Chronic Toxicity of Commercial Chlorpyrifos to Earthworm Pheretima peguana Ptumporn Muangphra,1 Kampanat Tharapoom,1 Nongnuch Euawong,1 Suluck Namchote,1 Ravi Gooneratne2 1
Biology Department, Faculty of Science, Silpakorn University, Nakhon Pathom 73000, Thailand
2
Faculty of Agriculture and Life Sciences, PO Box 85084, Lincoln University, 7647, Canterbury, New Zealand
Received 21 August 2014; revised 8 April 2015; accepted 11 April 2015 ABSTRACT: A chronic toxicity study was conducted in earthworms (Pheretima peguana) exposed to soil spiked with chlorpyrifos at concentrations of 0, 0.1, 1, 10, and 100 mg/kg soil dry matter for 7, 14, and 28 days. The integrity of the coelomocyte lysosomal membrane, nervous system, and male reproductive tissue was monitored using, respectively, the neutral-red retention assay, acetylcholinesterase (AChE) enzyme assay, and histomorphology of spermatogenic cells in the seminal vesicles and cocoon production (at 28 days after 28 days’ exposure). Chlorpyrifos decreased the coelomocyte neutral-red retention time (NRRT) significantly (p < 0.05) at concentrations > 0.1 mg/kg soil as early as day 7 of exposure and was dose- and time-dependent. Chlorpyrifos inhibition of AChE activity was greater at day 7 than at day14 (p < 0.05) indicating possibly nerve recovery. Chlorpyrifos induced concentration-dependent damage to spermatogenic cells and cytophores in premature stages. The number and size of premature, maturing, and fully mature spermatogenic stages were increased at low concentrations (50% of the total number of cells or at 60 min. This time period was recorded as the neutral-red retention time (NRRT).
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UltraTurrax (T25 basic) homogenizer. The homogenates were then centrifuged at 10,000 3 g for 10 min and the resultant supernatant was recentrifuged at 10,000 3 g for 10 min in a refrigerated centrifuge (Sanyo, Harrier 18/80). All enzyme preparations were carried out at 4 8C. The supernatants were used to determine AChE activity. The AChE assay was performed according to the method of Ellman et al. (1961). Briefly, the assay mixture consisted of 2.8 mL of 0.1 M, pH 7.2 phosphate buffer, 50 mL of 0.16 mM DTNB (5,50 -dithiobis-2-nitrobenzoic acid), 50 mL of worm supernatant, and 100 mL of 0.05, 0.065, 0.1, or 0.2 mM acetylthiocholine iodide as substrate at 37 8C. The rate of thiocholine production was measured using a spectrophotometer (Shimudzu, UV-160A) following the reaction of thiol with DTNB to produce the yellow anion of 5-thio-2-nitrobenzoic acid for 6 min at 412 nm. The activity was calculated as mmol/mg protein per minute. Enzyme activity was expressed graphically using double-reciprocal plots of Lineweaver and Burk (1934) transformations. The total protein content in the homogenate was measured by the method of Lowry et al. (1951).
Histology of Spermatogenic Stages From the earthworms exposed to 0, 1, 10, and 100 mg/kg chlorpyrifos used in the AChE assay, the 7th213th segments were taken for histomorphology and histomorphometry studies at l4 and 28 days. Briefly, the sections from these segments were fixed in Bouin’s fluid for 24 h, washed in running tap water for 1 h, and preserved in 10% formalin for tissue wax preparations using routine standard laboratory methods. Earthworm tissues were processed by dehydration using different alcohol grades (70, 90, and 100% absolute ethyl) and xylene, and embedded in paraffin wax as described by Gray (1958). Serial sections of 6–7 mm of earthworms were cut in a rotary microtome and stained with hematoxylin and eosin for histology. Fifty serial sections per earthworm were examined and a number of spermatogenic stages counted under a light microscope (Olympus CH20i attached to a Magus Micro Image Projection System) at 10003 magnification. The sizes of immature, premature, maturing, and fully mature spermatogenic stages in seminal vesicles (Rolando et al. 2007) were measured using DinoCapture 2.0 Version 1.4.2.D at 4003 magnification.
AChE Activity Earthworms (five per dose) exposed to each of four chlorpyrifos concentrations (0.1, 1, 10 and 100 mg/kg) for 7 and 14 days (not 28 days because, according to the literature, AChE activity is only a short-term response; Booth et al., 1998) used. The earthworms were partially narcotized (to avoid excessive muscle contractions) by placing them in a refrigerator for 10 min. The anterior parts (first to sixth segment) of each group of five earthworms were separately homogenized (10% w/v) in 0.1 M, pH 7.5, cold phosphate buffer using an
Cocoon Production and Viability The test endpoint was the number of cocoons hatching following worm exposure to chlorpyrifos for 28 days. For this study, an additional 10 earthworms per dose (in a box) were exposed to chlorpyrifos concentrations of 0, 0.1, 1, 10, and 100 mg/kg at 25 8C (62 8C) under a 12:12 h dark:light photoperiod. At 28 days, cocoons from each treatment were hand-sorted, separated from adults and weighed. Six cocoons from each concentration were placed on damp filter
Environmental Toxicology DOI 10.1002/tox
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MUANGPHRA ET AL.
TABLE I. Effects of chlorpyrifos on NRRT in Pheretima peguana coelomocytes following 7, 14 and 28 days’ soil exposure. NRRT (min; results are expressed as mean 6 SEM)
Exposure period (d) 0 7 14 28
0 (mg/kg) a,A
32.00 6 1.15 30.67 6 0.07a,AB 29.33 6 1.76a,AB 25.33 6 1.33a,B
0.1 (mg/kg) a,A
32.67 6 1.76 23.67 6 0.33b,B 17.33 6 0.67b,C 6.67 6 1.76b,D
1 (mg/kg)
10 (mg/kg) a,A
29.33 6 1.76 13.33 6 1.76c,B 6.67 6 0.67c,C 2.67 6 0.67b,C
a,A
34.00 6 1.15 11.33 6 1.33c,B 2.67 6 067c,C 2.00 6 0b,C
100 (mg/kg) 32.05 6 1.33a,A 5.23 6 0.67d,B 1.47 6 0.67c,B 1.29 6 0b,B
Within a column, data marked with the same upper case letter are not significantly different (p > 0.05) from each other. Within a row, data marked with the same lower case letter are not significantly different (p > 0.05) from each other.
paper in a Petri dish at 25 8C (62 8C) for another 28 days. Cocoon hatchings were examined every 2 days from day 28 to day 56, using a stereoscopic microscope (Olympus VMZ). Three replicates were used for each treatment.
Statistical Analysis Results are expressed as the mean plus or minus the standard error of the mean (SEM). Data were analyzed using SPSS Software (version 11.5) and significant differences between different treatment groups were determined using one-way analysis of variance and Tukey’s multiple comparisons test. For all statistical tests, differences were considered significant if p < 0.05.
