Arch. Environ. Contam. Toxicol. 51, 79–85 (2006) DOI: 10.1007/s00244-005-0072-1

Cypermethrin Toxicity to Aquatic Life: Bioassays for the Freshwater Prawn Palaemonetes argentinus P. Collins,1,2 S. Cappello1 1 2

Instituto Nacional de Limnologa, Jos Maci 1933, 3016 Santo Tom, Santa Fe, Argentina Escuela Superior de Sanidad ‘‘Ramo´n Carrillo,’’ Fac Bioq y Cs Biol, Universidad Nacional del Litoral, Sante Fe, Argentina

Received: 23 May 2005 /Accepted: 19 September 2005

Abstract. Cypermethrin (CY) effects were evaluated in freshwater prawn Palaemonetes argentinus, a common member of the aquatic fauna in the vegetated littoral of lotic and lentic environment of La Plata basin. LC50 was calculated, and oxygen uptakes together with ammonia-N excretion were related to biocide concentrations. Behavioral and growth changes were analyzed, and a unique application of CY was evaluated in P. argentinus micropopulations. LC50 and their 95% confidence limit were 0.0031 lg CY L)1 (0.0023– 0.0039) for 24 h and 0.0020 lg CY L)1 (0.0014–0.0027) for 96 h. Oxygen uptake and ammonia-N excretion increased in the prawns kept in CY solutions. The behavioral effect was hyperactivity. Although prawns in biocide groups have null or negative growth, the intermolt period was 246% larger than in the control group. Moreover, the mortality in the second cycle was 100%. A unique application of pyrethoid provoked high mortality after 50 days. The low concentrations of this biocide affected the survival and altered the prawn metabolic activity, behavioral and ecdysis cycle. These results suggest that juveniles of P. argentinus are much more sensitive to CY pollution than other crustaceans, fish, and tadpoles.

Pesticide pollution is frequently found when drinking water sources of surface and ground water are monitored in Europe (Schiavon et al. 1995) and probably may occur in other parts of the world. This situation is due to the use of insecticides in intensive agriculture without observing the basic norms of care and protection of the nature on the part of man. Cypermethrin (CY), (RS)-alphacyano-3-phenoxybenzyl (1RS)-cis-, trans-3-(2,2 dichlorovenil)-2,2-dimethylcyclopropane carboxilate, is a commonly used pesticide in urban and agricultural environments. It is a highly active synthetic type II pyrethroid and is considered a good pest control agent in agriculture, horticulture, and aquatic systems with low toxicity to nontarget animals (Casida 1980).

Correspondence to: P. Collins; email: [email protected]

However, investigators have shown that the biocidal action produces alterations in nontarget animals through their action in the ion conductance of nerve cell membranes by increasing transmembrane sodium influx and inhibiting ion-dependent ATPases of arthropods and amphibians (Berlin et al. 1984; Salibian and Marazzo 1995). Moreover, it has recently been postulated that it induces apoptosis in mammals (rats) and amphibian tadpoles (El-Gohary et al. 1999; Izaguirre et al. 2000). Possibly CY could affect decapod crustacean in the same way (Williner and Collins 2003). Palaemonetes argentinus is an abundant nonmigratory freshwater prawn (Crustacea, Decapoda, Palaemonidae) of La Plata System, a region in Argentina that is characterized by intensive agriculture and urbanization. Moreover, this prawn, as others, integrates the diet of several groups of aquatic and terrestrial animals such as fish (Bonetto et al. 1963; Cabrera et al. 1973; Oliva et al. 1981), reptiles, birds (Beltzer 1983; Beltzer and Paporello 1984; Beltzer 1984; Navas 1991; BE and Darrieu 1993; Lajmanovich and Beltzer 1993; Navas 1993, 1995), and mammals (Massoia 1976; Bianchini and Delupi 1993). The decrease of this trophic resource by biocide could affect the survival and abundance of its predators, as well as significantly impact the aquatic food web. The aim of this work was to examine, under laboratory conditions, the acute toxicity of CY to P. argentinus juveniles and to determine the effect of the insecticide on oxygen uptake and nitrogen excretion. Moreover, sublethal effects on growth and behavior were determined, and the action of a unique application of CY on a population of P. argentinus was evaluated.

