Distribution, fate and histopathological effects of ethion insecticide on selected organs of the crayfish, Procambarus clarkii

Food and Chemical Toxicology 52 (2013) 42–52

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Distribution, fate and histopathological effects of ethion insecticide on selected organs of the crayfish, Procambarus clarkii Mahmoud M.A. Desouky a,b,⇑, Hassan Abdel-Gawad c, Bahira Hegazi c a

Department of Zoology, Faculty of Science, Zagazig University, Egypt Department of Biology, Faculty of Science, Ha’il University, Saudi Arabia c Applied Organic Chemistry Department, National Research Centre, Dokki, Cairo, Egypt b

a r t i c l e

i n f o

Article history: Received 10 March 2012 Accepted 23 October 2012 Available online 2 November 2012 Keywords: Procambarus clarkii 14 C-Ethion Bioaccumulation Histopathology Hepatopancreas Gills

a b s t r a c t This study aims to investigate the fate and histopathological effects of ethion on selected organs of the crayfish, Procamabrus clarkii. Crayfish were exposed to 1 mg l1 14C-ethion and the concentrations of ethion and its possible degradation products were measured in water and different organs of the crayfish over both the exposure and recovery periods. Chromatographic analysis revealed that ethion was degraded into ethion monooxon, ethion dioxon, O,O-diethyl phosphorothioate, O-ethyl phosphorothioate and one unknown compound. At the end of exposure period, ethion was accumulated in different organs of the crayfish especially in the hepatopancreas and gills. Following the transfer of crayfish to clean water for seven days, the concentration of insecticide residues were decreased in both the hepatopancreas and gills suggesting that these organs play an important role in elimination of ethion. On the other hand, the exposure of the crayfish to 1=4 96 h-LC50 (0.36 mg l1) of ethion caused extensive ultrastructural alterations to both hepatopancreas and gill epithelial cells. In the hepatopancreas, the most notable pathological features included vacuolation, degradation and distinct cell lysis. In the gill epithelium, the histopathological alterations included infiltration of hemocytes, cytoplasmic vacuolation and a decrease in the number of basal plasma membrane infoldings. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Environmental pollution is an undesirable spinoff of human activities and represents a problem of repeated occurrences (Dowd et al., 1985). Indubitably, pesticides attract public concern due to their potential transport from one environmental compartment to another and their effects on non-target biota (Klassen, 1986). In Egypt, it is common to observe the intensive use of insecticides in agricultural areas. The application of these contaminants near an aquatic environment may cause hazards to the organisms inhabiting those areas including non-target organisms such as fish, crab, and shrimp (Tronczynski, 1990). Organophosphorus insecticides are used in large quantities and repeatedly because of their rapid breakdown in the environment. The effects of these contaminants on aquatic crustaceans include a widespread disturbance in general physiological processes (Chang et al., 2006) and histopathological changes in a number of different organs (Heiba, 1999). Ethion (O,O,O0 ,O0 -tetraethyl-S,S0 -methylene-bis phosphorodithioate)) is one of the widely used organophosphorus insecticides (OPIs), which have been identified as contaminants in many components of the global ecosystem. It is used for control of aphids, spiders ⇑ Corresponding author. Tel.: +966 507573909. E-mail address: [email protected] (M.M.A. Desouky). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.10.029

mites and insects on a wide variety of food, fiber, and crops (Heiba, 1999; Abdel-Gawad et al., 2011). A high concentration (0.4– 1.0 mg l1) of this OPI is environmentally relevant in some Egyptian freshwater environment (Nile Basin Initiative, 2005). The red swamp crayfish, Procambarus clarkii (Girard, 1852) is an autochthonous species from the Northeast of Mexico and South Central USA. It was introduced worldwide and has become the dominant freshwater crayfish in almost all areas where it lives. It was introduced to River Nile, Egypt through a commercial aquaculture in Giza (Manial-Shiha), in the early 1980’s (Fishar, 2006). They greatly spread all over the River Nile and its tributaries. P. clarkii is a hardy warm water freshwater crayfish that is typically found in marshes, rivers, slow flowing water, reservoirs, irrigation systems, and rice fields. It has the ability to tolerate extreme and polluted environments, and has been used as an indicator of metal pollution in numerous studies of aquatic environments (Schilderman et al.,1999; Serrano et al., 2000). It is not known if ethion is bioaccumulated in crayfish tissues or metabolized to less toxic components. This prompted our investigation of the possibility of ethion accumulation and metabolism by crayfish tissues. The aims of this study were to (a) determine the levels of ethion residues in water and different organs of P. clarkii, (b) identify the degradation products of the insecticide in water and the organs of the investigated species, and, (c)

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elucidate the histopathological effects of ethion on the hepatopancreas and gills using both light and electron microscopic examination.

S C H2

O

H3C

C H2

O

2.1. Chemicals

S P

C H2

S

Adult male red swamp crayfish, P. clarkii, weighing 25.7 ± 4.8 g were collected from rice fields (water temperature, 25 °C; pH 7.4) from Abbasa, Sharkia Governorate, Egypt and transferred to the laboratory. They were kept (30 animals per each aquarium) in three rectangular glass aquaria (45  70  35 cm) containing 4 cm deep static aged tap-water. They were allowed to acclimate for ten days before the beginning of the experiment. The conditions were maintained at 25 °C and a 12:12 h light–dark regime. Water was changed every second day and crayfish were fed with beef liver prior to the water change. Only four individuals died during transport and acclimation.