RESULTS All earthworms exposed to chlorpyrifos concentrations survived except at the highest concentration (100 mg/Kg) at which only 85% of the earthworms survived.
tested doses (Fig. 1). At concentrations of 0.1, 1, 10, and 100 mg/kg, the respective AChE activities were 47.06, 29.41, 17.65, and 11.76% of the control at day 7, and 71.43, 57.14, 42.86, and 28.57% at day 14. IC50 values of chlorpyrifos at day 7 and day 14 to P. peguana (as calculated from Fig. 1) were 0.09 and 6.5 mg/kg, respectively.
Cocoon Production and Cocoon Viability Chlorpyrifos had an adverse impact on cocoon production and cocoon viability (hatching) following exposure to chlorpyrifos for 28 days (Table II). Number of cocoons from 10 earthworms per treatment at chlorpyrifos concentrations of 0, 0.1, 1, 10, and 100 mg/kg were 52.33 6 4.33, 62.67 6 2.73, 54.67 6 3.18, 21.33 6 1.45, and 0, respectively. Cocoons were not detected in earthworms exposed to 100 mg/kg chlorpyrifos. At 10 mg/kg soil chlorpyrifos concentration, earthworms produced fewer cocoons per earthworm and hatchings compared with controls (p < 0.05; Table II). The mean cocoon weight was significantly less in earthworms exposed to all chlorpyrifos concentrations tested.
Neutral Red Retention Assay The effects of commercial chlorpyrifos on lysosomal membrane stability were evaluated by the NRR assay. Chlorpyrifos affected lysosomal membrane integrity with a progressive decrease in the NRRT of earthworm coelomocytes (Table I). The NRRT of earthworms exposed to chlorpyrifos for 7 days were in the range of 23.67 6 0.33 to 5.23 6 0.67 min, which steadily decreased significantly (p < 0.05) with increasing chlorpyrifos concentration. The NRRT at concentrations of 0.1, 1, 10, and 100 mg/Kg decreased significantly (p < 0.05) also with increasing duration of chlorpyrifos exposure.
AChE Activity Chlorpyrifos significantly decreased AChE activity at 7 and 14 days’ exposure in a dose–response manner (Fig. 1). However, the relative AChE activity of earthworms exposed to chlorpyrifos for 14 days was higher than at day 7 for all
Environmental Toxicology DOI 10.1002/tox
Fig. 1. Relative AChE activity of earthworm following exposure to chlorpyrifos-spiked soil at 7 and 14 d. Results are expressed as mean 6 SEM. The vertical bars marked with the different letters at each exposure time are significantly (p < 0.05) different. (n 5 5). 190 3 254 mm (96 3 96 DPI).
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TABLE II. Effect of 28 days’ exposure to chlorpyrifos (0.1, 1, 10 mg/kg soil) on reproduction of Pheretima peguana
Concentration (mg/kg)
Mean Cocoon Weight (mg; n 5 10)
Mean Number of Cocoons per Earthworm (n 5 10)
Mean Number of Hatchings (n 5 10)
Hatching (%; n 5 10)
0 0.1 1 10
0.013 6 0.000a 0.009 6 0.002b 0.007 6 0.002b 0.005 6 0.001b
5.23 6 0.43a 6.27 6 0.27a 5.47 6 0.32a 2.13 6 0.15b
4.33 6 0.33a 5.00 6 0.58a 4.67 6 0.33a 2.33 6 0.33b
72.22 83.33 77.78 38.89
Results are expressed as mean 6 SEM. Within a column, data marked with the same letter are not significantly different (p > 0.05) from each other.
Histomorphology Study of Male Reproductive Tissue Spermatogenic morula (cluster) development in seminal vesicles was classified into four developmental stages [Stage 1 or immature (having approximately 4–16 cells with center cytoplasmic bridge, cytophore), Stage 2 or premature (32–64 cells), Stage 3 or maturing (64–128 cells) and Stage 4 or fully mature (128 cells) stages] depending on the size of spermatogenic morulae and number of cells per morula [Fig. 2(B)]. Exposure to chlorpyrifos for 14 and 28 days affected spermatogenesis. The nuclei of spermatogenic cells were condensed, morula numbers were decreased, and there was damage to morulae with uneven arrangement of cells around the cytophores. In addition, vacuolization and lysis of cytophores in the morulae were observed [Fig. 2(E–H)]. In the control group, normal testicular histology with uniform spermatogenic cells and a central and well-defined hilus was evident [Fig. 2(A)]. No abnormalities were observed in seminal vesicle histology and spermatogenesis, including immature, premature, maturing and fully mature stages in a sac at low-dose chlorpyrifos (1 mg/kg) [Fig. 2(C,D)]. The number of premature, maturing and fully mature stages in the morulae increased significantly at the low chlorpyrifos dose (1 mg/kg) (Table III), but the number of immature-stage morulae decreased significantly. The number of all stages of morulae decreased significantly at and above the 10 mg/kg concentration (Table III). At the highest exposure dose (100 mg/kg), severe degenerative changes with marked damage to cytophores, condensed nuclei, damaged spermatogenic cells and vacuolization of premature stages [Fig. 2(G,H)] were evident in the seminal vesicles. At the highest exposure concentration (100 mg/kg), both maturing and fully mature stages were absent. The results of our study of spermatogenisis in P. peguana following exposure to chlorpyrifos are shown in Table IV. Chlorpyrifos affected the length of morulae at most of the development stages. The length of premature and maturing stages increased significantly at chloropyrifos concentrations of 1 and 10 mg/kg. The width of maturing stages increased significantly at the 10 mg/kg concentration. Maturing and fully mature stages were not evident in the group exposed to chlorpyrifos at 100 mg/kg concentration.
DISCUSSION In this study, P. peguana earthworms were exposed to chlorpyrifos concentrations of 0.1, 1, 10, 100 mg/kg for 7, 14 and 28 days. The highest chlorpyrifos concentration, 100 mg/kg soil, is the 28-day LC50 of chlorpyrifos to the tropical earthworm Perionyx excavates (De Silva et al., 2010). However, in our study, over the same exposure period, only about 15% of P. peguana died at 100 mg/kg concentration, indicating either that P. peguana are probably more resistant to chlorpyrifos than Perionyx excavates or the bioavailability of chlorpyrifos in the soil used in our study was less. Exposure of P. peguana to sublethal chlorpyrifos concentrations induced significant changes in all the biomarkers studied but to different degrees. The NRRT, which is a reflection of lysosomal membrane stability, is regarded as a sensitive and reliable tool for the assessment of environmental pollutants in aquatic and terrestrial systems (Zhao et al., 2011). The capacity of lysosome to retain the neutral red depends on the maintenance of a low internal pH (5, compared with cytosol pH of 7.2) and the efficiency of membrane-bound proton pumps (Seglen, 1983). If the lysosomal membranes or the H1 ion pumps are destabilized, the neutral red will leak into the cytosol of the cell and the cytosol color changes to pink (Moore, 1990). A significant decrease in NRRT of coelomocytes was observed even at the lowest chlorpyrifos concentration (0.1 mg/kg) on day 7 and this was the most sensitive biomarker tested in this study. This is in agreement with Booth and O’Halloran (2001) who reported that the NRRT is a more sensitive indicator than effects on growth of A. caliginosa exposed to the insecticides, diazinon and chlorpyrifos. Similarly, Robidoux et al. (2004) reported that NRRT was significantly reduced in Eisenia andrei exposed to explosive-contaminated soils in both field and laboratory studies. Muangphra et al. (2013) reported that NRRT was significantly decreased in P. peguana earthworms exposed to cypermethrin in filter paper tests and concluded that NRRT was easier to conduct and a more sensitive indicator of exposure to pesticides. AChE activity has been routinely used as a tool to monitor nerve impulse transmission in non-target animals exposed to insecticides (Rao and Kavitha, 2004; Eamkamon
Environmental Toxicology DOI 10.1002/tox
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MUANGPHRA ET AL.
