Iranian Journal of Fisheries Sciences 13(3) 702- 718 2014

Histopathological effects and toxicity of atrazine herbicide in , frisii kutum, fry

Khoshnood Z. 1*; Jamili Sh. 1; Khodabandeh S.2; Mashinchian Moradi A.1; Motallebi Moghanjoghi A.A.3

Received: June 2012 Accepted: May 2014

Abstract This study aimed to investigate the toxic effects of atrazine herbicide on the fry of Caspian Kutum (Rutilus frisii kutum, Kamensky, 1901). First the 96-h LC50 of the fry were exposed to atrazine at the concentration of 24.95 ppm was determined. Then the toxicity of this herbicide on Caspian kutum fry exposed to the concentration of 12.47ppm (1/2 LC50), for four days was measured and compared with a control group. Comparison of the length, weight and condition factor showed no significant differences between atrazine exposed and control group. The concentration of Na+, K+, Ca2+, Mg2+ and Cl- in the whole body of fry in control and atrazine exposure groups were as the following order: Ca2+>K+> Na +> Cl- >Mg2+ and Ca2+>Na+>K+>Mg2+>Cl-, respectively. Results showed that the concentration of all these ions were higher in atrazine exposure group than control group, except for Cl-, and the only significant differences was found in Na+ concentration. Major histopathological effects of atrazine on the gills were hyperplasia and thickening of the filaments, separation of the pavement cells of the lamellae epithelium from the pillar cells and swelling of the epithelial cells. Results of the present study showed that atrazine could affect the ion composition of the body, and caused major damages in gill epithelium even at sublethal concentration and acute exposure, but had no effects on the growth parameters.

Keywords: Atrazine, Rutilus frisii kutum, Toxicity, Ion, LC50

1-Department of Marine Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. 2-Faculty of Environmental Resources and Marine Sciences, Tarbiat Modares University, Noor, Iran. 3-Iranian Fisheries Researches Organization, Tehran, Iran. *Corresponding author email: [email protected]

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Introduction osmoregulation and stress response Atrazine (2-chloro-4-ethylamino-6- (Bisson and Hontela, 2002). isopropylamino-s-triazine) is a pre- In , gills are vital for respiration emergent herbicide first approved for use and osmoregulatory functions, and in the US in 1958, where it is used respiratory distress is one of the early primarily on corn, sorghum and sugar cane symptoms of pesticide intoxication (Solomon et al., 1996). Atrazine inhibits (Jayachandran and Pugazhendy, 2009). In electron transport in Photosystem II, which recent years considerable histopathological results in a disruption of photosynthesis studies have been conducted on fishes and in turn leads to death from starvation exposed to sublethal concentrations of in broad-leaf plants (Giddings et al., different pesticides and herbicides 2004). (Alazemi et al., 1996; Wood, 2001; Cengiz Herbicides are generally applied in and Unlu, 2006). As a result tissue changes spring or early summer, which often are the functional responses of organisms coincide with the breeding season of many which provide information on the nature of species. Some of these fishes breed in the toxicant. The present study was an aquatic habitats receiving the runoff attempt to investigate the histopathological drained from the cultivation fields. alterations in the gill of Rutilus frisii kutum Atrazine has low volatility, but its fry exposed to atrazine. moderate water solubility (33 mg/L at 25 Early developmental stages of the fish life ◦C) makes it relatively mobile in soil and cycle are considered to be the most aquatic environments, where it tends to sensitive stages to the toxic effects of partition into the water column rather than chemical contaminants (Weis and Weis, sorbing to sediments (Giddings et al., 1987). Short-term sublethal effects on 2004). growth, behavior or osmotic control may Several recent laboratory studies have affect these critical stages and impact shown that environmentally realistic recruitment (Houde, 1987; Sclafani et al., concentrations of atrazine have significant 1997; Alvarez and Fuiman, 2005). For toxic effects on fish. For example, low example, loss of osmotic control altering concentrations of atrazine (1 µg/L) altered water content may influence larval density olfactory-mediated endocrine function in and buoyancy. The vertical position of male Atlantic salmon (Salmo salar) larvae in the water column affects their (Moore and Lower, 2001). At 100 µg/L, patterns of drift and their interactions with atrazine altered the Na+, K+-ATPase preys or predators. Thus, a temporary loss activity in common carp (Cyprinus carpio) of osmotic control in fish larvae may held in fresh water, indicating increase their susceptibility to predation or osmoregulatory disturbances (Hanke et al., impair their feeding abilities (Sclafani et 1983). In addition, in vitro studies in fish al., 1997). Disruption of normal cortisol have shown that atrazine may affect the secretion in early life stages may also secretion of cortisol, involved in affect their survival by reducing the ability to cope with acute stressful situations and