Materials and Methods Palaemonetes argentinus juveniles were collected from Salado River in the Paran River floodplain (31
39¢ S; 60
41¢ W), Santo Tom, Argentina. The prawns were acclimatized for 7 days in glass tanks with artificial water (APW): pH 8.1, conductivity 410 lmhos cm)1, dissolved oxygen (DO) concentration 5.5 mg L)1, hardness 83 mg L)1 as CO3Ca at 25 € 2
C and 14–10 h light–dark photoperiod. Juveniles were fed ad libitum during the laboratory acclimation period with pelletized diet (protein: 36%; lipid: 10%) (Collins and Petriella 1996).

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Acute Dose The 96-h acute toxicity test of CY was conducted according to USEPA (1975) standard methods, with juvenile prawns (Boschi 1981) and without feeding scheduled. Glass tanks (35 cm diameter and 30 cm high) filled with 4 L of water (APW) and 10 juveniles (average weight: 0.01 € 0.006 g) were used in the experiment. The assayed product was Sherpa, a commercial formulation of Bayer containing 25% of CY in xylene. In the final acute toxicity survival test, the concentrations used were 0.0250, 0.0125, 0.0062, 0.0031, 0.0012, and 0.0006 lg CY L)1. Controls were conducted in APW with or without vehicle (xylene) during the same period. Both control and test solutions were made by triplicate. Mortality was recorded every 24 h. The controls exposed to xylene did not show a significant difference from the controls maintained in water without xylene in survival, oxygen uptake, ammonia excretion, and behavior.

Oxygen Uptake and Ammonia-N Excretion Analyses of oxygen uptake and ammonia-N excretion experiments were conducted in Pyrex glass bottles (250 ml € 1.5 ml volume) filled with air-saturated CY solution. One prawn was placed in each bottle, covered, and placed in a 25 € 2
C room by 24 h. There were three CY solutions: 0.0002, 0.0025, and 0.0250 lg CY L)1, and a control without pesticide. Each treatment had 10 replicates. Measurements of dissolved oxygen (DO) were taken at the beginning of the experiment and at each hour during the first 3 h. DO was measured with a YSI Model 57 DO meter and electrode probe. Ammonia-N was determined at the beginning and end of the experiment, and water samples were analyzed according to the Nessler Method (Rodier 1981) (Test Kit Model FF)2 of Hatch). These two parameters, oxygen consumption (mg g)1 h)1) and ammonia-N excretion (lg g)1 h)1), were calculated by multiplying the observed difference of DO and ammonia-N by the water volume in each bottle, and dividing the result by the wet body weight and time elapsed (h).

fed with pelletized diet (Collins and Petriella 1996), and the water with and without CY was renovated every 2 days [for more considerations of handling see Collins and Petriella (1999)].

Unique Application Effects Concentration shown to be effective in previous exposures was used to show the effect to the population level exposed only in one time. Prawns were kept in eight 0.9-m2 plastic containers with a density of 55 ind m)2. A continuous flow of water without biocide of 1 ml s)1 was used in all containers during 15 days. Afterwards, the solutions (0.0001, 0.001, and 0.01 lg CY L)1) were applied in the same flow rate during 1 day. Later, the flow returned again to water without CY. Daily the prawns were fed with fish muscle ad libitum and the mortality was registered in all containers. The exposure lasted 45 days. At the end, ecdysis phase according to the setogenesis in uropod (development of setas of the new exoskeleton) was observed in each prawn survivor (Drach and Tchernigovtzeff 1967).

Data Analysis The LC 50 with confidence limits (p < 0.05) were estimated by using a Probit Analysis Program (Finney 1971). Data from control and experimental groups were analyzed by one-way analysis of variance (ANOVA) in conjunction with Tukey test (Zar 1996). Differences in oxygen uptake and ammonia-N excretion were evaluated with ANOVA. ANOVA was also used to determine the significance of each behavioral response. The isolated growth was represented by CLtþ1ðpostmoltÞ ¼ a þ b CLtðpremoltÞ

ð1Þ

where a is CLt+1 intercept and b is the constant of growth. All the correlation coefficients were tested for significance using ANOVA. Prawn survival in the experiment of a unique dose was analyzed by ANOVA in conjunction with Tukey test (Zar 1996).