2.3.1. Acute toxicity of ethion In order to establish crayfish tolerance limits to ethion, a preliminary short-term lethal concentration (LC50) toxicity test was carried out according to the methods described by the American Public Health Association et al. (1985). Probit method was used to calculate LC50. Adult crayfish were kept in glass aquaria (40  60  30 cm) containing different concentrations of unlabeled ethion solution (0, 0.1, 0.2, 0.4, 0.6, and 0.8 mg l1). Each aquarium contained ten animals, and the test solution was renewed daily. Exposure conditions were as described in acclimation period. Triplicate determinations were run for each test solution with a total number of 30 crayfish (10 per replicate) for each test solution. Water temperature was maintained at 25 °C. Observations were made at 24-h intervals up to 96 h. Death was assumed when crayfish were immobile and showed no response when touched with a glass rod.

* 4 C2H5OH

+

P2S5

S

OC2H5

HS P

2

OC2H5 Et3N

CH2Cl2 S

O

* C CH3 H2

O

* C H2

P H3C

* C H2

O

H3C

* C H2

O

S P

C H2

S

S

Fig. 1. Pathway of

14

C-ethion synthesis.

CH3

C H2

O

C CH3 H2

S

O

C H2

O

O

C C H3 H2

O

C H2

O P H3C H3C

C H2

O

C H2

O

S P S

C H2

C H2

O

H3C

C H2

O

O

H3C

C H2

O

OH

H3C

C H2

O

S P

C H3

(II)

P H3C

C H3

(I)

C H2

S

C H3

(III) OH

S P

P HO

S (IV )

C2H5

2.3. Methods

O

S

14

2.2. Crayfish

C C H3 H2

P H3C

2. Materials and methods

C-ethion labeled at carbon atom of ethyl groups (Fig. 1) was prepared using 14 C-ethanol (Sp. Act. 37 MBq, Amersham, UK) according to Abdel-Gawad et al. (2011). The 14C-ethion had a specific activity 0.19 mCi/g or 7.4 MBq/g and the radiometric purity was greater than 98%. The non-labeled insecticide and some of its degradation products were prepared for identification purposes, specifically ethion (I), ethion monooxon (II), ethion dioxon (III), O,O-diethyl phosphorothioate (IV), O-ethyl phosphorothioate (V) and O,O-diethyl S-hydroxymethyl phosphorodithioate (VI). The structures of these compounds are shown in Fig. 2 and the possible pathway for the degradation route of 14C-ethion in water and crayfish tissues is shown in Fig. 3. Degradation routes include oxidation to ethion monooxon and ethion dioxon, and hydrolysis to O,O-diethyl phosphorothioate (IV) and O-ethyl phosphorothioate (V). Hydrolysis of ethion monooxon leads to the formation of O,O-diethyl S-hydroxymethyl phosphorodithioate (VI).

O

O

C H2

C H3

(V ) O

S

C H2O H

P C2H5

S

O (V I)

Fig. 2. Main degradation products of ethion; Ethion (I), ethion monooxon (II), ethion dioxon (III), O,O-diethyl phosphorothioate (IV), O-ethyl phosphorothioate (V) and O,O-diethyl S-hydroxymethyl phosphorodithioate (VI).

2.3.2. Exposure experiment The exposure studies were carried out in October, 2010 at the Radioisotope Department, Atomic Energy Authority, Egypt. Thirty individuals of the test crayfish were exposed to either clean water (control) or added 1 mg l1 14C-ethion for 8 days in rectangular glass aquaria containing insecticide solution. Exposure conditions were as described in acclimation period. Water was changed and redosed with the labeled insecticide every second day. The animals were then transferred into clean water for further 7 days as a recovery period. During depuration period, water was changed every three days but the used water was collected for radioactivity assay at the end of the depuration period.

2.3.3. Sample collection Three water samples were collected each second day of the exposure period and at the end of depuration period. Three crayfish were removed from the test solutions at five and eight days of exposure as well as at the end of depuration period. Animals were sedated in ice-cold water containing 1% chloroform for 20 min. Three replica of selected organs (hepatopancreas, gills, abdominal muscle and carapace) were quickly removed, weighed, and stored at 20 °C till analysis.

2.3.4. Extraction of ethion residues Ethion residues were extracted according to the method of Abou-Arab et al. (1995) Two hundred ml of water (pH 8) were successively extracted with dichloromethane (3  50 ml), chloroform (50 ml) and hexane (50 ml). The organic phases were combined, dried using anhydrous sodium sulfate and concentrated by using a rotary evaporator. The conjugated products in the aqueous phases were acidified with 2 M HCl, then extracted with chloroform and identified by thin layer chromatography (TLC).

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OC2H5

S P C2H5

O

S

C H2

S

(I)

P C2H5

O

OC2H5

S

oxidation

hydrolysis

OC2H5

O P C2H5

O

S P

C2H5

C H2

OC2H5

S

(II)

S

O

hydrolysis hydrolysis

oxidation

OC2H5

O

C2H5

O

P C2H5

O

S P

C2H5

O

S OC H 2 5 O PH OC 2H5 (IV)

C H2

S

S P

OC2H5

C2H5

O

O (III)

C H2

S OH

OH

O PH

S (VI) Further metabolism, conjugation

(V)

OC2H5

Fig. 3. Possible degradation route of ethion in water and tissues of crayfish.