Fig. 2. Histomorphology of spermatogenic morula in control and chlorpyrifos treated earthworms. A,B: Control; In A is a normal testis with a uniform conformation of spermatogonia with a central hilus (h) at day 14. In B is normal developing of spermatogonial morula in seminal vesicles at day 28. C,D: Earthworm exposed to 1 mg Kg-1 chlorpyrifos at 14 (C) and 28 d (D), respectively. In C is normal nuclear division (arrow). In D is normal spermatogenesis. E,F: Exposed to 10 mg kg21 chlorpyrifos at 14 and 28 d, respectively. In E is cytophore (c) beginning to vacuolize (arrow). In F, increased vacuolization (v) and uneven of spermatogenic cells in a premature stage (arrow). G,H: Exposed to 100 mg kg21 chlorpyrifos at 14 and 28 d respectively. In G is increased vacuolization in premature stages and swollen cells (cs). In H is increased vacuolization with damaged spermatogenic cells. (1: Immature stage; 2: Premature stage; 3: Maturing stage; 4: Fully matured stage). 190 3 254 mm (96 3 96 DPI). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
et al., 2012; Martinez-Morcillo et al., 2013, Qi et al., 2013). Inhibition of AChE activity at the nerve-to-nerve and nerveto-muscle junctions will cause both muscarinic and nicotinic
Environmental Toxicology DOI 10.1002/tox
effects and are interpreted as signs of neurotoxicity that profoundly affects the survival of organisms (Rao and Kavitha, 2004). In this study, chlorpyrifos significantly inhibited
EARTHWORM CHLORPYRIFOS TOXICITY
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TABLE III. Effect of chlorpyrifos on the number of spermatogenic stages in Phreretima peguana exposed to chlorpyrifos-spiked soils for 28 days
Concentration (mg/kg soil) 0 1 10 100
Mean number of spermatogenic stages per earthworm N 3 3 3 3
Immature
Premature a
32.33 6 1.45 20.67 6 0.89b 13.33 6 1.20bc 9.67 6 0.33c
Maturing b
52.67 6 2.96 78.33 6 1.86a 22.67 6 1.20c 13.66 6 0.88d
Fully mature b
44.66 6 1.45 62.33 6 1.86a 19.00 6 1.53c 0
21.67 6 2.40b 51.33 6 2.03a 12.67 6 1.20c 0
Results are expressed as mean 6 SEM. Within a column, data marked with the different letters are significantly different (p < 0.05).
AChE activity compared with the controls even at the lowest exposure dose, showing that it is highly neurotoxic to earthworms. Similar results have been reported in A. caliginosa earthworms exposed to chlorpyrifos at 0.51 and 10 mg/kg for 21 days with 84% and 97% AchE inhibition respectively (Sanchez-Hernandez et al., 2014). Booth et al. (1998) reported AChE inhibition of up to 87% by chlorpyrifos even on day 1 of exposure, and remained low for 14 days. In our study, relative AChE activity (Vmax, indicative of total enzyme activity) in earthworms exposed for 7 and 14 days decreased significantly in a dose-dependent manner. The percentage of inhibition was higher at 7 days (52.94, 70.59, 82.35, and 88.23%) than at 14 days’ exposure (28.57, 42.86, 57.14, and 71.43%). The greatest inhibition of AChE was at the highest exposure dose of 100 mg/kg at 7 days, and at 14 days AChE inhibition was less pronounced, 0.46-, 0.39-, 0.31-, and 0.19-fold reduced from at 7 days. It appears there is recovery of AChE activity after 7 days, but activity is still below control values at 14 days. This is in contrast to the findings of Booth et al. (1998) who reported that AChE is an excellent biomarker of short-term chlorpyrifos exposure because the highest inhibition was observed at day 1 and it remained low for up to 14 days. The recovery of AChE in our study may be due to a breakdown of of chlorpyrifos in soil. In aerobic soils, the half-life of chlorpyrifos varied from 11 to 141 days depending on soil texture (loamy sand or clay), pH (5.4–7.4), and climate (USEPA, 1989). Reproduction tests demonstrated that chlorpyrifos has an adverse effect on both cocoon production and cocoon viability. Following 28 days’ exposure, the number of cocoons per earthworm and cocoon hatching decreased significantly at 10 mg/kg soil exposure concentration compared with the controls (Table II). This is in agreement with Zhou et al. (2007) who reported similar results in Eisenia fetida andrei exposed to chlorpyrifos for 28 days. In our study, the number of cocoons per adult following a 28-day chlorpyrifos exposure was 60% less than in the control group (Table II). Zhou et al. (2007) reported a slightly higher decline in cocoon production (70%) in Eisenia fetida exposed to chlorpyrifos at 20 mg/kg soil concentration. The significant reduction in cocoon production and hatching is probably a direct result of damage to reproductive tissue by chlorpyrifos because
vacuolization and lysis of spermatogenic cells were observed in our study even at 14 days’ exposure to 100 mg/kg chlorpyrifos (Fig. 2). Such changes can lead to marked reproductive consequences and subsequent reduction in population density. Pheretima peguana a protandric hermaphrodite and fertilizes its eggs in a cocoon secreted by the clitellum. The reproductive segments are located in the anterior portion of the worm, between the 8th and 13th segments. Spermatogonia are produced in the testis and are then transferred to seminal vesicles grouped as a 4- celled morula, where spermatogenesis and spermiogenesis occur. This morula reaches a maximum of 128 germ cells after meiosis due to their synchronous development. Our histomorphological study showed that the spermatogenesis of P. peguana is similar to that in Eisenia fetida (Espinoza-Navarro and BustosObregon, 2004). These morulae have different numbers of cells, which may grow in geometric progression to morulae of spermatogonia, spermatocytes, or spermatids. In all cases, the germinal cells are attached through cytoplasmic bridges to a central anucleate mass of cytoplasm rich in organelles, the cytophore. The only independent cells are the spermatozoa, which are detached from the cytophore once spermiogenesis is completed (Anderson et al., 1967; Rolando et al., 2007). The transverse section of the testes and seminal vesicles of treatment groups indicated damage to the morula structure in seminal vesicles. It was evident from the histological study that morula structure was disrupted with vacuolization of the cytophore and later condensation of nuclei of spermatogenic cells in the premature stage, followed by DNA damage of spermatogenic cells in morulae, necrosis and cell lysis [Fig. 2(G,H)]. All earthworms exposed to chlorpyrifos exhibited elevated numbers of abnormal spermatogenic cells in morulae. Sorour and Larink (2001) reported that pesticides could damage spermatozoa by altering the ultrastructure of the cytoskeleton of the cytophore and spermatogonial morula, thereby affecting the normal development of spermatogenesis and spermiogenesis. Espinoza-Navarro and Bustos-Obregon (2004) reported that malathion affects tissue disorganization, vacuolization, and the presence of small and hyperchromatic nuclei in spermatogonial morulae indicative
Environmental Toxicology DOI 10.1002/tox
18.33 6 0.082ab 16.08 6 0.053a 20.63 6 0.133b 0 27.61 6 0.115 30.85 6 0.093a 31.33 6 0.191a 0 13.12 6 0.056 14.94 6 0.005ab 16.04 6 0.123b 0 20.82 6 0.090 27.02 6 0.102b 28.08 6 0.173b 0
a
W
Environmental Toxicology DOI 10.1002/tox
Results are expressed as mean 6 SEM. Within a column, data marked with the different letters are significantly different (p < 0.05).