Behavioral Changes Variation in the locomotion activity of prawns was measured with several CY concentrations (control, 0.0002, 0.0025, 0.0250 lg CY L)1). Prawns were individually observed for swimming and walking behavior. Ten plastic tanks with one prawn were filled with 3 L of CY solutions of each concentration. The observations lasted 5 minutes and were carried out twice for every aquarium with a lapse among each observation of 10 minutes. A bout was defined as a single movement or a series of similar uninterrupted movements. The behavior was measured as the number of bouts of different movements that occurred during each observation.

Individual Growth In another experiment, three solutions—0.0001, 0.001, 0.01 lg CY L)1—and control without CY were used to show the pyrethroid effects on growth (or ecdysis cycle) of this species. P. argentinus were kept individually in glass container (1 L) and acclimatized (25 € 2
C and 14:10 L:D photoperiod) for 15 days before the beginning of the trial. The first molt was discarded and 3 days after the second molt, in intermolt stages, the prawns were placed into the bottles with the solutions. Each treatment had 10 replicates. Isolated growth was analyzed with pre- and postmolt length data (CL). The prawns were

Results Acute Dose There was no mortality in the control and xylene groups of P. argentinus. Within the range 0.0006 – 0.0012 lg CY L)1, the survival rate for 24 h was 100%. At 0.0031, 0.0062, 0.0125, and 0.0250 lg CY L)1, the survival rate showed significant differences when it was compared with the control group (ANOVA F = 225.68; p < 0.0001) (95% TukeyÕs confidence intervals) (Fig. 1). The LC50 diminished with time, and the 24-LC50 differed from the 96-LC50 by 35.5%, but the minimum and maximum values indicated a nonsignificant difference (ANOVA F = 3.21; p = 0.1477). The 96-LC50 of CY for P. argentinus juveniles was 0.0020 lg CY L)1 (Fig. 2).

Oxygen Uptake and Ammonia-N Excretion Oxygen uptake in control prawns was similar during the observation periods, whereas prawns in CY solutions varied in

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Table 1. ANOVA table of oxygen consumption during 3 h and ammonia–N excretion determined at the beginning and the end of the experiment of Palaemonetes argentinus exposed to different cypermethrin (CY) concentrations Oxygen consumption Among hours Control 0.0002 (lg CY L)1) 0.0025 (lg CY L)1) 0.0250 (lg CY L)1) Ammonia-N excretion Among CY concentrations a

Df

Sum of squares

F

p

20 20 20 20

4.42 50.44 170.14 141.95

1.90 7.35 4.29 4.65

0.1778a 0.0046 0.0299 0.0236

40

0.2596

100.69

<0.0001

Values no-significant difference.

Fig. 2. Relationships of LC50 to the exposure period for Palaemonetes argentinus (n total = 240)

intervals), whereas in the other prawn groups the ammonia-N excretion increased according to CY concentrations (Fig. 4).

Fig. 1. Survival curves for Palaemonetes argentinus juveniles exposed to cypermethrin during 96 h

its consumption (Table 1; Fig. 3). In the first hour, the oxygen uptake increased significantly in the treatment groups with pyrethroid, but the minor concentration (0.0020 lg CY L)1) was shown to decrease in the second hour. The same facts occurred in the other concentrations during the third hour, with the differences among control and treatments not statistically significant (ANOVA F = 1.09; p = 0.3771). The nitrogen excretion measured as ammonia-N was different among treatment and control groups (Table 1). However, the control and the minor concentration (0.0020 lg CY L)1) values were similar statistically (95% TukeyÕs confidence

Behavioral Changes The different bouts considered in P. argentinus were stationary, walking, and swimming. The most important and visible neurotoxic effect of CY upon this freshwater prawn was hyperactivity (walking and swimming). This happened immediately after they were placed in the pyrethroid solution. In the aquariums, the prawns swam inclined and in a circle, although for short distances they walked forward or backward on their pereiopods. Comparison among control and treatments groups revealed significant differences in the activity (ANOVA F = 17.33; p = 0.0093). The swimming increased with the highest concentration, whereas 0.0002 and 0.0025 lg CY L)1 groups have similar activity level (95% TukeyÕs confidence intervals), being more mobile than prawn control (Fig. 5).