Selected crayfish organs (hepatopancreas, gills, abdominal muscle and carapace) were homogenized by mechanical tearing with acetonitrile and then diluted with water. Ethion residues were extracted into petroleum ether three times; the organic layer was dried using anhydrous sodium sulfate, evaporated using a rotary evaporator and radioassayed. The non-extractable tissues were air dried and digested using 1 ml Solusol (tissue and gel solubilizer), 1 ml 30% H2O2 and 70 ll glacial acetic at 40–50 °C. 2.3.5. Isolation and characterization of 14C-residues Analyses of radioactive extracts of water samples and different crayfish organs were carried out using TLC on silica gel plates (Merck-silica gel 60 F254). The solvent systems used were. System 1: toluene: xylene System 2: dioxane: xylene: petroleum ether System 3: n-hexane: ethyl acetate

20:20 10:20:20 98:2

(v/v) (v/v/v) (v/v)

three crayfish were freshly dissected out after one, three, five and seven days of exposure and then fixed for three h in 2% glutaraldehyde in 0.1 M phosphate buffer. Post-fixation was completed in 1% OsO4 using the same buffer for 60 min. on a rotating plate at room temperature. Samples were rinsed in buffer, and then dehydrated in ascending ethanol solutions (70%, 80%, 95%, and 100%). Samples were placed on a rotating plate for a total of 30 min. for each progressive ethanol solution. Samples were cleared in acetone, placed in a 1:1 mixture of epon:araldite in ethanol (100%) for 1 h, then in a 1:3 mixture of epon: araldite in acetone for 1 h, and finally placed in 100% epon: araldite and polymerization was completed at 60 °C for 24 h. Semi-thin (1 lm) and ultra-thin (70–80 nm) sections were cut with glass and diamond ultramicrotome knives, respectively. The semi-thin sections were stained with 0.5% toluidine blue for light microscopy (LM). The ultra-thin sections were contrasted with uranyl acetate, lead citrate stains, and examined using a JOEL JTM.1200 EXII transmission electron microscope (TEM) at Ain Shams University, Cairo, Egypt.

3. Results The conjugated metabolites in aqueous layers were liberated by heating the mixture with 2 M HCl for 2 h at 100 °C. After cooling, the mixture was extracted three times with chloroform. The combined chloroform was dried over anhydrous sodium sulfate, filtered, evaporated under vacuum and identified by TLC analysis. The parent and derivatives standards used for calibration were run alongside as reference and spots were visualized using a UV-lamp at 254 nm. After a preliminary spray with PdCl2 solution, the plates were subjected to I2 vapor for detection of the compound by color. Rf values were determined after UV visualization. 2.3.6. Radioactivity assay The radioactivity of water samples and tissues during different times of exposure as well as at the end of the recovery period, were determined using a Packard liquid-scintillation spectrometer (Model TRI-CARB 2300 TR) in vials containing a dioxane-based scintillation cocktail. The radioactivity in crayfish organs (80– 120 mg) was determined by digestion using H2O2 and Solusol followed by liquid scintillation counting for total 14C-residues. Thin layer plates were divided into 1 cm increments, scraped out into vials, covered with scintillator and counted. Radioactivity levels were all corrected for quenching using an internal standard.

3.1. Acute toxicity of ethion When ethion was applied, the initial responses of the crayfish were hyperreactivity to external stimuli, increased aggressiveness, ‘‘body jerk’’ and loss of equilibrium for 15 min. The crayfish activity then became normal. After three days of exposure, the toxicity of ethion was characterized by lethargy and ‘legs up’ position and also by antennae vibrations. All crayfish survived in control group for all exposure times. However, the 24-, 48-, 72- and 96 h, LC50 values and their 95% confidence limits in mg l1 of ethion for

Table 1 Total 14C-ethion residuesa in water samples (n = 3 ± SD) during the experimental period (no radioactivity was detected in the control group). Days

2.3.7. Statistical analysis Statistical significance at (P < 0.05) for each variable was tested using factorial analysis of variance. 2.3.8. Light and electron microscopy For this purpose, crayfish were exposed to either clean water or 1=4 96 h-LC50 of unlabeled ethion for seven days following the same protocol as for exposure experiment. At different exposure periods, small pieces of the hepatopancreas and gills of

Exposure period

Depuration period A

2 4 6 8 15

14

C-ethion residues in water (mg l1)

Total (mg l1)

Organic layer (mg l1)

0.746 ± 0.035 0.686 ± 0.023 0.786 ± 0.025 0.732 ± 0.016 0.356 ± 0.030

0.696 ± 0.042 0.611 ± 0.031 0.714 ± 0.026 0.625 ± 0.019 0.225 ± 0.016

Detection limit = 23 dpm or 14 lg l1 of ethion.

