Effect on enzymes and histopathology in earthworm (Eisenia foetida) induced by triazole fungicides

Effect on enzymes and histopathology in earthworm (Eisenia foetida) induced by triazole fungicides

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Effect on enzymes and histopathology in earthworm (Eisenia foetida) induced by triazole fungicides Minling Gao, Wenhua Song ∗ , Jinyang Zhang, Jing Guo School of Environmental and Chemical Engineering, Tianjin Polytechnic University, No. 399 Binshui Western Road, Xiqing District, Tianjin 300387, China

a r t i c l e

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a b s t r a c t

Article history:

Earthworms are an ideal biological model in toxicity assays and environment monitor-

Received 24 July 2012

ing studies, especially for the toxicity of pesticides on soil ecosystem. However, There are

Received in revised form

very little data on the toxicity of triazoles on earthworms despite the fact that such data

28 January 2013

are critical in assessing their fate and potential toxic effects in soil organisms. To address

Accepted 2 February 2013

this issue, earthworms were exposed to triazoles (triadimefon, triadimenol, difenoconazole

Available online 13 February 2013

and propiconazole) to study biochemical and histopathological examination. The results showed protein content significantly increased in treatment of difenoconazole compared


to control. There were no significant differences between controls and triadimefon treated

Triazole fungicides

groups, while the glutathione peroxidase (GSH-Px) activity is significantly lower than con-

Enzymatic activity

trol. Other triazoles also had an inhibitory effect on GSH-Px activity at higher concentration.

Histopathological examination

The histopathological examination showed the epidermis and the epidermis cell of earth-


worm was ruined at lower triazoles concentration. The arrangement of smooth muscle layer disordered, and some cell disintegrated with concentration increasing of pesticides. Cell pyknosis, cytoplasm deep stained, nucleus concentrations were observed in the treated group with propiconazole. © 2013 Elsevier B.V. All rights reserved.



Triazoles are widely used as clinical drugs and agricultural pesticides, including the applications for the treatment and protection of cereals, soybeans, and a variety of fruits (Konwick et al., 2006). Triazoles are designed to inhibit cytochrome P450-mediated ergosterol biosynthesis and are used as a systemic foliar fungicide (Steven and James, 1999). After treatment, triazoles can widely distribute into soil (Wang et al., 2008; Bermúdez-Couso et al., 2007), crop (GonzálezRodríguez et al., 2008; Soler et al., 2007) and water (Zhou et al., 2007; Sampedro et al., 2000). They cause adverse effect to aquatic and mammal animals, due to its neurotoxicity

(Crofton, 1996), physiology and hereditary toxicity of biology (Rockett et al., 2006; Tully et al., 2006). Consequently, concern has been raised to the toxicity of triazoles on aquatic organisms and mammalian. Goetz et al. (2009) studied the inhibition of triazole antifungals to rat and human steroidogenesis and found that myclobutanil, propiconazole and triadimefon were weak inhibitors of testosterone production in vitro. However, in vivo exposure of rats to triazoles, it resulted in the increase of serum and intratesticular testosterone levels (Goetz et al., 2009). The impact of propiconazole, fluconazole, trazodone and myclobutanil on serum and liver of rats were also studied. The results showed triazole had apparent effect on the expression of numerous cytochrome P450 (CYP) genes in rat liver and testis, including

Corresponding author. Tel.: +86 022 83956667; fax: +86 022 83956667. E-mail addresses: [email protected] (M. Gao), [email protected] (W. Song). 1382-6689/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2013.02.003


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Table 1 – Physical and chemical properties of triazole fungicides. Pesticide

Molecular weight

C log P (Song et al., 2009)