Iranian Journal of Fisheries Sciences 13(3) 2014 704 by inducing adverse secondary effects on range finding test was carried out prior to osmoregulation, growth, development and the definitive test to determine the immune function (Benguira et al., 2002; concentration of the test solution. For the Gravel et al., 2005; Kennedy and Farrell, test, the atrazine (80% WP, Hangzhuou 2005). Ruijiang Chemical Co. Ltd., China) was Caspian kutum is an important dissolved in distilled water, and added to commercial fish species in the the aquarium (20L) following the method in Iran. The sharp decline in its annual of Pluta (1989). In the definitive test, a set catch observed in 1970s and early 1980s of 10 fish specimens were randomly (Ghaninejad and Abdulmaleki, 2007) had exposed to each of the atrazine prompted the Iranian government to concentrations (viz. 20, 22, 24, 28, and 30 launch its restocking project in 1984. mgL-1) and the experiment was set in This study is the first attempt to to triplicate to obtain the LC50 value of the assess the toxicity of a commercial herbicide for the species. The LC50 value formulation of the herbicide atrazine on of test chemical in R. frisii kutum was some biochemical indices, of Caspian determined by Probit analysis method kutum, a commercially and economically (Finney, 1971) for 12, 24, 48, 72 and 96 important species of cyprinid fishes in hours Based on the 96h LC50 value, one northern Iran. The information obtained sublethal test concentration of atrazine was may be useful for the management and determined and the fish specimens were monitoring of atrazine contamination in exposed to this concentration for the the environment. assessment of its toxic effects on the osmoregulatory system. Material and Methods Experimental design Fish and sampling After the determination of the LC50, one Caspian kutum, fry, were obtained from sublethal concentration of atrazine was the Shahid Ansari Fish Proliferation and determined as 1/2LC50 (Ramesh et al., Culture Center (Rasht, Iran), in July 2011. 2009). Three aquaria (100L) each Total length (cm) and Body weight (g) containing 50 Caspian kutum fry were were measured, and based on the length exposed to this sublethal concentration for and weight, the Condition Factor (CF) was 4 days, and the same number of aquaria calculated using Williams (2000) method: and fry in clean water (no atrazine) were K= (100×w) L3. held as control group. Sampling begun Determination of LC50 and sub-lethal after 24hrs of exposure and continued concentration every 24 h until the end of the experiment. Acute toxicity was conducted to determine During the experiment water factors: pH, the 96 h LC50 value of atrazine with temperature and dissolved oxygen (DO) definitive test in semi-static system in were measured using Eutech instruments, laboratory as per standard methods pcd650, and fish were not fed during the (APHA, AWWA, WPCE, 2005). The experiment.

705 Khoshnood et al. Histopathological effects and toxicity of atrazine herbicide in Caspian Kutum

Measuring the Concentration of Ions Table 1: Physico-chemical properties of the test For measuring the concentration of the water. Characteristics Unit Mean Range ions, fish samples were frozen in liquid 22.8- + + Air Temperature ˚C 23.4 nitrogen. The concentration of Na , K , 24.3 2+ 2+ Ca and Mg were determined by Atomic Water 17.9- ˚C 18.1 Absorption Spectrometry (Flame atomic Temperature 19.1 Dissolved 7.11- absorption spectrometry GBS Avanta PM), mg·L-1 7.20 Oxygen 8.01 and the Cl- concentration was measured by pH - 7.8 7.7-7.9 flame spectrophotometry (UV-Vis HACH