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Table 2 Concentration (n = 3 ± SD) and Rf values of 14C-ethion and its degradation products in the organic layer of water samples at the end of both exposure and recovery periods (no radioactivity was detected in the control group). Metabolites

Ethion (I) Ethion monooxon (II) Ethion dioxon (III) O,O-diethyl phosphorothioate (IV) O-ethyl phosphorothioate (V) Unknown a b

Rf values in various solvent systemsa

Concentration of ethion residues (mg l1)

1

2

3

At the end of exposure period

At the end of recovery period

0.75 0.88 0.69 0.33 0.24 0.1

0.80 0.92 0.56 0.23 0.50 0.15

0.75 0.65 0.55 0.42 0.71 0.0

0.0795 ± 0.0073 0.048 ± 0.0041 n.db 0.3195 ± 0.0251 0.0315 ± 0.0022 0.0645 ± 0.0054

0.024 ± 0.0018 0.0975 ± 0.0082 0.0975 ± 0.0082 0.024 ± 0.0088 n.d 0.147 ± 0.0137

1, 2 and 3: solvent systems (see materials and methods). n.d: not detected, detection limit = 23 dpm or 14 lg l1 of ethion.

P. clarkii were 3.831 (0.8561, 1.122), 2.812 (0.93261, 1.1132), 2.2697 (0.5321, 0.4233), and 1.4555 (0.2442, 0.4820), respectively. These values are obtained by extrapolation. 3.2. Distribution of ethion residues in water and tissues Table 1 shows the total amount of 14C-residues recorded at different times in water samples dosed with 14C-ethion. After 48 h of application of the insecticide, total 14C-residues ranged between 0.686–0.786 mg l1. Upon transfer of the animals to clean water for seven days, the total 14C-residues decreased to 0.356 mg l1. Table 2 shows the Rf values and the average concentrations of 14 C-ethion and its degraded products residues in water samples at the end of both exposure and recovery periods. In addition to the parent compound, ethion monooxon (II), ethion dioxon (III), O,O-diethyl phosphorothioate (IV), O-ethyl phosphorothioate (V) and one unknown compound were found as free metabolites. However, O,O-diethyl S-hydroxymethyl phosphorodithioate (VI) was found in water samples as a conjugated metabolite and was liberated by acid hydrolysis in the aqueous layer with 2 M HCl at 100 °C. Its concentration was 0.059 and 0.061 mg l1 at the end of exposure and depuration periods, respectively. The distribution of 14C-ethion residues (as total 14C activity) in selected crayfish organs during exposure and recovery periods are shown in Table 3 and Fig. 4. The results show that upon exposure to ethion for eight days, the rank of crayfish organs according to their ability to accumulate ethion residues was hepatopancreas > carapace > gills > muscles. The highest amounts of ethion residues were retained by hepatopancreas and gills after five days of exposure (0.57 and 0.36 lg g1, respectively). Thereafter, tissue concentration of ethion increased significantly (P < 0.05) to 0.89 and 0.53 lg g1 after eight days of exposure. The behavior of ethion accumulation in the muscle followed the same trend. Interestingly, only about 0.1 lg g1 of ethion was accumulated in the carapace after five days of exposure, but after eight days a high amount (0.58 lg g1) of ethion was accumulated. Following the transfer of crayfish to clean water for seven days, 34% of ethion residues was lost from the hepatopancreas. The percentage loss of ethion residues from the other tissues was lower ranging from 7.6% to 11.5%. The average concentrations of 14C-ethion and its degraded products residues in different crayfish organs at the end of both exposure and recovery periods are shown in Fig. 5. These data show that upon exposure of crayfish to ethion for eight days, both ethion and ethion monooxon (II) were found mainly in the hepatopancreas and gills (0.101 and 0.194 lg g1, respectively). A moderate amount of other degradation products were recorded in the hepatopancreas, gills and carapace. A high level of unidentified labeled metabolite was observed mainly in hepatopancreas and gills (0.265 and 0.178 lg g1, respectively). Upon transfer of crayfish to clean water for seven days, about 45% of ethion and its degraded

products were depurated from the hepatopancreas. However, a great proportion of ethion monooxon (II) was found in the hepatopancreas while O,O-diethyl phosphorothioate (IV) and an unidentified fraction were deposited into the carapace (0.24 and 0.23 lg g1, respectively) at the end of the recovery period. 3.3. Histopathological effect of ethion on the hepatopancreas 3.3.1. Normal hepatopancreas The hepatopancreas consists of numerous blind ended tubules separated by connective tissue (Plate 1A). The lumina of the tubules are lined with simple epithelial cells which can easily be recognized as three main types: absorptive (R) cells, fibrillar (F) cells and secretory (B) cells. The absorptive (R) cell is the most numerous cell type which occurs along the whole length of the tubules except at the distal tip. These cells are characterized by the presence of lipid material (Plate 1B). The nucleus is centrally or basally located and the cytoplasm contains a number of mitochondria, some of which are concentrated below the apical plasma membrane (Plate 2A). Apically, the cell resembles a vertebrate intestinal absorptive epithelium with a dense brush border and an organelle-free region lying below the microvilli (Plate 2A). A straight filament extends downwards from each microvillus into the apical cytoplasm. Rough endoplasmic reticulum and Golgi complex occur throughout the cell (Plate 2B). Fibrillar (F) cells are smaller than R cells. The nucleus is normally oval, but may be lobed and has a prominent nucleolus (Plate 1C). The cytoplasm has an extensive rough endoplasmic reticulum with long, narrow ribosome studded cisternae (Plate 2C). This gives the cell a fibrillar appearance in the light micrographs (Plate 1B). A distinctive feature of some F cells is the presence of a supranuclear body containing electron-dense merged granules, small vesicles and membranes (Plate 2D). The secretory (B) cells are concentrated mainly in the distal region of the tubules. B cell is the largest cell type, and is characterized by the presence of a large oval vacuole which may occupy about 80% of the cell volume (Plate 1C). The presence of the vacuole, which contains electron-lucent and flocculent material, forces the cytoplasm into a thin layer around the periphery of the cell. The nucleus is basal and compressed but the surrounding cytoplasm is similar in appearance to that of an F cell, having granular endoplasmic reticulum (Plate 2E). The apex of the cell has microvilli, but the underlying cytoplasm is extremely vacuolar and electron-dense. 3.3.2. Ethion-treated hepatopancreas Both light and electron microscopic examinations showed many cytological alterations of the hepatopancreatic tissues of crayfish exposed to 1=4 96 h-LC50 of ethion. Upon 24 h of exposure, the lumina of the hepatopancreas had irregular or branched shape

