Investigation of acute toxicity of (2,4-dichlorophenoxy)acetic acid (2,4-D) herbicide on crayfish ( Astacus leptodactylus Esch. 1823)

Pesticide Biochemistry and Physiology 88 (2007) 296–299 www.elsevier.com/locate/ypest Investigation of acute toxicity of (2,4-dichlorophenoxy)acetic acid (2,4-D) herbicide on crayWsh (Astacus leptodactylus Esch. 1823) A. Çaflan Karasu Benli a, Rabia SarÂkaya b, Aylin Sepici-Dincel c, Mahmut Selvi d, Duygu oahin c, Figen Erkoç d,¤ a Turkish Republic, The Ministry of Agriculture and Rural AVairs, GDAPD (General Directorate of Agricultural Production and Development), Department of Aquaculture, Eskioehir Yolu 9. Km Lodumlu, Ankara, Turkey b Department of Primary School Education, Gazi University, Teknikokullar, 06500 Ankara, Turkey c Department of Medical Biochemistry, Faculty of Medicine, Gazi University, 06510 Ankara, Turkey d Department of Biology Education, Gazi University, Teknikokullar, 06500 Ankara, Turkey Received 16 November 2006; accepted 4 January 2007 Available online 11 January 2007 Abstract The acute 96 h LC50 of (2,4-dichlorophenoxy)acetic acid (2,4-D), a widely used agricultural herbicide, was determined on crayWsh (Astacus leptodactylus Esch. 1823). CrayWsh of 23.5 § 1.49 g mean weight and 9.6 § 0.21 cm mean length were selected for the bioassay experiments. The experiments were repeated three times, in 10 L tap water. The data obtained were statistically evaluated by the use of the E.P.A computer program based on Finney’s probit analysis method and the 96 h LC50 value for crayWsh was calculated to be 32.6 mg/L in a static bioassay test system. 95% lower and upper conWdence limits for the LC50 were 15.10–327.16. In conclusion, 2,4-D is highly toxic to crayWsh, a non-target organism in the ecosystem. Water temperature was 23 § 1 °C. Behavioral changes of crayWsh were recorded for all herbicide concentrations.  2007 Elsevier Inc. All rights reserved. Keywords: (2,4-D); (2,4-Dichlorophenoxy)acetic acid; CrayWsh; Astacus leptodactylus; Acute toxicity; LC50 1. Introduction Among herbicides widely used, with potential toxicity against humans, are phenoxy compounds such as (2,4dichlorophenoxy)acetic acid (2,4-D); (2,4,5-trichlorophenoxy)acetic acid (2,4,5-T); (4-chloro-2-methylphenoxy)acetic acid (MCPA) and their respective esters. Among these 2,4D is the most widely used herbicide in the world [1]. 2,4-D is a systemic herbicide and is used to control many types of broadleaf weeds. It is used in cultivated agriculture, in pasture and rangeland applications, forest management, home, garden, and to control aquatic vegetation. 2,4-D functions by maintaining high levels of the plant hormone auxin, * Corresponding author. Fax: +90 312 2228483. E-mail address: erkoc@gazi.edu.tr (F. Erkoç). 0048-3575/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2007.01.004 resulting in overstimulation of plant growth and ultimately death. Historically, the product Agent Orange, used extensively throughout Vietnam, was about 50% 2,4-D [2]. Data on the aquatic toxicity of 2,4-D on non-target organisms is either incomplete or lacking. Although some formulations of 2,4-D were reported highly toxic to Wsh; others were less so. Limited work have been carried out on ecotoxicology concerning invertebrates of the ecosystem. There are studies in the literature concerning the accumulation of 2,4-D, its derivatives, and other agricultural chemicals in tissues [3]. Oruc and Uner [4] studied the combined eVects of 2,4-D and azinphosmethyl on antioxidant enzymes for clarifying mode of action of these chemicals. Farah et al. [5] studied acute (96 h) toxicity and stress behavior of 2,4-D on freshwater Wsh (Heteropneustes fossilis, Clarias batrachus, Channa punctatus) as LC50 values as 81, 122, 107 mg/L; while they calculated 48 h LC50 to mosquito 297 A.