Toxic Bioassays: LC50 Sediment Testing of the Insecticide Fipronil with the Non-Target Organism, Hyalella azteca

The use of the insecticide fipronil has dramatically increased in recent years, yet few studies have been performed compared to other popular pesticides. Although fipronil binds readily to organic carbon in soil, past studies have focused on fipronil toxicity in water but not the effects of fipronil toxicity in sediment. This study addresses the need for a broader range in fipronil toxicity research by using LC50 sediment testing to determine the acute toxicity of fipronil and its metabolite degradates, fipronil sulfone and fipronil sulfide, on the non-target organism Hyalella azteca. All pesticides were tested on two sediment combinations from different areas within California. Results showed that 9-day LC50 estimates were 227μg/kg for fipronil, 113μg/kg for fipronil sulfone, and 385μg/kg for fipronil sulfide in a San Pablo-Lake Anza sediment mixture. LC50 estimates for fipronil and its degradates tested in Ingram-Pacheco Creek sediment were 385μg/kg, 203μg/kg, and 485μg/kg, respectively. Although fipronil degradates are generally considered to be more toxic than its parent compound to freshwater invertebrates, only the LC50 for fipronil sulfone was found to be more toxic than the parent fipronil (by two-fold) while fipronil sulfide was found to be less toxic. LOEC and NOEC values support the conclusion that fipronil sulfone is toxic at lower concentration levels than the other compounds. This unexpected variability indicates that even within taxonomic groups, fipronil sensitivity may differ, underscoring the importance of exploring different variables such as sediment toxicity testing and use of different test species. Susan Ma Fipronil Toxicity May 8 2006 p. 2 Introduction Fipronil, an insecticide in the phenylpyrazole class, was first marketed in the U.S. in 1996, replacing several organophosphates in urban pesticide use (University of Minnesota 2005; TDC Environmental 2005a). It acts as a neurotoxin, disrupting the central nervous system by targeting GABA receptor-regulated chloride channels (Hainzl 1998). Although fipronil toxicity is highly selective towards arthropods, it also appears to bioaccumulate in fish, indicating a potential adverse effect to animals in higher trophic levels and to the health of aquatic ecosystems as a whole (US EPA 1996). Formulated in bait and granular form, and as seed treatment in agriculture, fipronil use has dramatically increased in recent years (TDC Environmental 2005b). However, the number of environmental toxicity tests that have been published to date are few compared to other widely used pesticides (e.g. Roundup and organophosphates such as chlorpyrifos and diazinon). Additionally, past studies have focused on fipronil toxicity in water, although fipronil binds readily to organic carbon in soil. Because many of the non-target organisms it affects are detritivores that eat organic debris, the additional dietary exposure of fipronil may result in higher toxicity levels than previous studies suggest (Schlenk et al. 2001; Chaton et al. 2002). Results of fipronil toxicity vary in the current body of scientific literature. A recent study done by Stark and Vargas (2005) found that fipronil causes lethal and sublethal effects to the water flea, Daphnia pulex, with LC50 estimates at 16 μg/L – similar to a Schlenk et al. study (2001) which established LC50 values at 14 and 19 μg/L for two species of crayfish. Chandler et al. (2004) found that even trace concentrations of fipronil significantly affected reproduction and development of the estuarine copepod, Amphiascus tenuiremis, with male copepods exhibiting higher acute sensitivity (male=3.5μg/L and female=13.0μg/L). In contrast to the Stark and Vargas (2005) study, Chaton et al. (2002) found that D.pulex was insensitive to the range of fipronil concentrations used in laboratory tests. While both papers examined acute toxicity in aqueous medium, they used different grades of fipronil – formulated and unformulated (technical grade) respectively. Specifically, formulated grades represent commercial products such as Regent 4SC (Stark and Vargas 2005) and Icon 6.2 FSTM (Schlenk et al. 2001) in which fipronil is the active ingredient. Technical grade is 98% pure fipronil as identified by gas chromatography (Schlenk et al. 2001; Chandler 2004). 