G

0.49 ± 0.01 0.45 ± 0.13 (91.8%) 0.03 ± 0.005 (6.1%) (97.9%)

C

0.52 ± 0.021 0.47 ± 0.16 (90.4%) 0.03 ± 0.015 (5.8%) (96.2%)

c

b

Total recovery

Non extractable

Results are expressed as mean ± SD for three determinations for each sample. The total recovery % = extractable + non-extractable residues/total 14C-activity  100. M: muscles, C: carapace, H: hepatopancreas, G: gills. b

a

H

0.55 ± 0.021 0.48 ± 0.32 (87.2%) 0.023 ± 0.003 (4.2%) (91.4%) 0.26 ± 0.021 0.23 ± 0.09 (88.5%) 0.02 ± 0.015 (7.6%) (91.1%)

M G

0.53 ± 0.031 0.47 ± 0.07 (88.6%) 0.03 ± 0.02 (5.6%) (94.2%) 0.58 ± 0.036 0.56 ± 0.10 (96.5%) 0.013 ± 0.006 (2.24%) (98.7%)

C H

Fig. 4. Total 14C-ethion and its degradative products, n = 3 ± SD (lg g1) in different organs of the crayfish during exposure and recovery periods. ⁄ Significantly different (p < 0.05) from day 5 of exposure in case of day 8 and from day 8 of exposure in case of recovery period.

0.89 ± 0.017 0.86 ± 0.24 (96.6%) 0.022 ± 0.007 (2.47%) (99.1%) 0.15 ± 0.016 0.13 ± 0.03 (86.6%) 0.009 ± 0.01 (6.0%) (86.1%) 0.36 ± 0.015 0.31 ± 0.07 (86.1%) 0.03 ± 0.02 (8.3%) (94.4%) 0.095 ± 0.015 0.080 ± 0.03 (84.2%) 0.008 ± 0.004 (8.4%) (92.6%)

M G C H

c

0.570 ± 0.02 0.530 ± 0.18 (92%) 0.013 ± 0.01 (2.28%) (95.18%) Total14C-activity Extractable

At the end of recovery period 8 days C-ethion residues (lg g1 wet weight)a 14

5 days

C-ethion residues in different crayfish organs at different times of exposure period and at the end of recovery period (no radioactivity was detected in the control group).

Average Fraction

14

Table 3 Distribution of

0.23 ± 0.025 0.21 ± 0.06 (91.3%) 0.01 ± 0.015 (4.4%) (91.8%)

M.M.A. Desouky et al. / Food and Chemical Toxicology 52 (2013) 42–52

M

46

and tubules were widely separated by loose connective tissue (Plate 1D). Slight signs of cell degeneration were also detected in the form of slight cytoplasmic vacuolation (Plate 2F) and degeneration of the microvilli in some cells (Plate 1D). The yellow granular materials-containing vacuoles (lipid vacuoles) were reduced in size and number (Plate 1D). After three days of exposure, the damage of the tubules became more pronounced. The tubules became narrower, irregular and vacuolation greatly increased (Plate 1E). Although the cytoplasm of most F cells possessed an extensive RER, the cisternae became dilated and had disorganized arrangement (Plate 2G). Moreover, the nuclei of R cells presented a paler appearance and showed an irregular nuclear envelope. Heterochromatin appeared as dense masses scattered in the nucleoplasm and as electron dense aggregates along the inner nuclear envelope (Plate 2H). After five days of exposure, numerous areas of lyses throughout the cytoplasm gave a lucent appearance to some cells (Plate 1E). However, this phenomenon was confined to individual tubules and did not affect the entire hepatopancreas, so some intact glandular epithelium was present. Distinct alterations of the tissue structure could be best discerned in the hepatopancreas after seven days of exposure. Generally, degeneration of the hepatopancreatic epithelium was noted and it showed distinct cellular destruction. The cells exhibited a general loss of cytoplasmic density and were frequently characterized by the presence of damaged areas throughout the cytoplasm. Many vacuoles containing cytoplasmic debris were also being observed (Plate 1F). Furthermore, the degeneration of the microvilli border became more pronounced and the mitochondria lost their identity (Plate 2I). Again, this phenomenon was confined to individual tubules and did not affect the entire hepatopancreas.