Structure OH


CH Cl Triadimenol








C Cl






3.74 N N N Cl Cl


C3H7 O







Cl O







multiple Cyp2c and Cyp3a isoforms as well as other xenobiotic metabolizing enzyme and transporter genes. For some genes, such as Ces2 and Udpgtr2, all four triazoles had similar effects on expression, suggesting possible common mechanisms of action (Tully et al., 2006). Monod et al. (2004) showed that prochloraz, epoxiconazole and imazalil strongly potentiated the induction of oocyte maturation by gonadotropin in a dose-dependent manner. To date, few studies have addressed the effects of triazoles to earthworm. In fact, earthworms are common in a wide range of soil and may represent 60–80% of the total soil biomass (Li et al., 2009). It is well known that earthworms

affect chemical, physical and biologic aspects of soil systems, and play an important role in supporting soil homeostasis (Zeng, 2006). Several earthworm protocols have been developed to assess the effects of chemicals on earthworms (Eisenia foetida) among which the most well known is the OECD guideline 207, a 14-day artificial-soil test (Luo et al., 1999). Therefore, earthworms are an ideal biological model in toxicity assays and environment monitoring studies, especially for the toxicity of pesticides on soil ecosystem.The aim of this work is to investigate the antioxidant response and tissue morphology of E. foetida after experimental exposure to triazoles. This paper focused on the biochemical toxicity of triazoles in terms

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of effects on protein and GSH-Px, as well as the effects on histopathology, which will provide more information about the potential ecological risk of pesticides in soil ecosystems.

Materials and methods


Test chemicals and solutions

2.50 Protein content (g/L)


Triadimefon (95% purity) was purchased from Jinghong Chemical Co. Ltd. Triadimenol and propiconazole (95% purity) was purchased from Yancheng Limin Agriculture Co. Ltd. Difenoconazole (95% purity) was purchased from Suzhou Eagro Co. Ltd. Acetone was purchased from Concord Technology (Tianjin) Co. Ltd. All analytical standards and solutions were stored in darkness at approximately 4 ◦ C. Their structure and properties were shown Table 1.

Acute toxicological tests

Toxicity experiments were performed following the method described in the OECD (OECD, 1984) guideline for testing of chemicals no. 207. Filter paper was cut to the size of 7.9 cm × 8.4 cm (66.36 cm2 ), which was set against the wall of a flat-bottomed glass vial, which were 8 cm in length and 3 cm in diameter. 1 mL of acetone solution was added to each vial and evaporated to dryness under air. Then, 1 mL of distilled water was added to moist the filter paper in the vials. An earthworm was then set on the moist paper in the vial, and the vial was covered with plastic film with small ventilation holes. Tests were done in the dark at 20 ± 2 ◦ C for 48 h. Each treatment was run in ten replicates and each consisting of one worm. Blank tests were also performed. Histopathological examination of triazoles was assayed with five treatment levels in a geometric series. The concentrations of triadimefon and triadimenol all were 3.01, 6.03, 9.04, 12.06 and 15.07 ␮g/cm2 ; concentrations of propiconazole were 3.77, 7.53, 11.3, 15.07 and 18.84 ␮g/cm2 ; concentrations of difenoconazole were 15.07, 30.14, 45.21, 60.28 and 75.35 ␮g/cm2 , respectively.


1.50 1.00 0.50 0

0.30 0.60 1.20 2.40 4.80 Concerntation (µg/cm2)

Fig. 1 – Effect of triadimefon on protein content.

Biochemical assay


E. foetida were purchased from Tianjin Liming Jia Earthworm Farm Company, China. They were cultured under laboratory conditions, which fed on the excrement of cows. This culture was judged to be free from triazole pesticides. Adult earthworms were selected for testing, which possessed clitellum and had an individual wet weight of 300–500 mg. Earthworm was kept on moist filter paper for 24 h, then washed with distilled water and dried before use.




2.5. 2.2.