1 Lethal concentration for 50% mortality in an exposed population Susan Ma Fipronil Toxicity May 8 2006 p. 3 The variability in results – from highly toxic to no effect – can be attributed not only to the different factors (grade of fipronil, test medium, test species) within each study, but also to differences in GABA receptor structure across different species (Schlenk et al. 2001). Table 1 summarizes the different LC50 analyses within current fipronil literature and the factors used to determine those values. Table 1 – Past studies examining fipronil toxicity Study Test species Grade Medium Type of toxicity test Results Chandler et al. 2004a Amphiascus tenuiremis Technical Aqueous Acute and life-cycle 96hr LC50 = 6.8μg/L (male=3.5μg/L; female=13.0μg/L) Chandler et al. 2004b Amphiascus tenuiremis Technical Sediment Chronic Reproductive effects at 0.25-0.5μg/L for parent compound Chaton et al. 2002 Daphnia pulex Technical Aqueous Acute No significant effects to D. pulex Schlenk et al. 2001 2 species Procambarus Formulated (Icon 6.2 FSTM) Aqueous 96hr LC50 = 14-19 μg/L Stark and Vargas 2005 Daphnia pulex Formulated (Regent 4SC) Aqueous Acute 48hr LC50 = 16μg/L This variability in toxicity estimates for fipronil also exists in studies of fipronil degradates – sulfone, sulfide, and desulfinyl – compounds generally considered to be more toxic than the parent fipronil (U.S. EPA 1996; Connelly 2001; Schlenk et al. 2001). The level at which these degradates are classified as more toxic vary, with the U.S. EPA (1996) establishing the metabolite sulfone as 6.6 times more toxic and the metabolite sulfide and photodegradate desulfinyl as 2 times more toxic to freshwater invertebrates than the parent fipronil compound. The dramatic increase of fipronil use in recent years coupled with its varying effects across different species justifies establishing a wide range of toxicity data for a variety of species, both in the laboratory and in situ. To date there has been no research testing fipronil with Hyalella azteca, a standard specimen widely used in pesticide toxicity tests because of their sensitivity to pollutants and important ecological role in many aquatic food chains (US EPA 2000; Watts 2002). Similarly, few studies (Chandler et al. 2004b) have addressed the acute toxic effects of fipronil in sediment despite its high affinity for soil particles; particularly the sulfone and sulfide Susan Ma Fipronil Toxicity May 8 2006 p. 4 metabolites have OC K values that indicate a very high persistence in soil environments 2 (Connelly 2001). The purpose of this study is to determine the acute toxicity of fipronil and its metabolite degradates, sulfone and sulfide, to H. azteca by finding the lethal concentration necessary to kill 50% of an exposed population (LC50) and to find the lowest observable effect concentration (LOEC). I hypothesize that the sulfone and sulfide degradates will be at least two times more toxic, exhibiting lower LC50 and LOEC values, than the parent fipronil compound. The results of this project can be directed towards the development of more appropriate pesticide regulation, especially in areas of new urban development in which fipronil is frequently applied. Methods This experiment was designed to establish the LC50 and LOEC levels for Hyalella azteca, a freshwater amphipod, exposed to technical-grade fipronil obtained from ChemService (West Chester, PA, USA). 9-day LC50 tests were conducted according to the standard protocols for testing sediment toxicity with freshwater invertebrates as outlined in the EPA manual (U.S. EPA 2000). H. azteca is considered to be an ideal specimen in pesticide toxicity research because it is more sensitive to pollutants than other species; therefore if a pesticide is non-toxic towards H. azteca, it will also be non-toxic to other organisms (Ibid). In addition to values for fipronil, this experiment will determine the LC50 and LOEC for the sulfone and sulfide metabolites of fipronil. Fipronil, fipronil sulfone, and fipronil sulfide toxicities were tested using two different sediment combinations, San Pablo-Lake Anza and Ingram Creek-Pacheco Creek. Both sediments have high total organic carbon (TOC) levels that are reflective of the soil environments in which fipronil would normally be applied. The combination of Lake Anza and San Pablo Dam sediment yielded a TOC of 2.09%, and the combination of Ingram Creek and Pacheco Creek sediments had a TOC level of 5.00% (higher pesticide concentrations were used in the latter to account for the higher TOC). Data was collected from two 9-day tests (one for 2 OC K : organic carbon-water partitioning coefficient. OC K for fipronil is 803, while OC K values for sulfone and sulfide are 4209 and 2719 respectively. 3 LC50 and LOEC estimates are common parameters used to develop regulations on pesticide toxicities. 4 Total organic carbon (TOC): pesticides bind to the organic carbon content in soil; therefore sediment with a higher TOC will have greater affinity for the pesticide. Susan Ma Fipronil Toxicity May 8 2006 p. 5 each sediment combination). Six different concentrations were tested for each pesticide based on results of a range finding test, and three replicate beakers were set up for the different sedimentpesticide groups with ten juvenile H. azteca in each sample beaker. Sediment and test organism preparation Test sediments were collected from San Pablo Dam (El Sobrante, CA), a drinking water reservoir devoid of agricultural runoff and other non point source pollutants (Amweg and Weston, pers. comm), and from Lake Anza (Berkeley, CA), a site similarly free of