Toxicity of Organophosphates on Morphology and Locomotor Behavior in Brine Shrimp, Artemia salina

Arch. Environ. Contam. Toxicol. 53, 227–232 (2007) DOI: 10.1007/s00244-006-0226-9

Toxicity of Organophosphates on Morphology and Locomotor Behavior in Brine Shrimp, Artemia salina J. Venkateswara Rao, P. Kavitha, N. M. Jakka, V. Sridhar, P. K. Usman Toxicology Unit, Biology Division, Indian Institute of Chemical Technology, 500 007, Hyderabad, India

Received: 3 February 2006 /Accepted: 11 February 2007

Abstract. The acute toxicity and hatching success of four organophosphorus insecticides—acephate (ACEP), chlorpyrifos (CPP), monocrotophos (MCP), and profenofos (PF)—was studied in a short-term bioassay using brine shrimp, Artemia salina. Fifty percent hatchability inhibition concentration and median lethal concentration (LC50) values were calculated after probit transformation of the resulting data. Among the insecticides tested, CPP is found to be the most toxic and also to inhibit hatching success of A. salina cysts in a concentration-dependent manner. In addition, the effect of these pesticides on locomotor behavior (swimming speed) and morphologic differences were studied in LC50-exposed nauplii after 24 hours. The in vivo effect of these insecticides on acetylcholinesterase (Enzyme commission number (EC 3.1.1.7) activity was also determined in LC50-exposed nauplii after 24 hours. Maximum percent decrease in their swimming speed and significant morphologic alterations were noticed in CPP-exposed brine shrimps. The order of toxicity was CPP > PF > MCP > ACEP in all the parameters studied.

Chemical pollution in the environment by organophosphorus (OP) insecticides has been increasing because of their extensive use in agriculture. OP insecticides are known to inhibit acetylcholinesterase enzyme (AChE EC 3.1.1.7), which plays an important role in neurotransmission at cholinergic synapses by rapid hydrolyzing the neurotransmitter acetylcholine to choline and acetate (Ozmen et al. 1999; Fulton & Key 2001). The inhibitory effects of OP insecticides are dependent on their binding capacity to the enzyme active site as well as their rate of phosphorylation. There is growing concern worldwide about the indiscriminate use of such chemicals, which result in environmental pollution and toxicity risk to nontarget organisms. Approximately 90% of pesticide application never reaches its target organisms; at the same time, they disperse through air, soil, and water (Moses et al. 1993). The largest input of OPs in the marine environment comes from the transport of these

Correspondence to: J. Venkateswara Rao; email: [email protected]

compounds to the sea by way of surface waters (Galassi 1991). The spillage of pesticides from the point of source (agricultural runoff) diffuses contamination into the environment, which affects larger areas and is more difficult to control. Residual concentrations of the more persistent OPs (i.e., chlorpyrifos) were also found in marine sediments of lagoon (Readman et al. 1992). Acute-toxicity tests involving aquatic invertebrates have been used for ecotoxicologic evaluation (Vittozi & Angelis 1991; Calleja et al. 1994). OPs toxicity to freshwater organisms is well known (Venkateswara Rao et al. 2003a, 2003b, 2004; De la Torre 2002), whereas only few studies have been reported for marine organisms (Sreenivasula et al. 1985); therefore, little information is available on marine aquatic organisms. Artemia salina, commonly known as brine shrimp, is a significant food source for fish, such as other zooplanktons (Sorgeloos 1980). The marine crustacean Artemia is a euryhaline species, so it has a great osmoregulation capacity, which contributes to a greater resistance to the toxic effects of OPs. Artemia spp. has gained popularity as a test organism because of its ease of culture, short generation time, cosmopolitan distribution, and commercial availability of its dormant eggs (cysts). Artemia salina hatching from cysts are of similar age, genotype, and physiologic condition; in addition, test variability is greatly decreased (Vanhaaecke et al. 1981). The activity of compounds on hatching of the cysts (encased embryos that are metabolically inactive) and toxicity on hatched nauplii has gained importance in toxicologic evaluations (Migliore et al. 1997). Locomotion has been found to be a consistently sensitive measure of toxic stress for a wide range of environmental contaminations (Little & Finger 1990). Studies on the effects of pesticides on aquatic invertebrates are lacking because of methodologic difficulties involved. However, because of the recent rapid development of computerized video-imaging techniques, it is now possible to make direct quantitative measurements of spontaneous locomotor behavior in test organisms. A computer-automated video-tracking system (Baatrup & Bayley 1993; Bayley 1995) was employed in this study to assess the locomotor behavior (velocity) of normal and toxicant-exposed nauplii of A. salina. The present study was undertaken to evaluate the median lethal concentration (LC50) and 50% hatchability inhibition concentration (EC50) of four OP pesticides to A. salina. Special emphasis was placed on the computerized video-tracking