3.4. Histopathological effect of ethion on the gills 3.4.1. Normal gill Light micrographs of untreated (control) gill lamella are shown in Plate 3A, B and C. Generally, the gills of control crayfish showed uniform arrangement of lamellae with uniform intralamellar spaces (Plate 3A). No structural abnormalities or abnormal gill lesions were observed in the gills of control crayfish. The gill lamellae are covered with a thick cuticula which is underlain by a single epithelial layer. In contact with the cuticula is a thin sheet of cytoplasm. From here the cells narrow towards the intralamellar space extending later to contain the nucleus (Plate 3B and C). Under TEM, the cells appear oval and with somewhat irregular nuclei. The

M.M.A. Desouky et al. / Food and Chemical Toxicology 52 (2013) 42–52

47

Fig. 5. Concentrations of 14C-ethion and its degradative products, n = 3 ± SD (lg g1) in different organs of the crayfish at the end of both exposure and recovery periods (C: carapace, G: gills, H: hepatopancreas, M: muscles).

cytoplasm contains elongated mitochondria and RER (Plate 4A). The apical side of the epithelial cell is covered with irregular microvilli. The irregularity of the frequently flattened microvilli is apparent in sections cut almost parallel to the epithelium surface. During fixation and embedding, microvilli are frequently torn from the epithelial cells (Plate 4B). The basal plasma membrane invaginates into the cytoplasm forming a dense membrane system which is in close contact with numerous mitochondria (Plate 4C). 3.4.2. Ethion-treated gills Gills of crayfish following 24-h exposure to 1=4 96 h-LC50 of ethion exhibited hemocytic infiltration in the hemocoelic space and swelling of the gill lamellae (Plate 3E). The tips of the gill lamellae appeared abnormal with peculiar malformations and hyperplasia resulted in the formation of clavate lamellae (Plate 3D). After three days of exposure, the epithelial cells of the gill filaments were disorganized. After five days of exposure, the damage increased where the cells became detached from the cuticula (Plate 3F and G). Examination of TEM ultrathin sections revealed many pathological changes in the gill epithelial cells of crayfish exposed to 1=4 96 hLC50 of ethion. After 24-h of exposure, electron-dense granules were found on the outer surface of the gill epithelia (Plate 4D). The exposure of the crayfish to ethion for three days caused furthermore ultrastructural alteration to the gill epithelial cells, including a decrease in the number of basal plasma membrane infoldings (and associated mitochondria) and cytoplasmic vacuolation. Moreover, the nuclei became hypotrophic and their heterochromatin became marginal (Plate 4E). After five days of exposure to ethion, the cells became severely degraded where many vacuoles-containing cytoplasmic debris were also observed throughout the cytoplasm (Plate 4F).

analysis of the extracts of water and different crayfish organs revealed the presence of ethion, ethion monooxon, ethion dioxon, O,O-diethyl phosphorothioate, O-ethyl phosphorothioate and one unknown compound. About 25% of the total nominal 14C-ethion residues were lost from the water during the exposure periods. According to USEPA (1994), volatility is not an important route of ethion dissipation. Therefore, this loss may be attributed to hydrolysis, oxidation, and photochemical degradation of the insecticide (Zhang and Pehkone, 2002). It may be also due to uptake of ethion by the crayfish. The metabolism of 14C-ethion in crayfish is similar to the pathways proposed for the rat, which are oxidative demethylation, hydroxylation and oxidation (Bhatti et al., 2010). While the basic features of ethion metabolism are known, detailed information is lacking. Like other organothiophosphate insecticides (chlorpyrifos, parathion), ethion may be converted in the liver via desulfuration by cytochrome P-450 enzymes to its active oxygen analog, ethion monooxon (Rao and McKinley, 1969). Our results demonstrated that ethion was metabolized in the crayfish into ethion monooxon (II) and ethion dioxon (III) by oxidation, and to O,O-diethyl phosphorothioate (IV) and O-ethyl phosphorothioate (V) by hydrolysis. Hydrolysis of ethion monooxon leads to the formation of O,Odiethyl S-hydroxymethyl phosphorodithioate (VI) which may be conjugated with glucuronic acid in animals (Gotoh et al., 2001). It is not known whether ethion monooxon can then be desulfurated to ethion dioxon. Ethion monooxon is a potent inhibitor of cholinesterases and exerts toxicity by reacting with an inhibiting neural acetylcholinesterase (Dewan et al., 2008). Abdel-Gawad et al. (2011) studied the distribution and elimination of 14C-ethion insecticide in chamomile flowers and oil. They revealed the presence of the parent compound together with four metabolites, which were identified as ethion monooxon, ethion dioxon, O,Odiethyl phosphorothioate and O-ethyl phosphorothioate.