12.51 6 0.037 12.22 6 0.033a 14.12 6 0.082a 12.54 6 0.087a 9.48 6 0.040 9.46 6 0.044a 9.45 6 0.054a 9.17 6 0.057a 0 1 10 100
a
L Concentration (mg/kg)
11.91 6 0.045 12.76 6 0.066a 13.01 6 0.095a 12.39 6 0.062a
W
a
L
a
19.14 6 0.047 20.59 6 0.051ab 22.94 6 0.126b 19.75 6 0.124ab
W
a
L
a
Maturing stage Premature stage Immature stage
Mean length (L) and width (W) of spermatogenic stages (mm)
TABLE IV. Biometric data of spermatogenic stages in Pheretima peguana exposed to chlorpyrifos-spiked soil at 28 days
L
a
W
MUANGPHRA ET AL.
Fully mature stage
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of possible apoptosis. This is similar to the findings of ElKashoury and Tag El-Din (2010) who reported that testes of chlorpyrifos-treated albino rats exhibit degeneration and atrophy of nonfunctional seminal tubules and a marked decline in sperm cells within the lumen of seminiferous tubules. Nishi and Hundal (2013) also reported that subchronic exposure to chlorpyrifos induces oxidative stress and marked pathology in the reproductive organs of female rats. Chlorpyrifos is toxic to earthworm reproduction and can subsequently lead to a population decline. In this study, all stages of spermatogenesis (i.e., maturing and fully mature spermatogonia or spermatozoa) were not visible in earthworms exposed to the highest chlorpyrifos concentration (100 mg/kg; Table III). This is in agreement with Landrigan et al. (1999) who reported that chlorpyrifos is highly toxic to developing fetuses, infants and children. Shittu et al. (2013) reported that chlorpyrifos decreased follicle-stimulating hormone (FSH) concentrations in male Wistar rats and this may be responsible for the low sperm count observed since FSH is actively involved in spermatogenesis. Brunninger et al. (1994) reported that fecundity in earthworms is sensitive to pesticides even though the population density of earthworms may not be immediately affected. In any case, the ongoing reproductive effects will result in a population decline in the longer term. Although no mortality was observed in earthworms exposed to 10 mg/kg chlorpyrifos, cocoon numbers per earthworm declined markedly up to 60%. Such a decline can ultimately lead to a marked population decrease. This is in agreement with Neuhauser (1990) who reported that exposure of earthworms to 50 mg/kg carbaryl in soil resulted in a decrease in cocoon numbers by more than 50% and this can result in a significant population decline a within two or three generations. It is known that low levels of toxicants can elicit an overcompensation response (Furst, 1987; Calabrese and Baldwin, 2002; Hackenberger et al., 2008) known as hormesis. In our study a low exposure concentration (1 mg/kg) initiated temporary overadaptation, as shown by an increase in the number of premature, maturing and fully mature stages of spermatogenic morulae in the earthworm P. peguana (Table III). But at higher chlorpyrifos concentrations, the number of spermatogenic morulae decreased significantly. In contrast, chlorpyrifos significantly increased the size of the maturing-morula spermatogenic stages at all concentrations except at the highest concentration (100 mg/kg) because at this concentration all maturing and fully mature stages were non-existent (Table IV). These results indicate a marked toxic effect of chlorpyrifos on the male reproductive system in earthworms. When assessing the toxicity of an insecticide, use of a suite of biochemical and histological biomarkers can provide a comprehensive understanding of its toxicity profile to an organism. The different biomarkers tested in this study showed different response profiles in earthworms exposed to chlorpyrifos. The cellular (NRRT) and biochemical (AChE activity) biomarkers were several-fold more sensitive, even
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at the lowest chlorpyrifos exposure concentration (0.1 mg/ kg) at 7 days, than histology and reproductive parameters. Choosing the most sensitive and ecologically relevant biomarker is important for accurate environmental risk assessment of chemicals.
De Silva PMCS, Pathiratne A, Van Gestel CAM. 2010. Toxicity of chlorpyrifos, carbofuran, mancozeb and their formulations to the tropical earthworm Perionyx excavates. Appl Soil Ecol 44: 56–60.
CONCLUSIONS
Eamkamon T, Klinbunga S, Thirakhupt K, Menasveta P, Puanglarp N. 2012. Acute toxicity of neurotoxicity of chlorpyrifos in black tiger shrimp, Penaeus monodon. Environ Asia 5: 26–31.
Pheretima peguana exposure to sublethal concentrations of chlorpyrifos induced significant changes in all the biomarkers studied but to different degrees. Cocoon production and viability and histomorphology of spermatogenic cells are useful biomarkers in the earthworm, but NRRT and AChE enzyme activity are more sensitive in detecting exposure to chlorpyrifos even at low dose and with short exposure periods. Although NRRT and AChE activity were the most sensitive of the biomarkers, cocoon production and cocoon viability could still be considered as diagnostic tools for monitoring low-dose long-term chlorpyrifos toxicity and evaluating population effects.