DR 5000. Toxic Stress and Poisoning Symptoms in Histology Fish during the LC50 Test Gill samples were immersed into Bouin’s Fish subjected to atrazine herbicide fixative for 24 hours, washed and displayed uncoordinated behavior. On dehydrated in ascending series of ethanol initial exposure, fish were alert, stopped and then embedded in Paraffin (Merck, swimming and remained in a static Germany). Following embedment in position in response to the sudden changes Paraffin, transversal and longitudinal in the surrounding environment. After sections of 6 µm were cut on a Leica some time they tried to avoid the toxic microtome (RM2255) and transferred on water by swimming quickly. Faster glass slides and stained with Haematoxylin opercula activity was observed as & Eosin (Mortoja and Mortoja-Pierson surfacing and gulping for air. In aquaria 1967; Khodabandeh et al., 2008). with higher concentrations of test Statistical Analysis herbicide, the fish swam erratically. They All the data were subjected to one-way secreted copious amounts of mucus from ANOVA using statistical software SPSS whole body continuously and soon a thick version 15.0. Independent sample t-tests layer of mucus was found deposited in the were used to determine the differences buccal cavity and gills. Ultimately, fish among treatment means at p<0.05. lost their balance, consciousness, engaged

in a rolling movement and became Results exhausted and lethargic. Lastly, they Physico-Chemical Parameters of the Test remained in a vertical position for a few Water minutes with their anterior side or terminal The physico-chemical characteristics of mouth up near the surface of the water, the test water are presented in Table 1. The trying to gulp for air and with their tails in water temperature varied from 17.9 to a downward direction. Soon they settled at 19.1˚C and the pH ranged from 7.7 to 7.9. the bottom of the aquaria, and after some The dissolved oxygen concentration time their bellies turned upward and the ranged from 7.11 to 8.01 mg·L-1. fish died.

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LC50, respectively following Finney’s Median Lethal Concentration (LC50) (1971) method and using SPSS (version Median lethal concentration (LC50) is the 15). A dose dependent increase and time most widely accepted basis for an acute dependent decrease were observed in toxicity test and it is the concentration of a mortality rate, such that as the exposure test chemical which kills 50% of the test time increases from 12 to 96 h, the median organisms after a particular length of concentration was reduced. It was exposure, usually 96 h. Generally in observed that as the concentration of the toxicity tests, death is a decisive criterion herbicide increased, fish mortality also because it is easy to determine and has increased, this indicates a direct obvious biological and ecological proportional relationship between significance. The LC50 values (with 95% mortality and concentration of atrazine confidence limits) of different herbicide. No mortality was observed in concentrations of atrazine (Table 2) were the control during the experimental period. found to be 29.22, 28.44, 27.27, 25.78 and 24.95 mg·L-1 for 12, 24, 48, 72 and 96 h

Table 2: Lethal concentrations (LC) of atrazine depending on exposure time (12-96 h) for R. frisii kutum. Different letters show significant differences and similar letters or numbers show no significant differences. Concentrations (mg·L–1) at various exposure times. Point (95% confidence intervals)

12h 24h 48h 72h 96h 21.43a 19.95b 18.76c 18.61c 19.02d LC 1 (18.76-22.92) (17.32-21.51) (16.25-20.33) (16.58-19.96) (12.49-21.36) 24.63a 23.39b 22.19c 21.54d 21.49d LC 10 (23.07-25.59) (21.76-24.43) (20.53-23.27) (20.12-22.51) (16.74-23.26) 29.22a 28.44b 27.27c 25.78d 24.95e LC 50 (28.20-30.90) (27.42-29.98) (26.34-28.51) (25.00-26.63) (23.92-25.02) 34.66a 34.57a 33.51b 30.85c 28.96d LC 90 (32.33-39.78) (32.18-39.50) (31.36-37.64) (29.40-33.28) (26.81-36.73)

Length, Weight and Condition Factor condition factor of the control and atrazine The mean body weight (BW) and mean exposure groups showed no significant total length (TL) of fry’s were: 0.26±0.01 differences (p>0.05) between these two g and 3.5±0.02 cm, respectively. experimental groups (Figs. 1 and 2). Measuring the length, weight and

707 Khoshnood et al. Histopathological effects and toxicity of atrazine herbicide in Caspian Kutum

Figure 1: Total Length of R. frisii kutum fry (A); Body weight of R. frisii kutum fry (B): No significant differences found between two experimental groups (p>0.05).