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system (Ethovision) for quantification of the effects on locomotor behavior and morphology, and target enzyme AChE interaction, in vivo.

Methods Test Chemicals All reagents used for the preparation of artificial seawater were of analytic grade and were used without further purification. Acetylthiocholine iodide and 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich. The test compounds—(1) acephate O, S-dimethyl acetyl phosphor-amidothioate, 2 chlorpyrifos (O, O- diethyl - 3, 5, 6 - trichloro-2-pyridyl phosphorothioate), 3 monocrotophos (dimethyl (E)-1-methyl-2-(methylcarbamoyl) vinyl phosphate), and 4 profenofos (O-(4-bromo-2-chlorophenyl)O-ethyl-S-propyl phosphorothioate)—used in the experiment were of 97% purity. They were synthesized in our laboratory work aimed to transfer newer technologies for the bulk production of these potential insecticides.

Test Organisms Brine shrimp cysts (eggs) were purchased from Ocean Star International (Snowville, USA). The cysts were hatched within 24 hours (range 18 to 24) in 3.0% artificial synthetic seawater at 27°C (pH 8.6) with continuous illumination and aeration.

Brine Shrimp Lethality Test The brine shrimp lethality test was performed according to the method described by Meyer et al. (1982). Earlier reports proved that 72-hour-old nauplii are a better indicator of carbamate insecticide potency than either 24- or 48-hour-old nauplii (Barahona & Sanchez-Fortun 1999). Therefore, in the present experiments, 48hour-old nauplii were exposed for 24 hours (thereby attaining the age of 72 hours). Briefly, 10 48-hour-old nauplii were transferred to each sample vial containing 4.95 ml freshly prepared artificial seawater along with 50 ll required test concentration in acetone. Corresponding control experiments were also performed with 4.95 ml artificial seawater and 50 ll acetone alone (carrier solvent). The predetermined concentrations (after range-finding tests) of CPP (0.2, 0.4, 0.6, 0.8, and 1.0 mg/L), PF (1, 10, 25, 50, and 75 mg/L), MCP (225, 250, 275, 300, and 325 mg/L), and ACEP (2000, 2250, 2500, 2725, and 3000 mg/L) were used to determine LC50s. The lethality experiments were carried out with 5 replicates/each concentration, and their average percent mortalities were recorded after 24 hours of exposure. During the exposure, the nauplii were not fed because they can feed on their yolk sacs (Pelka et al. 2000) and can survive up to 72 hours without food (Lewis 1995). Percent mortalities were corrected for natural mortality in controls using Abbots formula, p = PI – C/1 – C, where PI denotes the observed mortality rate, and C is natural mortality. LC50s were calculated using probit analysis (Finney 1953).

diluted stock concentration in acetone to 250 ml freshly prepared artificial seawater. Control experiments were also performed by the addition of carrier solvent (100 ll) alone. Precisely, 200 € 5 numbers of cysts, equal to 1 mg (weighed by AFCOSET analytic balance), were added to all of the control and test solutions respectively. The predetermined concentrations (after range-finding tests) of CPP (0.075, 0.1, 0.15, 0.2, 0.25, and 0.3 mg/L), PF (0.08, 0.12, 0.16, 0.2, 0.24, and 0.28 mg/L), MCP (125, 150, 175, 200, 225, and 250 mg/L) and ACEP (750, 1000, 1250, 1500, 1750, 2000, and 2250 mg/L) were used to determine EC50s. The hatchability experiments were carried out with 5 replicates/each concentration, and percent hatching success was recorded after 24 hours of exposure. The percent hatchability– versus–test concentrations were used to estimate the effective concentration (EC50 = EC50 taken as the concentration required to produce a decrease of 50% in hatching control experiments) by a computerized programme of probit analysis (Finney 1953).