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Fig. 3. Oxygen uptake of Palaemonetes argentinus exposed to several concentrations of cypermethrin during 3 h

Fig. 5. Percentage of swimming prawns exposed to several concentrations of cypermethrin

Growth and Intermolt Period Isolated prawns adapted well to the culture system, with 100% survival in the control group. However, the 0.0001, 0.001, and 0.01 lg CY L)1 groups had 10, 40, and 70% of mortality, respectively, during the first ecdysis cycle; thereafter, mortality was 100% in all CY groups. The growth expressed as length increase occurred in the control group, with a slope of 1.02 between premolt and postmolt cephalothorax length, but the slopes between premolt and postmolt length in CY groups were 0.53, 0.63, and 0.71, indicating a prawn size reduction (Fig. 6A). The intermolt period was directly proportional to size. Furthermore, the ecdysis cycle was significantly longer (ANOVA F = 118.80; p < 0.0001) in prawns reared within CY solutions (22.7 € 4.44 days) (Fig. 6B) than in control groups (9.2 € 1.32 days). On the other hand, the pyrethroid affected the growth rate. Treated prawns showed null or negative values corresponding to long elapsed intermolt time (Fig. 6C), whereas the prawn controls had a rate mean of 12.6 € 1.87 mm with 9.2 € 1.32 days of ecdysis cycle.

Unique Application Effects

Fig. 4. Ammonia-N excretion between beginning and end of the experiment of Palaemonetes argentinus exposed to several concentrations.

A high percentage of mortality (33.1 € 18.04%) occurred during the first 2 days after the unique application of three CY solutions (0.0001, 0.001, and 0.01 lg CY L)1) (Fig. 7). The values were significantly different among control and

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Discussion

Fig. 6. Growth in Palaemonetes argentinus exposed to several cypermethrin concentrations. (A) Relationships between CL premolt and postmolt; (B) relationships between intermolt period and CL; (C) relationships between growth rate and intermolt period (¤) p = 0, ()) p = 0.0001, (d) p = 0.001, (s) p = 0.01

0.001 lg CY L)1, control and 0.01, and 0.0001 and 0.01 lg CY L)1 (95% TukeyÕs confidence intervals). After 50 days, mortality significantly increased in all CY treatments compared to controls (ANOVA F = 7.23; p = 0.0115). Survivor prawns of all treatments were in initial premolt stage, whereas control prawns were in premolt and postmolt at similar amounts (20–30%), and the intermolt stage was the most abundant (approximately 50%).