Ç.K. Benli et al. / Pesticide Biochemistry and Physiology 88 (2007) 296–299 larvae (Culex pipiens fatigans) 302 mg/L. Fish displayed behavioral changes such as restlessness, swimming at the surface or abnormal swimming behavior, vigorous jerks of body, loss of balance, myotonia and anorexia. Breathing diYculties and respiratory problems were also encountered both in Wsh and mosquito larvae. Papaefthimiou et al. [6] studied physiological mechanism of toxic action of 2,4-D in frog, honeybee and beetle heart and reported heart of frog and honeybee to be extremely sensitive. Potential genotoxic eVects have been reported by various workers: mice in vivo [7]; cultured mammalian cells [8]; freshwater Wsh C. punctatus with micronucleus test [9]; induction of micronuclei and erythrocyte alterations catWsh, C. batrachus [10]; micronucleus test in human lymphocytes [11]; apoptic eVects and DNA degradation in walking catWsh, C. batrachus [12]. In addition to its mechanism of action on plant growth hormones, it is known that 2,4-D provokes changes in the animal nervous system based on complex formation with acetylcholine, and thus inhibition of acetylcholinesterase (AChE) activity and also increase of the level of another neurotransmitter serotonin. Decreased acetylcholinesterase activity of human erythrocytes (in vitro) due to indirect membrane modiWcation and increased reactive oxygen species has been reported by Bukowska et al. [13]. This study was conducted to determine the acute toxicity of 2,4-D, most widely used herbicide in the world, on a widely distributed, important invertebrate in many aquatic systems: the narrow clawed crayWsh (Astacus leptodactylus Esch. 1823) using the static test system. 2. Materials and methods CrayWsh (A. leptodactylus Esch. 1823) were obtained from a local breeder. The test organisms (average weight 23.5 § 1.49 g; average length 9.6 § 0.21 cm) were transported to the laboratory in appropriately wetted plastic containers and immediately transferred to the test aquaria (n D 6) and allowed to acclimatize for 48 h. Twenty liter-capacity aquaria containing 10 L water were used as test chambers. At the time of dosing air was turned oV; it was on at all times otherwise. Water temperature, dissolved oxygen, conductivity, pH, total hardness and NH3-N were 23 § 1 °C, 6.53 § 0.12 mg/L, 0.20 § 0.01 mS/cm, 6.83 § 0.05, 13.20 § 0.40 F and 0.001 § 0.00 mg/L, respectively. After 48 h of adaptation, diVerent concentrations of 2,4-D [(2,4-dichlorophenoxy)acetic acid; CAS Number: 94-75-7; stored at +4 °C] were added to the aquaria. During the adaptation period, and throughout the duration of the experiment, animals were not fed. Mortality was assessed at 24, 48, 72 and 96 h after the start of the tests. Dead individuals were removed immediately. Following the preliminary experiment, all determinations were repeated three times. Behavioral changes were followed closely. Control group was kept in tap water, no solvent was included since 2,4-D is soluble in water; all other conditions were same as experimental groups. The bioassay system was as described in standardized methods [14,15] and the national regulation [16]. LC50 and 95% conWdence limits were calculated by a computer program [17]. 3. Results and discussion The calculated 96 h acute LC50 value (95% conWdence limits) of technical 2,4-D, using a static bioassay system to crayWsh (Astacus leptodactylus) was 32.6 mg/L (95% conWdence limits: 15.10–327.16). Control mortality was zero. The results show that 2,4-D is toxic to crayWsh. CrayWsh is a recommended test organism according to the reference/ standard methods [14,15] and the Turkish national regulation [16]. Results appear in Table 1 and Fig. 1. Green and Abdelghani [18] investigated the toxicity of a mixture of 2,4-dichlorophenoxyacetic acid and monosodium methanearsonate to the red swamp crayWsh, Procambarus clarkii. According to the results, the herbicide mixture alone displayed half the toxicity of