4. Discussion 4.1. Chemistry of ethion

4.2. Accumulation and distribution of ethion in different organs of crayfish

Organophosphorous insecticides (OPIs) are widely used in Egypt and represent a potential threat to freshwater biota (Mansour et al., 2001). These compounds are quickly degraded in aquatic environments where the alkaline water accelerates their degradation (Saad et al., 1982). The present chromatographic

The measurement of distribution of 14C-ethion residues in different organs of the crayfish revealed that ethion was progressively accumulated over the exposure period. The hepatopancreas and gills account for most of the insecticide accumulation. Following transfer of animals to clean water for seven days, about 45% of

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Plate 1. Light micrographs of the hepatopancreatic tubules of crayfish stained with toluidine blue. (A): T.S. of control tubules with normal structures of the tubules and intertubular connective tissue (ct) (600). F, F-cell; R, R-cell. (B): L.S. of the control tubules showing different types of hepatopancreatic cells (900). F, F-cell; l, lipid granules; lu, lumen; R, R-cell. (C): T.S. of the control tubules showing different types of hepatopancreatic cells. Note the large vacuole (lv) which occupies most of B-cell (900). B, Bcell; F, F-cell; l, lipid granules; lu, lumen; R, R-cell. (D): T.S. of tubules after 24 h of exposure to 1=4 LC50 of ethion showing vacuolation (v) and necrosis of microvilli in some cells (arrows) (600). lu, lumen. (E): T.S. of tubules after three days of exposure showing greatly increased vacuolation (v), decrease of lipid granules (l) of R-cells (R) and narrowing of the lumen (lu). Note the presence of cytoplasmic lysis in some cells (c. ly) (600). (F) T.S. of tubules after seven days of exposure showing distinct cellular destruction (cd) and lysis of the cytoplasm. Note the presence of cell debris (cdb) (1200).

ethion and some of its degraded products were lost from the hepatopancreas and gills. This means that the hepatopancreas and gills may play an important role in elimination of ethion in crayfish. Similarly, Tomza-Marciniak and Witczak (2009) found that, OPI, DDT and its metabolites accumulate mainly in the hepatopancreas and gonads, while in the gills and muscles their content was several fold lower in crayfish. It is possible that ethion is stored in the hepatopancreas/digestive glands of these species because of the presence of specific detoxification or elimination entities including the relatively high perfusion rate, coupled with the presence of biotransformation enzymes responsible for the higher observed concentration and clearance. 4.3. Toxicity and histopathology of ethion Whilst the concentration of ethion used in the present study seems relatively high, it is relevant in some Egyptian freshwater

environments (Nile Basin Initiative, 2005). Moreover, for radioactivity assay, a higher dose of labeled ethion should be used to obtain a reasonable count. P. clarkii was found to have a high resistance to the toxic effects of both heavy metals (Del Ramo et al., 1987) and OPIs (Heiba, 1999). However, the present study revealed that ethion is highly toxic to crayfish compared to other OPIs such as diazinon (96-h LC50, 4.38 mg l1, Heiba, 1999). In fact, Organophosphate insecticide toxicity is due to inhibition of acetylcholinesterase (AChE) at cholinergic junctions of the nervous system. AChE hydrolyzes the neurotransmitter acetylcholine (ACh). Inhibition of AChE results in a buildup of ACh at nerve synapses and overstimulation at ACh receptors. (National Research Council, BEST, 1999; Chang et al., 2006). OPIs are also capable of inducing apoptosis by induction of multifunctional pathways (Carlson et al., 2000). In crustacean, there is a correlation between the physiological condition of the organism and the structure and appearance of

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Plate 2. Electron micrographs of the hepatopancreatic cells of crayfish. (A): Proximal region of R-cell of control crayfish loaded with lipid granules (l). Note the dense microvilli (mv), an organelle-free region (of) lying below the microvilli and the large number of mitochondria (M) (5000). (B) Distal region of R-cell of control crayfish with spherical nucleus (N), Golgi complex (GC), large mitochondria (M) and rough endoplasmic reticulum (RER) (8600). (C): F-cell of control crayfish with lobed nucleus (N) and an extensive rough endoplasmic reticulum (RER) that gives the cell its fibrillar appearance (4500). (D): F-cell of control crayfish containing supranuclear body (snb) that contains an electron-dense conglomeration of granules, small vesicles and membranes (4500). RER, rough endoplasmic reticulum. (E): B cell of control crayfish with a large vacuole of different constitutes which forces the cytoplasm into a thin layer around the periphery of the cell (5500). (F): F-cell after 24 h of exposure to 1=4 LC50 of ethion. Note the slight cytoplasmic vacuolations (v) (6000). N, nucleus; RER, rough endoplasmic reticulum. (G): F-cell after three days of exposure. Note that the vacuolations (v) increased and the cisternae of RER become dilated and had disorganized arrangement (dRER) (5500). N, nucleus. (H): R-cell after three days of exposure showing irregular nuclear envelope, the nuclei present a paler appearance and the heterochromatin appeared as dense masses scattered in the nucleoplasm (5000). N, nucleus. (I): Hepatopancreatic cells after seven days of exposure showing dramatic cell lysis, necrosis of brush border (dbb) and the mitochondria (M) had lost their identity. Note also the presence of cell debris (8500).

hepatopancreas (Popescu-Marinescu et al., 1997). The organ undergoes histological and histochemical modifications in response to different physiological demands (e.g., moulting,

reproduction, Al-Mohanna and Nott, 1989; Sousa and Petriella, 2001) and environmental change (e.g., physicochemical factors, pollution, Abdelmeguid et al., 2009; Woodburn et al., 2011). The