Figure 2: Condition Factor (CF) in R. frisii kutum fry. No significant ifferences found between two experimental groups (p>0.05).

Body Ions except for the Cl-, that had higher Measurement of the total body ions concentration in control group (Fig. 4). It showed that the highest concentration seemed that, exposure to atrazine has belongs to Ca2+ in the atrazine exposure increased the cations and decreased the group and the lowest belongs to the Mg2+ anions. On the other side, the in the control group (Fig. 4). Results also concentration of the ions in control group showed that all ions in both experimental was as the following order: Ca2+>K+>Na+ groups were increased during the > Cl- >Mg2+, but this order was different in experiment (Fig. 3). In atrazine exposed atrazine exposed group: group all the ions showed higher Ca2+>Na+>K+>Mg2+>Cl-. The results concentrations compared to control group, indicated that besides increasing and

Iranian Journal of Fisheries Sciences 13(3) 2014 708 decreasing the concentration of the ions, the K+ , Ca2+ , Mg2+, Cl- ions in control and atrazine has affected the ion composition atrazine exposure groups, but the of the body as well. Statistical analysis concentration of the Na+ is significantly showed that, there is no significant (p<0.05) higher in atrazine exposed group. difference between the concentrations of

Figure 3: Concentration of different ions in body of control fish during the experiment (A): All concentrations were increased during the experiment; Concentration of different ions in body of atrazine exposed fish during the experiment (B). All concentrations were increased during the experiment.

709 Khoshnood et al. Histopathological effects and toxicity of atrazine herbicide in Caspian Kutum

Concentration of different ions in two experimental groups

7000 6000 x x 5000 4000 Control 3000 Atrazine

2000 a 1 1

Concentration (mg/Kg) Concentration 1000 b * * I I 0 Na K Ca Mg Cl Ions

Figure 4: Comparison between mean concentrations of different ions in two experimental groups. The concentration of all ions were higher in atrazine exposed fish compared to control group, but the only significant difference found in Na+ concentration (p<0.05). Different letters or numbers show significant differences and similar letters or numbers show no significant differences.

Histopathology individual cells termed pillar cells (Fig. 5C Gill in R. frisii kutum larvae, as examined & D). The spaces around the pillar cells are made up of four gill arches in each side and between the two epithelial layers are of the head. Each arch is carrying two perfused with blood. The most significant rows of filaments, and the filaments alterations in gills caused by atrazine carrying two rows of lamellae. The main exposure were hyperplasia and thickening epithelial cells of the gill filaments and of the filaments, separation of the lamellae are simple squamus cells, called pavement cells of the lamellae epithelium the pavement cells. Each lamella is from the pillar cells and swelling of the essentially composed of two epithelial epithelial cells (Fig. 5A & B). sheets held apart by a series of

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Figure 5: Histological photograph of the gill in R. frisii kutum fry. Gill of the atrazine exposed fish (A & B), separation of the pavement cells of the lamellae, thickening of the filament and swelling of the epithelial cells were the major effects of the atrazine. Gill structure in control group (C & D), gill is made up of filaments carrying lamellae. F: Filament; L: Lamellae; PC; Pavement Cell; PiC: Pillar Cell. importance and can permit early detection Discussion of aquatic environmental problems Fish are often used as sentinel organisms (Lopez-Barea, 1996; Van Der Oost, et al., for ecotoxicological studies because they 2003). Acute toxicity data has been used to play a number of roles in the trophic web, derive water quality guidelines for accumulate toxic substances and respond regulatory measures (Sunderam, et al., to low concentrations of mutagens (Cavas 1994).The result of the LC50 (median and Ergene-Gözükara, 2005). Therefore, lethal concentration) for atrazine in the the use of fish biomarkers as indices of the present study at 96 h was 24.95 mg.L-1. effects of pollution are of increasing The results showed that the toxicity of