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Scott-Fordsmand JJ, Weeks JM, Hopkin SP. 2000. Importance of contamination history for understanding toxicity of copper to earthworm Eisenia fetida (oligochaeta: annelida), using neutralred retention assay. Environ Toxicol Chem 19:1774–1780. Seglen po. 1983. lnhibitors of lysosomal function. Methods Enzymol 96:737–764. Shittu M, Ambali SF, Ayo JO, Fatihu MY, Sualiman MM, Yaqub LS. 2013. Evaluation of chronic chlorpyrifos-induced reproductive toxicity in male wistar rat: Protective effects of vitamin C. J Exp Integr Med 3:23–30. Sorour J, Larink O 2001. Toxic effects of benomyl on the ultrastructure during spermatogenesis of the earthworm Eisenia fetida. Ecotoxicol Environ Safe 50:180–188. Steenland K, Jenkins B, Ames RG, O’Malley M, Chrislip D, Russo J. 1994. Chronic neurological sequelae to organophosphate pesticide poisoning. Am J Pub Health 84:731–736. Thompson CM, Richardson RJ. 2004. Anticholinesterase insecticides. In: Marrs TC, Ballantyne B, editors. Pesticide toxicology and international regulation. West Sussex, England: Wiely. p 89–127. Turner KJ, Barlow NJ, Struve MF, Wallace DG, Gaido KW, Dorman DC, Foster PMD. 2002. Effects of in utero exposure to the organophosphate insecticide fenitrothion on androgendependent reproductive development in the crl:CD(SD) BR rat. Toxicol Sci 68:174–183. USEPA (US Environmental Protection Agency). 1989. Registration standard (Second Round Review) for the reregistration of pesticide products containing chlorpyrifos. Washington, DC: Office of Pesticide Programs, US EPA. Zhao C, Li X, Luo S, Chang Y. 2011. Assessments of lysosomal membrane responses to stresses with neutral red retention assay and its potential application in the improvement of bivalve aquaculture. Afr J Biotechnol 10:13968–13973. Zhou S, Duan C, Fu H, Chen Y, Wang X, Yu Z. 2007. Toxicity assessment for chlorpyrifos-contaminated soil with three different earthworm test methods. J Environ Sci 19:854–858.
Biology Department, Faculty of Science, Silpakorn University, Nakhon Pathom 73000, Thailand
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Faculty of Agriculture and Life Sciences, PO Box 85084, Lincoln University, 7647, Canterbury, New Zealand
Received 21 August 2014; revised 8 April 2015; accepted 11 April 2015 ABSTRACT: A chronic toxicity study was conducted in earthworms (Pheretima peguana) exposed to soil spiked with chlorpyrifos at concentrations of 0, 0.1, 1, 10, and 100 mg/kg soil dry matter for 7, 14, and 28 days. The integrity of the coelomocyte lysosomal membrane, nervous system, and male reproductive tissue was monitored using, respectively, the neutral-red retention assay, acetylcholinesterase (AChE) enzyme assay, and histomorphology of spermatogenic cells in the seminal vesicles and cocoon production (at 28 days after 28 days’ exposure). Chlorpyrifos decreased the coelomocyte neutral-red retention time (NRRT) significantly (p < 0.05) at concentrations > 0.1 mg/kg soil as early as day 7 of exposure and was dose- and time-dependent. Chlorpyrifos inhibition of AChE activity was greater at day 7 than at day14 (p < 0.05) indicating possibly nerve recovery. Chlorpyrifos induced concentration-dependent damage to spermatogenic cells and cytophores in premature stages. The number and size of premature, maturing, and fully mature spermatogenic stages were increased at low concentrations (50% of the total number of cells or at 60 min. This time period was recorded as the neutral-red retention time (NRRT).
3
UltraTurrax (T25 basic) homogenizer. The homogenates were then centrifuged at 10,000 3 g for 10 min and the resultant supernatant was recentrifuged at 10,000 3 g for 10 min in a refrigerated centrifuge (Sanyo, Harrier 18/80). All enzyme preparations were carried out at 4 8C. The supernatants were used to determine AChE activity. The AChE assay was performed according to the method of Ellman et al. (1961). Briefly, the assay mixture consisted of 2.8 mL of 0.1 M, pH 7.2 phosphate buffer, 50 mL of 0.16 mM DTNB (5,50 -dithiobis-2-nitrobenzoic acid), 50 mL of worm supernatant, and 100 mL of 0.05, 0.065, 0.1, or 0.2 mM acetylthiocholine iodide as substrate at 37 8C. The rate of thiocholine production was measured using a spectrophotometer (Shimudzu, UV-160A) following the reaction of thiol with DTNB to produce the yellow anion of 5-thio-2-nitrobenzoic acid for 6 min at 412 nm. The activity was calculated as mmol/mg protein per minute. Enzyme activity was expressed graphically using double-reciprocal plots of Lineweaver and Burk (1934) transformations. The total protein content in the homogenate was measured by the method of Lowry et al. (1951).
Histology of Spermatogenic Stages From the earthworms exposed to 0, 1, 10, and 100 mg/kg chlorpyrifos used in the AChE assay, the 7th213th segments were taken for histomorphology and histomorphometry studies at l4 and 28 days. Briefly, the sections from these segments were fixed in Bouin’s fluid for 24 h, washed in running tap water for 1 h, and preserved in 10% formalin for tissue wax preparations using routine standard laboratory methods. Earthworm tissues were processed by dehydration using different alcohol grades (70, 90, and 100% absolute ethyl) and xylene, and embedded in paraffin wax as described by Gray (1958). Serial sections of 6–7 mm of earthworms were cut in a rotary microtome and stained with hematoxylin and eosin for histology. Fifty serial sections per earthworm were examined and a number of spermatogenic stages counted under a light microscope (Olympus CH20i attached to a Magus Micro Image Projection System) at 10003 magnification. The sizes of immature, premature, maturing, and fully mature spermatogenic stages in seminal vesicles (Rolando et al. 2007) were measured using DinoCapture 2.0 Version 1.4.2.D at 4003 magnification.
AChE Activity Earthworms (five per dose) exposed to each of four chlorpyrifos concentrations (0.1, 1, 10 and 100 mg/kg) for 7 and 14 days (not 28 days because, according to the literature, AChE activity is only a short-term response; Booth et al., 1998) used. The earthworms were partially narcotized (to avoid excessive muscle contractions) by placing them in a refrigerator for 10 min. The anterior parts (first to sixth segment) of each group of five earthworms were separately homogenized (10% w/v) in 0.1 M, pH 7.5, cold phosphate buffer using an
Cocoon Production and Viability The test endpoint was the number of cocoons hatching following worm exposure to chlorpyrifos for 28 days. For this study, an additional 10 earthworms per dose (in a box) were exposed to chlorpyrifos concentrations of 0, 0.1, 1, 10, and 100 mg/kg at 25 8C (62 8C) under a 12:12 h dark:light photoperiod. At 28 days, cocoons from each treatment were hand-sorted, separated from adults and weighed. Six cocoons from each concentration were placed on damp filter
Environmental Toxicology DOI 10.1002/tox
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MUANGPHRA ET AL.
TABLE I. Effects of chlorpyrifos on NRRT in Pheretima peguana coelomocytes following 7, 14 and 28 days’ soil exposure. NRRT (min; results are expressed as mean 6 SEM)
Exposure period (d) 0 7 14 28
0 (mg/kg) a,A
32.00 6 1.15 30.67 6 0.07a,AB 29.33 6 1.76a,AB 25.33 6 1.33a,B
0.1 (mg/kg) a,A
32.67 6 1.76 23.67 6 0.33b,B 17.33 6 0.67b,C 6.67 6 1.76b,D
1 (mg/kg)
10 (mg/kg) a,A
29.33 6 1.76 13.33 6 1.76c,B 6.67 6 0.67c,C 2.67 6 0.67b,C
a,A
34.00 6 1.15 11.33 6 1.33c,B 2.67 6 067c,C 2.00 6 0b,C
100 (mg/kg) 32.05 6 1.33a,A 5.23 6 0.67d,B 1.47 6 0.67c,B 1.29 6 0b,B
Within a column, data marked with the same upper case letter are not significantly different (p > 0.05) from each other. Within a row, data marked with the same lower case letter are not significantly different (p > 0.05) from each other.
paper in a Petri dish at 25 8C (62 8C) for another 28 days. Cocoon hatchings were examined every 2 days from day 28 to day 56, using a stereoscopic microscope (Olympus VMZ). Three replicates were used for each treatment.