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Plate 3. Light micrographs of the gill lamellae of crayfish, stained with toluidine blue; (A): L.S. of normal (control) gill lamellae (gl) showed uniform arrangement of lamellae with uniform intralamellar spaces (is) (280). cu, cuticula. (B): L.S. of normal gill lamellae showed uniform arrangement of gill epithelial cells (ec) (600). cu, cuticula. (C): T.S. of normal uniform gill lamella with uniform intralamellar spaces (is) (280). ec, epithelial cell. (D): L.S. of gill lamella of crayfish exposed to 1=4 96 h-LC50 of ethion for 24 h exhibited hemocytic infiltration (hc) in the hemocoelic space and swelling of the gill lamellae (350). (E): L.S. of gill lamella of crayfish exposed to 1=4 96 h-LC50 of ethion for 24 h. Note the aggregation of granular hemocytes (hc) inside the intralamellar space and some degeneration (arrows) in the cuticula. (600) (F): L.S. of gill lamella of crayfish exposed to 1=4 96 h-LC50 of ethion for three days. Note that the epithelial cells become disorganized (dc) and the outer surface of cuticula becomes uneven (600). (G): T.S. of gill lamella of crayfish exposed to 1=4 96 h-LC50 of ethion for five days. Note that most of epithelial cells become detached from the cuticula (350). is, intralamellar space.

fine structural observations of the hepatopancreatic cells of crayfish exposed to 1=4 96 h-LC50 in this study revealed severe pathological changes including vacuolation, degeneration and distinct cellular damage. All these dramatic histopathological alterations, may be responsible for the high toxicity of ethion to the crayfish. Similar histopathological alterations were also reported in the hepatopancreatic tubules of P. clarkii upon exposure to other OPIs including malathion (Garo et al., 1998); diazinon (Heiba, 1999) and fenthion (Aly, 2000). These changes are probably due to accumulation of the insecticides in the cells of hepatopancreas (Jaiswal and Sarojini, 1990) or due to increasing the activity of lysosomal enzymes which are capable of destroying cell organelles (Sharma and Sastry, 1979). Crustacean gills are a vital organ as they play an important role in diffusion and transport of respiratory gases and regulation of osmotic and ionic balance. Since, for most pollutants, uptake from water is the most important route, gills are a primary target organ and may be one of the first organs to exhibit symptoms of sublethal toxicity. OPIs such as fernitrothion and trichlorfon were found

to reduce the oxygen consumption and disrupt the osmoregulatory function of gills of the shrimp (Lignot et al. 1997; Chang et al., 2006). It is likely that factors influencing these physiological processes will also affect gill ultrastructure. The present study demonstrates that extensive ultrastructural changes in the gill epithelium of the investigated crayfish occur upon exposure to sublethal concentration of OPI ethion. The most notable alterations are infiltration of hemocytes, cytoplasmic vacuolation and a decrease in the number of basal plasma membrane infolding (the main place of ion transport) and associated mitochondria. Haemocytes infiltration within the gill lamella of crustaceans following exposure to toxicants has been documented previously (Bubel, 1976; Lawson et al., 1995). Haemocytes may infiltrate the gill cells in order to phagocytose pollutants, or to release sugar from their granules or glycogen stores, for use as an alternative energy source by the cell (Bubel, 1976). According to Lawson et al. (1995) vacuolation may be produced from plasma membranes that may be rendered ‘leaky’ after structural disorientation. Vacuolation of epithelial cells will potentially affect the physiological processes in the gill by

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Plate 4. Electron micrographs of the gill epithelia of crayfish. (A): Gill epithelia of untreated (control) crayfish with thick cuticula (cu), underlay by thin sheet of cytoplasm (cy sh) and irregular nucleus (N) (4000). (B): Gill epithelia of untreated (control) crayfish. Note the presence of thick cuticula (cu) and irregular microvilli (mv). N, nucleus (7500). (C): Gill epithelia of untreated (control) crayfish. Note that the basal plasma membrane invaginates into the cytoplasm forming a dense membrane folding (mf) which is in close contact with numerous mitochondria (M). bm, basement membrane; N, nucleus (4500). (D): Gill epithelia of crayfish exposed to 1=4 96 h-LC50 of ethion for 24 h having electron-dense granules (dg) (4500). N, nucleus. (E): Gill epithelia of crayfish exposed to 1=4 96 h-LC50 of ethion for three days. Note the decrease in the number of basal plasma membrane infolding (mf) and associated mitochondria (m) and cytoplasmic vacuolation (v). N, nucleus (4500). (E): Gill epithelia of crayfish exposed to 1=4 96 h-LC50 of ethion for five days. The cells become severely degraded with many vacuoles containing cytoplasmic debris (cdb) (4500). N, nucleus.

increasing the diffusion distance for respiratory gases and ions. Moreover, a decrease in the number of basal plasma membrane infoldings which is the main site of ion transport may be another factor which disrupts the osmoregulatory function of gills upon exposure to OPIs. A decrease in the number of associated mitochondria would also result in a reduction of available energy for ionic regulation and respiratory gas exchange. The present histological observations revealed important information about ethion effects on another level of biological organization and provides better understanding of the toxicological profile of OPIs in freshwater. The extent of the histopathological damage in the crayfish was in line with the bioaccumulation of ethion

determined in the soft tissues and appeared to be exposure timedependent as evidenced by increasing vacuolization and the tendency of the cells to disintegrate. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements We would like to thank Professor Hedaya Kamel, the Radioisotope Department, Atomic Energy Authority, Egypt, for his

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