711 Khoshnood et al. Histopathological effects and toxicity of atrazine herbicide in Caspian Kutum

atrazine for R. frisii kutum is both time and surface to bottom movement were similar concentration dependent, thus, accounting to the observations of Hussein et al. for differences in LC values obtained at (1996); Pandey et al. (2005) and Chandra different concentrations and time of (2008). exposure. However, some other The length-weight relationship of fish researchers have shown that exposure time is an important fishery management tool. is not significant in LC50 determination Its importance is pronounced in estimating for fish (Lakota et al., 1989). The LC50 the average weight at a given length group value obtained for R. frisii kutum in this (Beyer, 1987) and in assessing the relative study is higher than that reported by Bathe well being of a fish population (Bolger and et al. (1973), Neškovic et al. (1993), and Connoly, 1989). Consequently, length- Hussein et al. (1996), who reported LC50 weight studies on fish are extensive. values of 16.0, 18.8 and 9.37 mgl-1 for Notable among these are the reports Lepomis macrochirus (Bluegill sunfish), Shenouda et al. (1994), for Chrysichthys C. carpio and Oreochromis niloticus, spp. from the Southernmost part of the respectively, exposed to atrazine. Toxicity River Nile (Egypt), Alfred-Ockiya and of chemicals to aquatic organisms has Njoku (1995) for mullet in New Calabar been shown to be affected by age, size and River, Ahmed and Saha (1996) for carps in health of the species (Abdul-Farah et al., Lake Kapital, Bangladash, King (1996) for 2004). Physiological parameters, and Nigeria fresh water fishes, Hart (1997) for water quality, temperature, pH, dissolved Mugil cephalus in Bonny Estuary; Diri oxygen and turbidity, the, amount and kind (2002) Tilapia guineensis in Elechi creek. of aquatic vegetation, concentration and Condition factor compares the formulation of the chemical and its wellbeing of a fish and is based on the exposure also greatly influence such hypothesis that heavier fish of a given studies (Gupta et al., 1981; Young, 2000). length are in better condition (Bagenal and Fish exposed to atrazine were stressed Tesch, 1978). Condition factor has been progressively with time before death. The used as an index of growth and feeding respiratory impairment due to the toxic intensity (Fagade, 1979). Condition factor effect of atrazine on the gills of R. frisii decrease with increase in length (Fagade, kutum is similar to the reports of Abdul- 1979); and also influences the reproductive Farah et al. (2004); De Mel and Pathiratne cycle in fish (Welcome, 1979). Condition (2005); Tilak et al. (2007) and Ayoola factors of different species of cichlid fishes (2008) that pesticides impair respiratory have been reported by Siddique (1977), organs. Death could have, therefore, Fagade (1978, 1979, and 1983), Dodzie occurred either by direct poisoning or and Wangila (1980), Arawomo (1982) and indirectly by making the medium Oni et al. (1983). Some condition factors unconducive for the fish or even by both. reported for other fish species include; The abnormal behavior observed during Alfred- Ockiya (2000), Chana chana in the exposure period like restlessness and fresh water swamps of Niger Delta and

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Hart (1997), M. cephalus in Bonny circuit current of the ventral skin of frog estuary, Abowei and Hart (2007), ten fish (Rana esculenta), resulting in stimulated species from the lower Nun River, and Na+ absorption. Increases of plasma Na+ at Abowei and Davies (2009), Clarotes intermediate doses of atrazine may be a lateceps from the fresh water reaches of compensatory response to moderate the lower Nun river. In present study no damage of ion regulatory tissue. In some significant differences were found in previous studies, exposure to different atrazine exposed and control group, this levels of atrazine caused elevation in result showed that all experimental fish plasma Na+, K+, Ca2+, Mg2+ and Cl- levels were in the same growth condition and (Nieves-Puigdoller et al., 2007). atrazine at sublethal concentration and The high mortality induced by atrazine acute condition have no effects on growth reported in Waring and Moore (2004) is all or condition factor of the R. frisii kutum the more unexpected given the acute fry. toxicity values for atrazine exposure in Several studies have tested the effects freshwater fish. These range from a 96-h of atrazine on survival and various LC50 of 4300 μg/L for the guppy measures of iono-regulatory performance (Poecilia reticulata) to a 96-h LC50 of in different fishes (Moore et al., 2003; N100,000 μg/L for the carp (Carassius Waring and Moore, 2004; Nieves- carassius) (Giddings et al., 2004). In the Puigdoller et al., 2007). The present study case of salmonids, the 96-h LC50 was differs from many in the literature in that 13,000 μg/L for rainbow trout the results did not reveal any significant (Oncorhynchus mykiss) and 12,000 μg/L effects of atrazine on survival, body for coho salmon (O. kisutch). Both short weight, and condition factor or term (4-day; present study) and longer ionoregulatory performance in R. frisii term (21 day, Nieves-Puigdoller et al., kutum fry. 2007) exposures to atrazine had no effects Plasma and whole body electrolyte on body weight. levels, Na+/K+-ATPase activity and muscle Many studies showed the effects of water content are commonly measured as atrazine on gills of the fish species, indicators of iono-regulatory performance including increase of epidermal thickness in fishes. and lamellar width, fusion of secondary In the present study, atrazine (12.47 lamellae, hyperplasia, club-shaped mgl−1) elevated whole body Na+ levels cartilaginous tissue, aneurysm, and significantly, while not affecting other necrosis in epithelium region, (Alazemi et ions. These results are similar to those of al., 1996; Cengiz and Unlu, 2006;Velisek Waring and Moore (2004) and that et al., 2006 ;Yang et al., 2010). Previous atrazine elevated plasma Na+ and had no studies suggested edematous changes in effects on plasma Cl- levels. Cassano et al. the gill were most probably due to the (2006) demonstrated that doses as low as increase in capillary permeability. 2µg.L−1 atrazine can stimulate the short- Hyperplasia was considered as a protective