Locomotor Behavior and Morphology In a separate set of experiments, approximately 1000 48-hour-old nauplii were exposed to LC50 concentrations of each test compound for 24 hours. The survivors were used to study the effect of toxicant on their locomotor behavior (swimming speed and distance travelled per unit time) and morphologic abnormalities. A computerized videotracking system (Ethovision; Noldus Information Technology, Wageningen, The Netherlands) was used for automation of behavioral experiments. Briefly, 25 nauplii/each exposure were taken and placed individually into a 96-well culture plate to record their behavior by using a state of-the-art video-tracking system. The morphologic abnormalities of exposed nauplii in each toxicant were observed under magnification (330 x) using a Polyvar Compound Microscope attached to a CCD camera (Sony) with an aid of software (Easy-Grab; Noldus Information Technology).

Determination of AChE Activity Two hundred nauplii were taken from each control and treatment replicate (LC50) and were used for estimation of AChE activity. They were washed twice with ice-cold phosphate buffer (0.1M [pH 7.5]) to remove the externally attached toxicant and homogenized in 1 ml ice-cold phosphate buffer (0.1M [pH 7.5]) containing 10ml/ L Triton X-100 using a Potter–Elvehjam homogenizer fitted with a Teflon pestle. The homogenate was centrifuged at 5000 x g for 15 minutes at 4°C in a Beckman TLC tabletop Ultra Centrifuge (model no. TLX 361 544). The resultant supernatant was recentrifuged at 15,000 x g for 15 minutes at 4°C and used as enzyme source. Protein was estimated according to the method of Lowry et al. (1951), and AChE was assayed as described by Ellman et al. (1961). Briefly, 100 ll cocktail solution (0.2 mM substrate [acetylthiocholine iodide] plus 0.4 mM DTNB in phosphate buffer) was added to react with 50 ll crude enzyme in a 96-well plate with 5 replicates each. The rate of reaction was monitored in kinetic mode for 2 minutes using a spectrophotometer (molecular device supported by Soft proMax-3 software) at 412 nm. The enzyme activity was expressed as lmole thiocholine hydrolyzed per minute per milligram of protein.

Brine Shrimp Hatchability Test

Statistical Analysis

Hatchability tests were performed according to the modified method of Migliore et al. (1997). The predetermined five concentrations (mg/ L or ppm) of each toxicant were prepared by the addition of 100 ll

Generated data were analyzed by Student t test. Statistical comparisons were done between control and exposure data, and p < 0.001, p < 0.01 and p < 0.05 were accepted as levels of statistical significance.

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Table 1. Median Lethal concentrations of different Organophosphorus pesticides against brine shrimp (Artemia salina) Compound

Regression Equation  b Y = (Y x) + bx

Acute Toxicity range 98% confidence limit Upper (mg/L)

Chlorpyriphos Profenofos Monocrotophos Acephate

Y Y Y Y

= = = =

3.07+1.21x 1.77+1.11x )24.05+6.57x )40.56+8.48 x

0.67 14.59 296.59 2607.70

LC50 (mg/L)

Relative Toxicity

0.385 € 0.08 7.71 € 1.48 262.68 € 17.30 2350.07 € 131

1.00 20.02 682.28 6104.08

Lower (mg/L) 0.09 1.82 228.77 2093.77

Fig. 1. Percent hatching success of A. salina cysts incubated with different OP pesticides. The EC50 values are marked in the figure. Values are mean € SE of five individual experiments