Statistical Analysis Results are expressed as the mean plus or minus the standard error of the mean (SEM). Data were analyzed using SPSS Software (version 11.5) and significant differences between different treatment groups were determined using one-way analysis of variance and Tukey’s multiple comparisons test. For all statistical tests, differences were considered significant if p < 0.05.
RESULTS All earthworms exposed to chlorpyrifos concentrations survived except at the highest concentration (100 mg/Kg) at which only 85% of the earthworms survived.
tested doses (Fig. 1). At concentrations of 0.1, 1, 10, and 100 mg/kg, the respective AChE activities were 47.06, 29.41, 17.65, and 11.76% of the control at day 7, and 71.43, 57.14, 42.86, and 28.57% at day 14. IC50 values of chlorpyrifos at day 7 and day 14 to P. peguana (as calculated from Fig. 1) were 0.09 and 6.5 mg/kg, respectively.
Cocoon Production and Cocoon Viability Chlorpyrifos had an adverse impact on cocoon production and cocoon viability (hatching) following exposure to chlorpyrifos for 28 days (Table II). Number of cocoons from 10 earthworms per treatment at chlorpyrifos concentrations of 0, 0.1, 1, 10, and 100 mg/kg were 52.33 6 4.33, 62.67 6 2.73, 54.67 6 3.18, 21.33 6 1.45, and 0, respectively. Cocoons were not detected in earthworms exposed to 100 mg/kg chlorpyrifos. At 10 mg/kg soil chlorpyrifos concentration, earthworms produced fewer cocoons per earthworm and hatchings compared with controls (p < 0.05; Table II). The mean cocoon weight was significantly less in earthworms exposed to all chlorpyrifos concentrations tested.
Neutral Red Retention Assay The effects of commercial chlorpyrifos on lysosomal membrane stability were evaluated by the NRR assay. Chlorpyrifos affected lysosomal membrane integrity with a progressive decrease in the NRRT of earthworm coelomocytes (Table I). The NRRT of earthworms exposed to chlorpyrifos for 7 days were in the range of 23.67 6 0.33 to 5.23 6 0.67 min, which steadily decreased significantly (p < 0.05) with increasing chlorpyrifos concentration. The NRRT at concentrations of 0.1, 1, 10, and 100 mg/Kg decreased significantly (p < 0.05) also with increasing duration of chlorpyrifos exposure.
AChE Activity Chlorpyrifos significantly decreased AChE activity at 7 and 14 days’ exposure in a dose–response manner (Fig. 1). However, the relative AChE activity of earthworms exposed to chlorpyrifos for 14 days was higher than at day 7 for all
Environmental Toxicology DOI 10.1002/tox
Fig. 1. Relative AChE activity of earthworm following exposure to chlorpyrifos-spiked soil at 7 and 14 d. Results are expressed as mean 6 SEM. The vertical bars marked with the different letters at each exposure time are significantly (p < 0.05) different. (n 5 5). 190 3 254 mm (96 3 96 DPI).
EARTHWORM CHLORPYRIFOS TOXICITY
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TABLE II. Effect of 28 days’ exposure to chlorpyrifos (0.1, 1, 10 mg/kg soil) on reproduction of Pheretima peguana
Concentration (mg/kg)
Mean Cocoon Weight (mg; n 5 10)
Mean Number of Cocoons per Earthworm (n 5 10)
Mean Number of Hatchings (n 5 10)
Hatching (%; n 5 10)
0 0.1 1 10
0.013 6 0.000a 0.009 6 0.002b 0.007 6 0.002b 0.005 6 0.001b
5.23 6 0.43a 6.27 6 0.27a 5.47 6 0.32a 2.13 6 0.15b
4.33 6 0.33a 5.00 6 0.58a 4.67 6 0.33a 2.33 6 0.33b
72.22 83.33 77.78 38.89
Results are expressed as mean 6 SEM. Within a column, data marked with the same letter are not significantly different (p > 0.05) from each other.
Histomorphology Study of Male Reproductive Tissue Spermatogenic morula (cluster) development in seminal vesicles was classified into four developmental stages [Stage 1 or immature (having approximately 4–16 cells with center cytoplasmic bridge, cytophore), Stage 2 or premature (32–64 cells), Stage 3 or maturing (64–128 cells) and Stage 4 or fully mature (128 cells) stages] depending on the size of spermatogenic morulae and number of cells per morula [Fig. 2(B)]. Exposure to chlorpyrifos for 14 and 28 days affected spermatogenesis. The nuclei of spermatogenic cells were condensed, morula numbers were decreased, and there was damage to morulae with uneven arrangement of cells around the cytophores. In addition, vacuolization and lysis of cytophores in the morulae were observed [Fig. 2(E–H)]. In the control group, normal testicular histology with uniform spermatogenic cells and a central and well-defined hilus was evident [Fig. 2(A)]. No abnormalities were observed in seminal vesicle histology and spermatogenesis, including immature, premature, maturing and fully mature stages in a sac at low-dose chlorpyrifos (1 mg/kg) [Fig. 2(C,D)]. The number of premature, maturing and fully mature stages in the morulae increased significantly at the low chlorpyrifos dose (1 mg/kg) (Table III), but the number of immature-stage morulae decreased significantly. The number of all stages of morulae decreased significantly at and above the 10 mg/kg concentration (Table III). At the highest exposure dose (100 mg/kg), severe degenerative changes with marked damage to cytophores, condensed nuclei, damaged spermatogenic cells and vacuolization of premature stages [Fig. 2(G,H)] were evident in the seminal vesicles. At the highest exposure concentration (100 mg/kg), both maturing and fully mature stages were absent. The results of our study of spermatogenisis in P. peguana following exposure to chlorpyrifos are shown in Table IV. Chlorpyrifos affected the length of morulae at most of the development stages. The length of premature and maturing stages increased significantly at chloropyrifos concentrations of 1 and 10 mg/kg. The width of maturing stages increased significantly at the 10 mg/kg concentration. Maturing and fully mature stages were not evident in the group exposed to chlorpyrifos at 100 mg/kg concentration.