the individual herbicides, but the mixture with surfactant was twice as toxic as the individual herbicides. However, no LC50 values were reported. Paul et al. [19] reported the eVects of 2,4-D on several aquatic species: Brook trout (Salvelinus fontinalis), walleye (Sander vitreus), fathead minnow (Pimephales promelas), and the amphipod (Hyallela azteca) in static acute toxicity tests in the laboratory. Our results are in agreement with their reported mg/L ranges. The 96-h LC50 for brook trout, walleye, and fathead minnow were respectively 0.76, 0.66 and 2.22 mg/L. The 48-h LC50 for H. azteca was 0.60 mg/L. However, we here report toxicity of 2,4-D to crayWsh for the Wrst time in the literature. Potential environmental eVects of 2,4-D currently draw attention and further studies are needed to get a better picture of ecotoxicological impacts. Along this line, estrogenic activities of aquatic herbicides and surfactants using rainbow trout were evaluated by Xie et al. [20]. 2,4-D and triclopyr caused signiWcant induction of Vitelogenin. Concentration-response studies demonstrated that the lowest observed eVect concentrations (LOECs) for 2,4-D and triclopyr were 0.164 and 1 mg/L, respectively. Inhibitory eVect of heavy metals on oxygen consumption due to increase in temperature was reported by Khan et al. [21] in juvenile crayWsh (Orconectes immunis); indicating that rising global temperatures due to climate change have the potential of Table 1 Acute 96 h toxicity of 2,4-D to crayWsh (Astacus leptodactylus) Point Concentration (mg/L) Slope § SE Intercept § SE LC 1.00 LC 5.00 LC 10.00 LC 15.00 LC 50.00 LC 85.00 LC 90.00 LC 95.00 LC 99.00 3.06 6.08 8.78 11.24 32.6 91.06 116.62 168.27 334.69 2.281 § 1.079 1.566 § 1.583 Note. Control group (theoretical spontaneous response rate) D 0.0000. 298 A.Ç.K. Benli et al. / Pesticide Biochemistry and Physiology 88 (2007) 296–299 Fig. 1. Plot of adjusted probits and predicted regression line for 2,4-D to crayWsh (Astacus leptodactylus). increased sensitivity of aquatic animals to heavy metals in their environment. Observations of behavioral response of crayWsh were conducted at 1–8 h, and every 12 h during the acute toxicity tests. The control group showed normal behavior during the test period. In the aquarium crayWsh usually walked along the wall, stopped at every corner and searched for a shelter. The 10 and 20 mg/L concentrations had close to normal behavior of the control group. At 30, 40 and 50 mg/ L, behavioral changes started 1 h after dosing. CrayWsh moved with diYculty and frequently stood at the corners of the aquaria. When standing, the crayWsh with its claws and abdomen up, began rocking like a swing, or walked in circles in the middle of the aquaria. Some crayWsh attempted to climb the vertical walls of the aquaria. Others settled in the middle of the aquaria. After exposing to high concentrations of 2,4-D, the occurrence and frequency of the “moving backward” element has increased. Fighting was frequent. Before death, crayWsh lost equilibrium and showed turning tendency in the reverse direction. Then crayWsh Xipped over and turned upside-down on their backs. Similar behavioral changes were reported both in Wsh and mosquito larvae by Farah et al. [5]. Fish displayed behavioral changes such as restlessness, swimming at the surface or abnormal swimming behavior, vigorous jerks of body, loss of balance, myotonia and anorexia. Breathing diYculties and respiratory problems are also reported. Acknowledgment The authors thank the U.S. E.P.A. for making available the acute toxicity testing probit analysis computer program. References [1] R.D. Wauchope, T.M. Buttler, A.G. Hornsby, P.W.M. Augustijn Beckers, J.P. Burt, Pesticide properties database for environmental decision making, Rev. Environ. Contam. Toxicol. 123 (1992) 7–22, SCS/ ARS/CES. A.Ç.K. 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