Results and Discussion The acute toxicity of four OP insecticides—CPP, PF, MCP, and ACEP—were tested against brine shrimp, A. salina, and their lethal concentrations (LC50) are listed in Table 1. Experimental data (Table 1) revealed that CPP, with an LC50 value of 0.385 € 0.08 mg/L, was the most toxic of the insecticides, whereas ACEP was the least toxic (2350.07 € 131 mg/L). The other two insecticides remaining, PF and MCP, were intermediately toxic (7.71 € 1.48 and 262.68 € 17.30 mg/L, respectively). The order of toxicity was CPP > PF > MCP > ACEP. Similar results were obtained in our earlier experiments, which indicated that the CPP was highly toxic to an aquatic organism, Oreochromis mossambicus, compared with other OP insecticides (Venkateswara Rao et al. 2003a, 2003b, 2004). Based on the toxicity data, CPP was considered to be more highly toxic than the other insecticides used in the present study. Relatively, PF, MCP, and ACEP were 20, 682, and 6104 times, respectively, less toxic compared with CPP (Table 1). Along with the toxic nature of compound, the percent hatching success were also estimated, and their effective concentrations (EC50 = the concentration required for a decrease of 50% in hatching control experiments) are presented

in Fig. 1. It is evident from the results that the CPP has once again been proved to be highly effective in inhibiting the hatching of brine shrimp, with an EC50 value of 0.123 € 0.005 mg/L. Although the order of hatchability appeared to be similar to the that of lethality, the effect on hatching property of the cysts varied among the toxicants. When compared with the EC50 values, it appeared that CPP and PF emerged as more highly potent to inhibit the hatching of cysts than other two toxicants. Among them, relatively, PF exhibited more deviation between the concentrations of LC50 and EC50, i.e., 46.16 times lower concentration of LC50 was enough to produce 50% inhibition of hatching (EC50), whereas 3.13, 1.50 and 1.4 lower concentrations showed the same effect for CPP, ACEP, and MCP respectively. In a separate set of experiment, 48-hour-old nauplii were exposed to LC50 concentrations of the toxicants for 24 hours, and their altered locomotor behavior (distance moved in meters per minute and swimming speed in centimeters per second) was studied by automated video-tracking system. The locomotor behavior of nauplii surviving exposure to lethal concentrations of the toxicants was severely impaired compared with controls as shown in Fig 2. Nauplii in the control experiments moved a distance of 1.617 € 0.13 m/min, whereas

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Fig. 2. Sample tracking distance moved (in meters per minute) of A. salina after 24-hour exposure to LC50 concentrations of different OP pesticides, and control, as recorded for 1 minute by computerized video-tracking system. *p <0.001; **p <0.01 (significantly different from control values) R = replicate

90

80

80

2.0

60 40 20

A

1.6

* 1.2

0 ACEP

MCP

PF

CPP

*

0.8

*

0.4

*

0.0 Control

ACEP

MCP

PF

CPP

Percent Inhibition in AChE activity

2.4 Swimming speed (cm/sec)

100

Percent Reduction

2.8

* *

70

*

60 50

**

40 30 20 10 0 CPP

PF

MCP

ACEP

Fig. 3. Swimming speed (in centimeters per second) by A. salina 24hour exposure to LC50 concentrations of different OP pesticides as recorded for 1 minute by computerized video-tracking system. Inset A: Percent decrease in swimming speed. *p <0.001 (significantly different from control values)

Fig. 4. In vivo effect of OP pesticides on AChE activity of nauplii of A. salina. Values are mean € SE of 3 enzymatic determinations/replicate. *p < 0.001; **p < 0.01 (significantly different from control values)

