Cytogenetic biomarkers
In the present study, the high MI observed in all treatments were appropriate for the analysis of chromosomal aberrations in root cells of B. laevis, and similar to that found in previous reports using this wetland macrophyte (Pérez et al. 2011, 2014), and to the international standard protocol with Allium cepa (Rank 2003). These results indicate that the different AZX treatments induced no changes in the mitotic cycle (Table II).
The results showed that there were no statistically significant differences between the negative controls (Co-1 and Co-2), indicating that the aliquot of DMSO (0.004 %) used in plants exposed to Co-2 did not affect the basal frequency of chromosomal aberrations in mitotic root cells of B. laevis (Table III).
Moreover, there were statistically significant differences between the negative controls and the positive control, indicating that MMS at 10 mg/L induced an increase in the total CA in somatic root cells of B. laevis (Table III). In addition, it is important to denote that MMS induced spindle disturbances at cytogenetic level in this plant, evidenced by higher frequencies of laggards and vagrants, as described in previous studies (Pérez et al. 2011, 2014).
Rank (2003) reported the clastogenic effect of MMS in Allium cepa, whereas Pérez (2012) reported that MMS (10 mg/L) induced DNA fragmentation in B. laevis, assessed by the comet assay, indicating the clastogenic effect of MMS on this plant.
Cytogenetic analyses are recognized as sensitive and reliable for the detection of mutagenic-genotoxic compounds. In particular, the chromosomal aberrations assay is a simple and low-cost technique that allows studying the mode of action of genotoxins in mitotic cells, depending on the different types of abnormalities observed.
In this sense, two types of chromosomal aberrations should be taken into account according to whether they are indicators of (1) spindle disturbance or aneunogenesis (including laggard or vagrant chromosomes) or (2) clastogenicity (including bridges, fragments and broken chromatids).
Other less frequent aberrations such as c-mitosis, multipolarity anaphases, polyploid cells, pulverized chromosomes and chromosome clumping, should also be scored (Rank 2003). The presence of fully condensed chromosomes randomly distributed in the cell or c-mitosis suggests a partial or total inhibition of spindle formation, which affects the metaphase-anaphase transition (Andrioli and Mudry 2011). Also, chromosomes not congregated in metaphase are indicators of spindle disturbance (Pérez et al. 2014).
In the present study, the results showed that the active ingredient of AZX increased the number of chromosomal aberrations in mitotic root cells at low concentrations from 0.1 to 100 µg/L in B. laevis (Table III).
The main types of chromosomal aberrations found were laggards in anaphase-telophase and chromosomes not congregated in metaphase, indicating that AZX induced spindle disturbances in mitotic root cells of B. laevis at environmentally relevant concentrations (Table III, Fig. 1).
The mechanism by which AZX induces spindle disturbances is still unknown. However, this could be elucidated by the analysis of the microtubule structure (immunoassay with labeled β-tubuline), as described by Andrioli et al. (2012) for the spindle disturbance induced by carbamate pesticides in A. cepa root cells.
Moreover, a recent study using the submerged aquatic macrophyte M. quitense exposed to the active ingredient of AZX showed that this fungicide causes genetic damage, as assessed at molecular level by the comet assay, since DNA fragmentation was found in plants exposed to 50 and 100 µg/L (Garanzini and Menone 2015).
In the present study, the basal frequencies of bridges and fragments did not change under AZX treatment, meaning that the AZX mode of action may not involve clastogenicity in B. laevis since plant responses to the active ingredient of AZX could vary according to the species.
The most relevant effect of AZX in B. laevis was mainly spindle disturbance, which can induce different types of abnormalities resulting in irreversible damage, because laggard chromosomes or chromosomes not congregated in metaphase can be lost in cells of the following generation. The genetic information contained in lost chromosomes or fragments can result in a valuable loss. Likewise, cell lineages that contain deficiencies will form deficient tissues, and then the viability and health of the plant in field conditions would decrease (Bickham et al. 2000).
This becomes more relevant when the affected lineages are part of the germinal cells (meiotic cells), because they may directly affect pollen viability and reproductive success. This last effect would lead to a demographic decline (demo-ecological effects) in the population (Bickham et al. 2000).
CONCLUSION
The present work showed the genotoxic effects of low (0.1 µg/L) and high (100 µg/L) concentrations of the strobilurin fungicide AZX on the mitotic root chromosomes of the wetland macrophyte B. laevis. The AZX mode of action in root cells of B. laevis was mainly by spindle disturbance, because the chromosomal aberrations most frequently found were laggards in anaphase-telophase and chromosomes not congregated in metaphase.
Physiological biomarkers related with the photosynthetic apparatus were less affected by AZX. The only significant effect was a decrease in Chlb levels, assessed by a traditional destructive chemical method, in plants exposed at the maximum AZX concentration tested. On the other hand, no effects of AZX were detected by non-destructive methods used to analyze ChlaF and SPAD units.
The results showed that the chromosomal aberrations in mitotic root cells constitute a more sensitive biomarker of early effects than the physiological biomarkers studied, at least for the acute exposure used in this work.