Toxicological tests were on earthworms were conducted in OECD filter paper. Enzymatic activities were assayed with five treatment levels in a geometric series, they were 1/32, 1/16, 1/8, 1/4 and 1/2 LC50 , respectively. After 48 h, earthworms were rinsed with distilled water and dried on filter paper. Earthworms were frozen rapidly with liquid nitrogen, weighed and homogenized for 2 min in normal saline in a 1/9 (w/v) ratio using homogenizer. Homogenates were centrifuged at 3000 rpm at 4 ◦ C for 5 min. The supernatant fluid was collected for the assay of enzyme activity and protein determination. Protein content of earthworms was determined using the Coomassie brilliant blue technique according to the manufacturer’s instructions. 0.05 mL protein standard solution was added to 3.0 mL Coomassie brilliant blue standard tubes, 3.0 mL Coomassie brilliant blue and 0.05 mL samples tubes, 0.05 mL distilled water was added to 3.0 mL Coomassie brilliant blue blank tubes, respectively. The reaction system was incubated for 10 min. The content of protein was determined according to the standard curve at 599 nm using a VIS-7220 spectrophotometer (Beijing, Ruili). The GSH-Px activities were determined according to the method of enzymatic reaction as described by manufacturer’s instructions. A small volume (0.2 mL) of 1 mmol/L GSH was added into test-tube containing 0.2 mL sample and

Histopathological examination

After acute exposure of the earthworms, they were rinsed with distilled water. The earthworms were fixed with 10% formalin, embedded into paraffin and sliced vertically for 4 ␮m thick with a freezing microtome. Sections were mounted on glass microscope slides using standard histopathological techniques. Sections were stained with hematoxylin–eosin (HE) and examined by an optical microscope.

Fig. 2 – Effect of triadimenol on protein content.


e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 427–433

Fig. 3 – Effect of propiconazole on protein content. Fig. 6 – Effect of triadimenol on GSH-Px.

Fig. 4 – Effect of difenoconazole on protein content.

the control-tube, respectively. Then they were incubated at 37 ◦ C for 5 min. A small volume (2 mL) reagent 1 was added into test-tube and control-tube, respectively, and mixtures were incubated at 37 ◦ C for 5 min. Then 2 mL reagent 2 was added into test-tube and control-tube, respectively, and 0.2 mL sample was added into control-tube. The mixtures were centrifuged at 3500 rpm for 10 min. One-milliliter reagent 3, 0.25 mL reagents 4 and 5 (reagents 1–5 were prepared according to enzymatic kits) were added into another control-tube and test-tube containing 1 mL supernatant fluid, respectively. The reaction system was incubated at 25 ◦ C for 10 min. The samples were tested according to the standard curve at 412 nm using a VIS-7220 spectrophotometer. One unit of GSH-Px activity is defined as the amount of enzyme activity of 1 mg protein,

Fig. 5 – Effect of triadimefon on GSH-Px.

Fig. 7 – Effect of propiconazole on GSH-Px.

which decreases GSH by 1 ␮mol/L/min through enzymatic reaction. The activity of GSH-Px is expressed in U/mg protein where 1 U = 1 ␮mol/L of GSH reduction min−1 mg−1 protein.


Statistical analysis

Differences between control and treated samples were analyzed by one-way ANOVA using SPSS 16.0 statistical software, taking P < 0.05 as significant according to the Duncan test.

Fig. 8 – Effect of difenoconazole on GSH-Px.

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Fig. 9 – Pictures of pathological section of earthworms: (1) epidermis; (2) epidermis cell; and (3) smooth muscle layer.


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Results and discussion


Effect of triazoles on protein content of earthworm

The changes of total soluble protein content are shown in Figs. 1–4. There were no significant differences between control and treated groups with the increasing exposure concentration of triadimefon. Total soluble protein content significantly decreased after the treatments with triadimenol (except for 0.19 ␮g/cm2 ) and increased significantly after the treatments with difenoconazole compared to control (P < 0.05). Total soluble protein content increased at low propiconazole concentration and decreased at the highest concentration of 5.2 ␮g/cm2 compared to control (P < 0.05). The results showed that the earthworm might be more sensitive to triadimenol. Determination of protein content is the most common and basic method in biochemical research (Goncalves et al., 2002). The change of quantity and qualitative of protein would have great value on disease diagnosis and effect observation. Total soluble protein content decreased significantly that indicated body function of earthworms and production source of protein were damaged, and protein synthesis was impeded (Hu and Lin, 2006; Jiao et al., 2002). However, protein content increased when earthworm exposed to lower propiconazole and difenoconazole in this study. It indicated the increase of protein content could be a contribution to the defense mechanism against stress for animals. But the defence mechanism of earthworms would be silenced by excessive propiconazole or difenoconazole concentration. Consequently, the synthesis of protein will be inhibited while disrupting existing proteins.