CPP-exposed nauplii exhibited a maximum decrease in distance (0.178 € 0.04 m/min) covered followed by those exposed to PF (0.409 € 0.06 m/min), MCP (0.606 € 0.07 m/ min), and ACEP (0.811 € 0.09 m/min). The order of percent decrease in their swimming speed (velocity) was CPP > PF > MCP > ACEP, which resulted in 89.00% € 3.32% (p <0.001), 74.68% € 2.13% (p <0.001), 62.48% € 1.87% (p <0.001), and 49.83% € 1.53% (p <0.001) inhibition, respectively (Fig 3). Earlier reports indicated that the decrease in velocity in other organisms could be caused by food deprivation, anoxic conditions, or inhibition of AChE enzyme (Nilsson et al. 1993; Westerterp 1977). In the present case, the exposed nauplii

were not facing either anoxic conditions or deficiency of food. Therefore, the altered locomotor behavior of nauplii exposed to the OP insecticides tested in this investigation may have been caused by the accumulation of acetylcholine (ACh), a neurotransmitter, at synaptic junctions, which interrupts coordination between the nervous and muscular junctions (neurotoxicity). Hence, the neurotransmitter enzyme (AChE) activity was studied in vivo in nauplii exposed to OP pesticides. AChE is known to be the site of action of anticholinesterase insecticides and their active intermediates. In the present experiments, the AChE enzyme activity of nauplii was measured in vivo 24 hours after exposure to LC50

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Fig. 5. Morphologic variations in brine shrimp (A. salina) after 24-hour exposure to LC50 concentrations of different OP pesticides

concentrations of four OP pesticides (Table 1), and the percentage inhibitions are presented in Fig 4. It is evident from Fig. 4 that AChE activity of nauplii exposed to LC50 concentrations of OP pesticides was significantly affected. Maximum AChE inhibition was 82% € 3.57% (p <0.001) in chlorpyrifos-exposed nauplii, whereas only 70.3% € 3.57% (p <0.001), 63.54% € 3.62% (p <0.001), and 36.9% € 2.47% (p <0.01) inhibition was observed in PF, MCP, and ACEP, respectively. Because AChE activity was inhibited by the toxicant (i.e., the OP) to result in accumulation of acetylcholine at neuromuscular junctions, it may have altered the locomotor behavior of the organism. A similar decrease in locomotor activity was observed in adult Carabid, Pterostichus cupreus, after copper treatment (Bayley et al. 1995) and in laboratory mice and five species of wild rodents (DellÕOmo et al. 2003) and termites (Venkateswara Rao et al. 2005) after exposure to an OP pesticide. Nauplii of brine shrimp undergo certain developmental changes, e.g., prominent growth as well as formation of one pair of mandibles and two pairs of antennae, of which the second pair consists of exopod, endopod, endite, swimming setae, etc, during the ages of 48 to 72 hours. At this crucial stage, 48-hour-old nauplii were exposed to lethal concentration of toxicants for 24 hours to study morphologic abnormalities appearing at the age of 72 hours. Significant morphologic differences were observed in all nauplii exposed to the four toxicants are presented in Fig. 5. Abnormalities included improper development of mandibles and underdeveloped endopod, endite, and the swimming setae of the second pair of antenna, which resulted in an imbalanced and irregular

swimming pattern. The degree of abnormalities, except for their size, was comparatively lower in ACEP-exposed nauplii.

Conclusion Unfortunately, only a limited number of studies has demonstrated a relation between OP pesticide occurrence and their toxicity on estuarine or marine species (Kuivila et al. 1995; Werner et al. 2000; DeLorenzo et al. 1999). However, given the effect of salinity on the toxicity of pesticides, differences in sensitivity could be expected between freshwater and marine species. It is obvious that pesticide residues are constantly entering into marine water, which may cause adverse effects to marine organisms. Therefore, a simple, reliable, and inexpensive bioassay with suitable biomarkers is essential to detect the adverse affects of OP pesticides on marine organisms. The present study proved that the brine shrimp assay could be a simple and accurate to assess the marine aquatic toxicity profile of any toxicant. It also demonstrated that OP insecticides alter the morphology and locomotor behavior of brine shrimp, A. salina. Among the toxicants, CPP exhibited more prominent adverse affects on the test organism than other three pesticides. Further experiments are warranted to study the effects of extensively used insecticides against different marine aquatic organisms. Acknowledgments. The investigators are thankful to the director of IICT for providing the facilities and constant encouragement through out the study. IICT Communication No. 040424.

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