DISCUSSION In this study, P. peguana earthworms were exposed to chlorpyrifos concentrations of 0.1, 1, 10, 100 mg/kg for 7, 14 and 28 days. The highest chlorpyrifos concentration, 100 mg/kg soil, is the 28-day LC50 of chlorpyrifos to the tropical earthworm Perionyx excavates (De Silva et al., 2010). However, in our study, over the same exposure period, only about 15% of P. peguana died at 100 mg/kg concentration, indicating either that P. peguana are probably more resistant to chlorpyrifos than Perionyx excavates or the bioavailability of chlorpyrifos in the soil used in our study was less. Exposure of P. peguana to sublethal chlorpyrifos concentrations induced significant changes in all the biomarkers studied but to different degrees. The NRRT, which is a reflection of lysosomal membrane stability, is regarded as a sensitive and reliable tool for the assessment of environmental pollutants in aquatic and terrestrial systems (Zhao et al., 2011). The capacity of lysosome to retain the neutral red depends on the maintenance of a low internal pH (5, compared with cytosol pH of 7.2) and the efficiency of membrane-bound proton pumps (Seglen, 1983). If the lysosomal membranes or the H1 ion pumps are destabilized, the neutral red will leak into the cytosol of the cell and the cytosol color changes to pink (Moore, 1990). A significant decrease in NRRT of coelomocytes was observed even at the lowest chlorpyrifos concentration (0.1 mg/kg) on day 7 and this was the most sensitive biomarker tested in this study. This is in agreement with Booth and O’Halloran (2001) who reported that the NRRT is a more sensitive indicator than effects on growth of A. caliginosa exposed to the insecticides, diazinon and chlorpyrifos. Similarly, Robidoux et al. (2004) reported that NRRT was significantly reduced in Eisenia andrei exposed to explosive-contaminated soils in both field and laboratory studies. Muangphra et al. (2013) reported that NRRT was significantly decreased in P. peguana earthworms exposed to cypermethrin in filter paper tests and concluded that NRRT was easier to conduct and a more sensitive indicator of exposure to pesticides. AChE activity has been routinely used as a tool to monitor nerve impulse transmission in non-target animals exposed to insecticides (Rao and Kavitha, 2004; Eamkamon
Environmental Toxicology DOI 10.1002/tox
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MUANGPHRA ET AL.
Fig. 2. Histomorphology of spermatogenic morula in control and chlorpyrifos treated earthworms. A,B: Control; In A is a normal testis with a uniform conformation of spermatogonia with a central hilus (h) at day 14. In B is normal developing of spermatogonial morula in seminal vesicles at day 28. C,D: Earthworm exposed to 1 mg Kg-1 chlorpyrifos at 14 (C) and 28 d (D), respectively. In C is normal nuclear division (arrow). In D is normal spermatogenesis. E,F: Exposed to 10 mg kg21 chlorpyrifos at 14 and 28 d, respectively. In E is cytophore (c) beginning to vacuolize (arrow). In F, increased vacuolization (v) and uneven of spermatogenic cells in a premature stage (arrow). G,H: Exposed to 100 mg kg21 chlorpyrifos at 14 and 28 d respectively. In G is increased vacuolization in premature stages and swollen cells (cs). In H is increased vacuolization with damaged spermatogenic cells. (1: Immature stage; 2: Premature stage; 3: Maturing stage; 4: Fully matured stage). 190 3 254 mm (96 3 96 DPI). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
et al., 2012; Martinez-Morcillo et al., 2013, Qi et al., 2013). Inhibition of AChE activity at the nerve-to-nerve and nerveto-muscle junctions will cause both muscarinic and nicotinic
Environmental Toxicology DOI 10.1002/tox
effects and are interpreted as signs of neurotoxicity that profoundly affects the survival of organisms (Rao and Kavitha, 2004). In this study, chlorpyrifos significantly inhibited
EARTHWORM CHLORPYRIFOS TOXICITY
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TABLE III. Effect of chlorpyrifos on the number of spermatogenic stages in Phreretima peguana exposed to chlorpyrifos-spiked soils for 28 days
Concentration (mg/kg soil) 0 1 10 100
Mean number of spermatogenic stages per earthworm N 3 3 3 3
Immature
Premature a
32.33 6 1.45 20.67 6 0.89b 13.33 6 1.20bc 9.67 6 0.33c
Maturing b
52.67 6 2.96 78.33 6 1.86a 22.67 6 1.20c 13.66 6 0.88d
Fully mature b
44.66 6 1.45 62.33 6 1.86a 19.00 6 1.53c 0
21.67 6 2.40b 51.33 6 2.03a 12.67 6 1.20c 0
Results are expressed as mean 6 SEM. Within a column, data marked with the different letters are significantly different (p < 0.05).
AChE activity compared with the controls even at the lowest exposure dose, showing that it is highly neurotoxic to earthworms. Similar results have been reported in A. caliginosa earthworms exposed to chlorpyrifos at 0.51 and 10 mg/kg for 21 days with 84% and 97% AchE inhibition respectively (Sanchez-Hernandez et al., 2014). Booth et al. (1998) reported AChE inhibition of up to 87% by chlorpyrifos even on day 1 of exposure, and remained low for 14 days. In our study, relative AChE activity (Vmax, indicative of total enzyme activity) in earthworms exposed for 7 and 14 days decreased significantly in a dose-dependent manner. The percentage of inhibition was higher at 7 days (52.94, 70.59, 82.35, and 88.23%) than at 14 days’ exposure (28.57, 42.86, 57.14, and 71.43%). The greatest inhibition of AChE was at the highest exposure dose of 100 mg/kg at 7 days, and at 14 days AChE inhibition was less pronounced, 0.46-, 0.39-, 0.31-, and 0.19-fold reduced from at 7 days. It appears there is recovery of AChE activity after 7 days, but activity is still below control values at 14 days. This is in contrast to the findings of Booth et al. (1998) who reported that AChE is an excellent biomarker of short-term chlorpyrifos exposure because the highest inhibition was observed at day 1 and it remained low for up to 14 days. The recovery of AChE in our study may be due to a breakdown of of chlorpyrifos in soil. In aerobic soils, the half-life of chlorpyrifos varied from 11 to 141 days depending on soil texture (loamy sand or clay), pH (5.4–7.4), and climate (USEPA, 1989). Reproduction tests demonstrated that chlorpyrifos has an adverse effect on both cocoon production and cocoon viability. Following 28 days’ exposure, the number of cocoons per earthworm and cocoon hatching decreased significantly at 10 mg/kg soil exposure concentration compared with the controls (Table II). This is in agreement with Zhou et al. (2007) who reported similar results in Eisenia fetida andrei exposed to chlorpyrifos for 28 days. In our study, the number of cocoons per adult following a 28-day chlorpyrifos exposure was 60% less than in the control group (Table II). Zhou et al. (2007) reported a slightly higher decline in cocoon production (70%) in Eisenia fetida exposed to chlorpyrifos at 20 mg/kg soil concentration. The significant reduction in cocoon production and hatching is probably a direct result of damage to reproductive tissue by chlorpyrifos because
vacuolization and lysis of spermatogenic cells were observed in our study even at 14 days’ exposure to 100 mg/kg chlorpyrifos (Fig. 2). Such changes can lead to marked reproductive consequences and subsequent reduction in population density. Pheretima peguana a protandric hermaphrodite and fertilizes its eggs in a cocoon secreted by the clitellum. The reproductive segments are located in the anterior portion of the worm, between the 8th and 13th segments. Spermatogonia are produced in the testis and are then transferred to seminal vesicles grouped as a 4- celled morula, where spermatogenesis and spermiogenesis occur. This morula reaches a maximum of 128 germ cells after meiosis due to their synchronous development. Our histomorphological study showed that the spermatogenesis of P. peguana is similar to that in Eisenia fetida (Espinoza-Navarro and BustosObregon, 2004). These morulae have different numbers of cells, which may grow in geometric progression to morulae of spermatogonia, spermatocytes, or spermatids. In all cases, the germinal cells are attached through cytoplasmic bridges to a central anucleate mass of cytoplasm rich in organelles, the cytophore. The only independent cells are the spermatozoa, which are detached from the cytophore once spermiogenesis is completed (Anderson et al., 1967; Rolando et al., 2007). The transverse section of the testes and seminal vesicles of treatment groups indicated damage to the morula structure in seminal vesicles. It was evident from the histological study that morula structure was disrupted with vacuolization of the cytophore and later condensation of nuclei of spermatogenic cells in the premature stage, followed by DNA damage of spermatogenic cells in morulae, necrosis and cell lysis [Fig. 2(G,H)]. All earthworms exposed to chlorpyrifos exhibited elevated numbers of abnormal spermatogenic cells in morulae. Sorour and Larink (2001) reported that pesticides could damage spermatozoa by altering the ultrastructure of the cytoskeleton of the cytophore and spermatogonial morula, thereby affecting the normal development of spermatogenesis and spermiogenesis. Espinoza-Navarro and Bustos-Obregon (2004) reported that malathion affects tissue disorganization, vacuolization, and the presence of small and hyperchromatic nuclei in spermatogonial morulae indicative
Environmental Toxicology DOI 10.1002/tox
18.33 6 0.082ab 16.08 6 0.053a 20.63 6 0.133b 0 27.61 6 0.115 30.85 6 0.093a 31.33 6 0.191a 0 13.12 6 0.056 14.94 6 0.005ab 16.04 6 0.123b 0 20.82 6 0.090 27.02 6 0.102b 28.08 6 0.173b 0
a
W
Environmental Toxicology DOI 10.1002/tox
Results are expressed as mean 6 SEM. Within a column, data marked with the different letters are significantly different (p < 0.05).