pesticide residues. Sediments were sieved through a 1mm sieve to ensure a roughly homogenous texture in sediment grain sizes, and then mixed to achieve a 20/80 combination of Lake Anza to San Pablo Dam sediment. Sediments from Ingram Creek (Stanislaus County, CA) and Pacheco Creek (Santa Clara County, CA) were also sieved and then combined in a 50/50 blend. Using data from a range-finding test, a control group, solvent-control group, and spiking concentrations ranging from 17-600μg/kg for San Pablo-Lake Anza and 61-2667μg/kg for Ingram-Pacheco Creek sediments were chosen to predict a theoretical survival range between 0 – 100%. The San Pablo-Lake Anza (SL) sediment and Ingram-Pacheco Creek (IP) sediment was separated into groups, each spiked with a different concentration of technical-grade fipronil dissolved in an acetone carrier for a stock dilution of 0.25μg/ml. The amount of solvent in each concentration group was normalized with =260μl of acetone in SL and =1316μl acetone in IP. The spiked sediment was then homogenized with a drill fitted with a steel auger and aged for 12 days at 4°C . The aging process allows the pesticide to combine with the organic carbon in the soil, therefore decreasing the bioavailability of the pesticide to the test organisms (reflecting conditions that are more realistic). Three days prior to commencement of the test, juvenile H. azteca were removed from mature cultures using a 500 μm sieve and retained on a 350μm sieve. Retained juveniles were incubated at 23°C and fed a mixture of trout chow, yeast, and cyanobacteria until initiation of the tests. Experimental procedure One day prior to commencement of the tests, three replicate 400mL beakers were set up for each concentration of fipronil-spiked sediment and for the control. 50ml sediment samples were added to each beaker along with 300ml of moderately hard 5 Range finding test: small-scale experiment testing wide range of pesticide concentrations from 1-1000μg/kg to yield preliminary data for further tests. 6 The half-life of fipronil under photolysis is slow, about 34 days (Connelly 2001). Sediment groups are kept at a constant temperature to ensure that fipronil does not degrade during the testing process. Susan Ma Fipronil Toxicity May 8 2006 p. 6 water (Milli-Q deionized water reconstituted with salts). The beakers were allowed to equilibrate overnight in a 24°C water bath. Samples of the sediment were also removed for chemical analyses to verify the actual concentration of fipronil within one of the groups. This same procedure was used for sediments spiked with fipronil degradates, sulfone and sulfide. At day zero, ten H. azteca juveniles were counted under a microscope and added to each beaker. The beakers were placed in the water bath for the duration of 10 days on a 16:8hr light:dark cycle. Beakers received automatic water changes twice a day and were fed daily with 1.0mL of trout chow-yeast-cyanobacteria slurry. Before water renewal, water samples were removed on the 2 and 10 day and water quality parameters were analyzed for dissolved oxygen, temperature, alkalinity, hardness, ammonia, pH, and conductivity. On the tenth day, the contents of each beaker were sieved through a 425μm screen, and surviving H. azteca counted. Data analysis Toxicity data was analyzed using ToxCalc 5.0 Software (Tidepool Scientific Software). LC50 values were determined using the trimmed Spearman-Karber method with Abbott’s correction and the significance of LOEC and no observable effect concentration (NOEC) values were determined using Dunnett’s one-tailed t-test. The trimmed SpearmanKarber method uses the input of pesticide concentration levels and mortality data to calculate LC50s with a 95% confidence interval (Hamilton 1977). Specifically, the alpha value represents the percent of extreme values to be trimmed from each end/tail of distribution (or maximum and minimum likelihood). Results LC50, LOEC, and NOEC 7 estimates were calculated using the mortality data at each of the different sediment-spiked concentration groups. Mortality results were generally consistent across the three replicate beakers of each sediment-pesticide group. Each data point in the population-level dose response graphs of Figures 1, 2, and 3 represent the average mortality data of the three replicates in San Pablo-Lake Anza sediment (Figures also indicative of IngramPacheco Creek response). A regression line was generated using these points, from which the LC50 estimate was then interpolated. LC50 analysis yielded zero survival at the highest concentrations and 80-100% survival at the lowest concentrations with some variability within the control. Due to the inconsistencies in 7 NOEC – No Observable Effect Concentration Susan Ma Fipronil Toxicity May 8 2006 p. 7 the control group but the high survival rates among the lower concentrations, these data points were treated as controls, and the trimmed Spearman-Karber method was chosen to analyze the rest of the data. 0 10 20 30 40 50 60 70 80 90 100