Effect of triazoles on the GSH-Px of earthworm

Effects of triazoles on GSH-Px activity of earthworm are shown in Figs. 5–8. The activity of GSH-Px decreased significantly when earthworms were exposed to triadimefon, triadimenol (except for 0.38 ␮g/cm2 ), difenoconazole (except for 2.94 ␮g/cm2 ) compared to control (P < 0.05). Moreover, the activity of GSH-Px decreased with increasing concentration of triadimefom. As for propiconazole treatments, 0.33 and 5.22 ␮g/cm2 of propiconazole increased significantly the activity of GSH-Px (P < 0.05). The results suggested GSH-Px might be more insensitive to propiconazole. Glutathione peroxidase is an important oxidoreductase in living creature that could scavenge free radical and lipid peroxide induced by • OH to protect the integration of membrane structure and function. The activity of GSH-Px increased with an increase of the concentration of enzyme substrate when GSH-Px could decompose toxic substance. However, the activity of GSH-Px decreased when the concentration of toxic substance is more than the capacity of decomposition GSHPx. The activity of GSH-Px decreased when the concentration of triazoles increased. It was possible that GSH-Px could not scavenge toxic chemicals.


extent of the poisoning and lesions of the cells were obtained according to hematoxylin–eosin (HE) technology. The movement functions of earthworms were impacted in the lower concentration. Moreover, the histopathology reports disclosed a variety of abnormal incidental lesions. The pathological section showed that the higher concentration of triazoles, the more serious image on earthworms. Pathological section of epidermis and smooth muscle layer of earthworms is shown in Fig. 9. The results showed that 3.01 ␮g/cm2 of triadimefon significantly increased hyperplasia and inflammatory response of epidermis that associated a lot of inflammatory cell infiltration. 6.02 ␮g/cm2 of triadimefon could lead to the atrophy and vacuole degeneration of epidermis. Dilatation and hyperemia were associated with the muscle layer disordered and slight necrosis in different concentration of triadimefon. The results indicate that epidermis cell of earthworm could be injured, and action of earthworm could be affected. Pathological morphology observation showed a muscle layer slight disorder, vacuole degeneration of epidermis, while no obvious inflammation was found in the treatments with triadimenol. Fibromuscular hyperplasia, breakage and asymmetric pigmentation and thickness of earthworm were observed in different concentration of propiconazole. The epidermis and muscular of earthworm was injured at higher concentration. Moreover, the results showed cell pyknosis, cytoplasm deep stained, nucleus concentration, vascular dilatation and engorgement, epidermis hyperplasia, vacuole degeneration and an increase in secretory cells were formed. The different degree hyperplasia and vacuole degeneration of epidermis was observed when the concentrations of difenoconazole were from 1.47 to 23.50 ␮g/cm2 . Moreover, muscle fiber broke significantly with increasing concentration of difenoconazole, hemangiectasis and hyperemia were associated with the necrosis of muscle layer was found in higher concentration.



The impact of triazoles on enzymes and histopathology in earthworm (E. foetida) was investigated in this paper. The study clearly showed a harmful effect of the triazoles on the biochemical metabolism of earthworm. For biochemical assays, triadimenol induced protein content decrease at all concentrations, suggesting that triadimenol has a high level of image on E. foetida. In contrast, exposure to difenoconazole led to the increase of the protein content. Moreover, triazoles induced GSH-Px content decrease at most concentrations. For histopathological assay, triazoles can seriously affected movement function of earthworm. On the other hand, higher propiconazole contamination induced cell pyknosis and cytoplasm deep stains, indicating there was cell apoptosis.

Histopathological examination

Conflict of interest statement Histopathological examination is a pathological observation of cellular level. The changes of physiological function, the

The authors declare that there is no conflict of interest.

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 427–433

Acknowledgement We acknowledge the National Natural Science Foundation of China (No. 21007045) for financial support.


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