12.51 6 0.037 12.22 6 0.033a 14.12 6 0.082a 12.54 6 0.087a 9.48 6 0.040 9.46 6 0.044a 9.45 6 0.054a 9.17 6 0.057a 0 1 10 100
a
L Concentration (mg/kg)
11.91 6 0.045 12.76 6 0.066a 13.01 6 0.095a 12.39 6 0.062a
W
a
L
a
19.14 6 0.047 20.59 6 0.051ab 22.94 6 0.126b 19.75 6 0.124ab
W
a
L
a
Maturing stage Premature stage Immature stage
Mean length (L) and width (W) of spermatogenic stages (mm)
TABLE IV. Biometric data of spermatogenic stages in Pheretima peguana exposed to chlorpyrifos-spiked soil at 28 days
L
a
W
MUANGPHRA ET AL.
Fully mature stage
8
of possible apoptosis. This is similar to the findings of ElKashoury and Tag El-Din (2010) who reported that testes of chlorpyrifos-treated albino rats exhibit degeneration and atrophy of nonfunctional seminal tubules and a marked decline in sperm cells within the lumen of seminiferous tubules. Nishi and Hundal (2013) also reported that subchronic exposure to chlorpyrifos induces oxidative stress and marked pathology in the reproductive organs of female rats. Chlorpyrifos is toxic to earthworm reproduction and can subsequently lead to a population decline. In this study, all stages of spermatogenesis (i.e., maturing and fully mature spermatogonia or spermatozoa) were not visible in earthworms exposed to the highest chlorpyrifos concentration (100 mg/kg; Table III). This is in agreement with Landrigan et al. (1999) who reported that chlorpyrifos is highly toxic to developing fetuses, infants and children. Shittu et al. (2013) reported that chlorpyrifos decreased follicle-stimulating hormone (FSH) concentrations in male Wistar rats and this may be responsible for the low sperm count observed since FSH is actively involved in spermatogenesis. Brunninger et al. (1994) reported that fecundity in earthworms is sensitive to pesticides even though the population density of earthworms may not be immediately affected. In any case, the ongoing reproductive effects will result in a population decline in the longer term. Although no mortality was observed in earthworms exposed to 10 mg/kg chlorpyrifos, cocoon numbers per earthworm declined markedly up to 60%. Such a decline can ultimately lead to a marked population decrease. This is in agreement with Neuhauser (1990) who reported that exposure of earthworms to 50 mg/kg carbaryl in soil resulted in a decrease in cocoon numbers by more than 50% and this can result in a significant population decline a within two or three generations. It is known that low levels of toxicants can elicit an overcompensation response (Furst, 1987; Calabrese and Baldwin, 2002; Hackenberger et al., 2008) known as hormesis. In our study a low exposure concentration (1 mg/kg) initiated temporary overadaptation, as shown by an increase in the number of premature, maturing and fully mature stages of spermatogenic morulae in the earthworm P. peguana (Table III). But at higher chlorpyrifos concentrations, the number of spermatogenic morulae decreased significantly. In contrast, chlorpyrifos significantly increased the size of the maturing-morula spermatogenic stages at all concentrations except at the highest concentration (100 mg/kg) because at this concentration all maturing and fully mature stages were non-existent (Table IV). These results indicate a marked toxic effect of chlorpyrifos on the male reproductive system in earthworms. When assessing the toxicity of an insecticide, use of a suite of biochemical and histological biomarkers can provide a comprehensive understanding of its toxicity profile to an organism. The different biomarkers tested in this study showed different response profiles in earthworms exposed to chlorpyrifos. The cellular (NRRT) and biochemical (AChE activity) biomarkers were several-fold more sensitive, even
EARTHWORM CHLORPYRIFOS TOXICITY
9
at the lowest chlorpyrifos exposure concentration (0.1 mg/ kg) at 7 days, than histology and reproductive parameters. Choosing the most sensitive and ecologically relevant biomarker is important for accurate environmental risk assessment of chemicals.
De Silva PMCS, Pathiratne A, Van Gestel CAM. 2010. Toxicity of chlorpyrifos, carbofuran, mancozeb and their formulations to the tropical earthworm Perionyx excavates. Appl Soil Ecol 44: 56–60.
CONCLUSIONS
Eamkamon T, Klinbunga S, Thirakhupt K, Menasveta P, Puanglarp N. 2012. Acute toxicity of neurotoxicity of chlorpyrifos in black tiger shrimp, Penaeus monodon. Environ Asia 5: 26–31.
Pheretima peguana exposure to sublethal concentrations of chlorpyrifos induced significant changes in all the biomarkers studied but to different degrees. Cocoon production and viability and histomorphology of spermatogenic cells are useful biomarkers in the earthworm, but NRRT and AChE enzyme activity are more sensitive in detecting exposure to chlorpyrifos even at low dose and with short exposure periods. Although NRRT and AChE activity were the most sensitive of the biomarkers, cocoon production and cocoon viability could still be considered as diagnostic tools for monitoring low-dose long-term chlorpyrifos toxicity and evaluating population effects.
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