Comparative Toxicity of Pyrethroid Insecticides to Two Estuarine Crustacean Species, Americamysis Bahia and Palaemonetes Pugio

Comparative Toxicity of Pyrethroid Insecticides to Two Estuarine Crustacean Species, Americamysis bahia and Palaemonetes pugio

Marie E. DeLorenzo,1 Peter B. Key,1 Katy W. Chung,1 Yelena Sapozhnikova,2* Michael H. Fulton1 1NOAA, National Ocean Service, Charleston, South Carolina, USA 2JHT Incorporated, contractor to NOAA, Charleston, South Carolina, USA

Received 21 September 2012; accepted 10 October 2012

ABSTRACT: Pyrethroid insecticides are widely used on agricultural crops, as well as for nurseries, golf courses, urban structural and landscaping sites, residential home and garden pest control, and mosquito abatement. Evaluation of sensitive marine and estuarine species is essential for the development of toxicity testing and risk-assessment protocols. Two estuarine crustacean species, Americamysis bahia (mysids) and Palaemonetes pugio (grass shrimp), were tested with the commonly used pyrethroid compounds, lambda-cyhalothrin, permethrin, cypermethrin, deltamethrin, and phenothrin. Sensitivities of adult and larval grass shrimp and 7-day-old mysids were compared using standard 96-h LC50 bioassay protocols. Adult and larval grass shrimp were more sensitive than the mysids to all the pyrethroids tested. Larval grass shrimp were approximately 18-fold more sensitive to lambda-cyhalothrin than the mysids. Larval grass shrimp were similar in sensitivity to adult grass shrimp for cypermethrin, deltamethrin, and phenothrin, but larvae were approximately twice as sensitive to lambda-cyhalothrin and permethrin as adult shrimp. Acute toxicity to estuarine crustaceans occurred at low nanogram per liter concentrations of some pyrethroids, illustrating the need for careful regulation of the use of pyrethroid compounds in the coastal zone. # 2013 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2013. Keywords: estuarine; crustacean; toxicity; pyrethroid insecticide; mysid; grass shrimp

INTRODUCTION 2008). Pyrethroids may enter aquatic ecosystems via spray drift, run-off, and wastewater treatment plant efﬂuent Pyrethroids are a widely used insecticide class applied in (USEPA, 2005). agricultural, turf grass, commercial, and residential settings Saltwater arthropods are more susceptible to pyrethroid to control a broad range of insect pests. Application of chemicals than freshwater arthropods (Solomon et al., synthetic pyrethroids in the United States is increasing as 2001). In particular, estuarine crustaceans are the most sen- they are used to replace other insecticides, such as organo- sitive saltwater species group for pyrethroid compounds phosphates and carbamates, in residential pest control and (Clark et al., 1989; DeLorenzo and Fulton, 2012). Estuarine in mosquito abatement programs (USEPA, 2005; Gan, crustaceans are also ecologically important as a food source for ﬁsh and in coastal nutrient recycling. Mysids (Ameri- camysis bahia) are small, shrimp-like crustaceans that *Present address: US Department of Agriculture, Agricultural Research occur in coastal estuaries from the Gulf of Mexico to Narra- Service, Eastern Regional Research Center, 600 East Mermaid Ln., Wynd- gansett Bay, Rhode Island (Verslycke et al., 2007). Their moor, Pennsylvania, 19038, USA. Correspondence to: M. DeLorenzo; e-mail: [email protected] life-cycle is approximately 17–20 days, and larval develop- Published online in Wiley Online Library (wileyonlinelibrary.com). ment takes place in a marsupium. Mysids feed on plankton DOI 10.1002/tox.21840 and detritus, and are consumed by a variety of estuarine