Pyrethroids are relatively nontoxic to birds and mammals but extremely toxic to aquatic organisms, including fish, amphibians, and invertebrates (Jolly et al. 1978; Holcombe et al. 1982; Little et al. 1993; Izaguirre et al. 2000). In laboratory tests, some authors mentioned that the ranges of 96 h LC50 were 0.01–5 lg CY L)1 for aquatic invertebrates, 0.4–2.8 lg CY L)1 for fish, and 129–1012 lg CY L)1 for amphibians (Pillai et al. 1989; WHO 1989; Izaguirre et al. 2000). CY toxicity in the freshwater prawn P. argentinus occurs in lower concentrations than those evaluated in previous works. Moreover, this study indicates that native populations of juvenile P. argentinus are affected by the CY application, even at a lower concentration than LC50 mentioned as reference (CASAFE 1995). There is very scarce information in the effects of the CY on the decapod crustaceans. In this study, lethal toxicity occurred at lower concentrations than the values reported for other freshwater decapods, e.g., Crangon septemspinosa (0.01 lg L)1) (McLeese et al. 1980) and Macrobrachium rosembergii (96 h LC50: 0.000031 ppm) (Pillai et al. 1989), a prawn that has a migrant behavior to saline water to complete larval development. Furthermore, marine shrimp are less sensitive, with LC50 values between 0.0012 lg L)1 and 0.11 lg L)1 (Clark et al. 1989; Cripe 1994). This wide range could be due to the stage of life or population variability. On the other hand, the LC50 in P. argentinus was as low as in Trichodactylus borellianus, a freshwater crab with abbreviated larval development (0.0097 lg CY L)1) (Williner and Collins 2003). Despite the great dispersion values reported for different species, all of them indicate a very high toxic level. The oxygen uptake increase in organisms exposed to CY is due to a higher metabolism. The increase of the ammonia-N excretion and heightened locomotor activity confirm this. Similarly, respiration rates of larval P. pugio exposed to toxic levels of the pesticide methoprene and fenvalerate were elevated along with elevated ammonia excretion (McKenney and Celestial 1995; McKenney et al. 1998), suggesting that the metabolism may be associated with the toxic mechanism of the pesticide (McKenney et al. 1998). Available data on oxygen consumption demonstrate a wide range of values under several CY concentrations and time. The highest metabolic rates occur during the first hours of the biocide concentrations, where the difference between control and treatment animals in the oxygen uptake will probably be a direct relationship between the pyrethroid presence and the locomotor activity. This biocide in the first hours provokes a constant activity, as swimming and walking, similar to the neurotoxic effects mentioned by Salibian (1992) for amphibians. In contrast, control prawns spend much of their time in an inactive state interrupted by short periods of locomotion. The quantity of ammonia excreted by crustaceans varies according to external and internal factors, including diet, injury, molting, temperature, presence of other animals (Parry 1960), but may also include swimming, nitrogen excretion, and CY concentrations. Furthermore, CY affected the ecdysis cycle, provoking a null or negative growth. Also, the intermolt time was severely altered, augmenting the period between molts. Alterations in

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and amphibians and may be an excellent indicator organism for aquatic stress provoked by pyrethroids.

Acknowledgments. The authors wish to thank the helpful suggestions of the anonymous reviewers that improved the text. These studies were supported by a grant PICTO UNL No. 01-13232 BID1201 grant.

References

Fig. 7. Mortality of Palaemonetes argentinus exposed to unique doses of several concentrations of cypermethrin. Black bar: 48 h after application; white bar: 50 days after application

Y-organ and seno gland may occur, affecting the production and storage of the inhibitory molt hormone, or more integrally, the neurohormonal system located in the eyestalks. CY being a neurotoxin could affect the synthesis process regulation in the nerve cells and especially upon the transmembrane sodium influx, mainly in the neurohormonal system. This indicates that, in spite of the fact that concentrations were lower than the lethal dose, CY affects the ecdysis cycle, increasing the mortality and possibly the reproductive events as well. Only one CY application affected the P. argentinus population in 48 h, but was still lethal after 50 days. Residual effect could be due to the alterations in the neurohormonal system located in the eyestalk. Similar effects occur in ablated eyestalk prawn, where the hormonal imbalances of inhibitory molt hormone and salt deposition hormone were the main reasons for mortality (Chu and Chow 1992; Collins et al. 1992; Collins 1996). On the other hand, the average rate of pyrethroid application to crops is 10–200 g of active ingredient (CY ha)1) (WHO 1992). In Argentina, the levels of application on farmland oscillate between 1.5 g and 120 g of active ingredient (CY ha)1) (CASAFE 1995), and its degradation rates were similar for both laboratory and field surveys (Roberts and Standen 1981). These applications may be washed into rivers and streams when there is heavy rainfall, and reach values that could be higher than the LC50 determined in this work. Where the CY enter the rivers, there may be a biological gradient downstream from the outfall (Walker et al. 2001) but P. argentinus population may reappear down stream, considering that the degradation rate is approximately 20% daily without aquatic ferns and the estimated half-life is 31 days (Lajmanovich et al. 2003). From an ecological point of view, the decapods are more sensitive to pyrethroid toxicity than fish

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