713 Khoshnood et al. Histopathological effects and toxicity of atrazine herbicide in Caspian Kutum

mechanism from environmental irritant by Journal of Applied Zoology and decreasing the respiratory surface and Environmental Biology, 9, 44-50. increasing the toxicant–blood diffusion Abowei, J.F.N. and Davies, A.O., 2009. distance (Meissner and Diamandopoulos, Some population parameters of 1977), and its intensification could result Clarotes laticeps (Rupell, 1829) from in the thickness of epithelial layers, which the fresh water reaches of the lower could be supported by the increases of river, Niger Delta, Nigeria. American epithermal thickness and lamellar width. Journal of Scientific Research, 2, 15- Thus, all the lesions found in the present 19. study would probably inhibit the Ahmed, K.K. and Saha, S.B., 1996. respiratory, secretory and excretory Length-weight relationship of major functions in the gill of R. frisii kutum. carps in Kaptai Lake. Bangladesh. Results of the present study showed NAGA. The ICLARM Q., 19(2), 28. that sublethal concentration of atrazine, Alazemi, B.M., Lewis, J.W. and even in acute and short term exposure can Andrews. E.B., 1996. Gill damage in alter the biochemical composition of the the freshwater fish Gnathonemus fish body and affect some behavioral petersii (family: Mormyridae) responses that could lead to failure of the exposed to selected pollutants: An surviving capabilities of the fish fry. ultrastructural study. Environmental Technology, 17, 225-238. Acknowledgments Alfred-Ockiya, J. F. and Njoku, D.C., We would like to thank all members of the 1995. A comparative analysis of the Shahid Ansari Fish Proliferation and length weight relationship and Culture Facility in Rasht, Iran for their condition factors of four species of great helps during this experiment, grey mullet (pisces/mugildae) from especially Mr. Darvishi, Mr. Nezafat, Mr. New Calabar River Rivers State, Sobhani, and Mr. Abedinzadeh. Nigeria. Journal of Technology and Design Education, pp. 5-10. References Alfred-Ockiya, J.F., 2000. The length- Abdul-Farah, M., Ateeq, B., Ali, M.N. weight relationship of snake head and Ahmad, W., 2004. Studies on (Chana chana) from the fresh water lethal concentrations and toxicity swamps of Niger Delta. Journal of stress of some xenobiotics on aquatic Aquatic Science, 15, 12-14. organisms. Chemosphere, 55, 257– Alvarez, M.d.C. and Fuiman, L.A., 265. 2005. Environmental levels of atrazine Abowei, J.F.N and Hart, A.I. ,2007. and its degradation products impair Size, Composition, age, growth, survival skills and growth of red drum mortality and exploitation rate of larvae. Aquatic Toxicology, 74, 229– Chysichthys nigrodigitatus from Nun 241. River, Niger Delta, Nigeria. Afr.

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