C 2013 Wiley Periodicals, Inc. 1 2 M. E. DELORENZO ET AL. organisms. Mysids have been used for decades in pyrethroids used for agriculture in California in 2005, with regulatory toxicity testing, since they can be obtained from reported uses of 37 550, 208 000, and 550 000 pounds AI commercial cultures, and are generally considered to be applied, respectively (Spurlock and Lee, 2008). more sensitive than many other test species (Verslycke et al., 2007). The grass shrimp, Palaemonetes pugio, is an abundant MATERIALS AND METHODS crustacean inhabiting tidal marshes along the US Atlantic and Gulf of Mexico coastlines (Anderson, 1985). Their Adult grass shrimp were collected from Leadenwah Creek, importance in the coastal ecosystem (serving as detritivores Wadmalaw Island, South Carolina (N 328 380 51.00@;W and as a prey item for many commercially and recreation- 0808 130 18.05@). Seawater used in the experiments was ally important fish species), along with the ease of supplied to the laboratory from the Charleston Harbor estu- obtaining multiple life stages for testing and demonstrated ary (N 328 450 11.52@;W798 530 58.31@), filtered (5 lm), sensitivity to numerous pesticides (Key et al., 2006), makes UV-sterilized, activated carbon filtered, and diluted with them an excellent toxicity test species. deionized water to adjust salinity. Field-collected grass The principal mechanism of action of pyrethroids is the shrimp were acclimated 7–14 days in a 76-L tank at 258C, disruption of sodium channel function in the nervous sys- 20 ppt salinity, and a 16-h light: 8-h dark photoperiod. tem (Miller and Salgado, 1985). Pyrethroids typically have 1 Adult shrimp were fed Tetramin fish flakes daily. Brood- low application rates and are usually co-formulated and ing traps were used to capture larvae released by ovigerous applied as mixtures. Pyrethroid formulations usually females. Grass shrimp larvae were fed cultured Artemia contain the synergist, piperonyl butoxide (PBO). PBO nauplii (24-h old) daily. Two life stages were used in the functions by inhibiting mixed-function oxidases, which are toxicity testing: adults (approximately 15–25 mm in length), involved in pyrethroid detoxification, and may enhance the and 24-h-old larvae (approximately 2–3 mm in length). toxicity of pyrethroids by 9- to 137-fold (Wheelock et al., The original stock culture of A. bahia, mysids, was 2004). Pyrethroids generally have low water solubility, obtained from Aquatic Indicators, St. Augustine, FL and high affinity for sediments and organic carbon, and gener- then cultured in 76-L aquaria (Lussier et al., 1988). Mysids ally have hydrolysis half-lives of days to weeks in aquatic were fed cultured Artemia nauplii (24-h old) daily. Mysid environments (Oros and Werner, 2005). Pyrethroids are culture conditions were as follows: temperature chiral compounds and have many isomers (Gan, 2008). 24.0–26.08C, dissolved oxygen 60% saturation, pH 7.6– Pyrethroids in the environment are subject to photochemi- 8.2, salinity 18–22 ppt, 16-h light:8-h dark photoperiod. cal transformation and sediment sorption. Degradation Tests were conducted using 7-day-old ‘‘young adults’’, not occurs primarily via microbial activity and photolysis sexually mature, and approximately 4–6 mm in length. (Cox, 1998). Reference tests were performed using sodium dodecyl Pesticide registration decisions are usually made using sulfate (SDS) to verify consistent responses in the batches data from only one estuarine crustacean species. Given the of grass shrimp and mysids used for testing. sensitive nature of this group of organisms to pesticides and the increasing use of pyrethroids, more data on different species and life stages are merited. The goal of this study Aqueous Toxicity Tests was to compare the sensitivity of adult and larval grass shrimp, and 7-day-old mysids to pyrethroid insecticides Aqueous static renewal 96-h bioassays were performed on using standard bioassay protocols for determining median two life stages of P. pugio, adults and larvae (1–2 days old), lethal concentrations (LC50 values). The pyrethroids tested and 7–day-old mysids. Stock solutions of each pesticide included lambda-cyhalothrin, permethrin, cypermethrin, (97.7% purity, Sigma-Aldrich Chemical Co.) were made deltamethrin, and phenothrin (sumithrin). Selection of these using pesticide-grade acetone. For each pyrethroid tested, chemicals was based on their frequent and diverse (agricul- there were five treatment concentrations plus a control. Test ture, mosquito control, and residential pest control) applica- solutions were made in 20 ppt salinity seawater, and an tions in coastal areas, and because they represent different equivalent acetone carrier concentration of 0.1% was chemical structures (permethrin and phenothrin are Type I administered for the control and each pyrethroid treatment. pyrethroids; cypermethrin, deltamethrin and lambda-cyha- For each pyrethroid tested, exposures were performed con- lothrin are Type II pyrethroids) and a range of chemical currently for most of the species and life stage comparisons. properties (water solubility values of 0.0002–0.0097 mg/L; Adult grass shrimp were exposed in 4-L glass jars with 2 log Kow values of 4.53–7.0 (Shamim et al., 2008; USEPA, L of test solution. There were three replicate jars per treat- 2008)). Total California synthetic pyrethroid sales in 2004 ment with 10 shrimp per jar. Adult grass shrimp were not were approximately 1.4 million pounds active ingredient fed during the exposure. Larval grass shrimp and 7-day-old (AI) (Spurlock and Lee, 2008). Lambda-cyhalothrin, mysids were tested in 600-mL beakers with a test volume cypermethrin, and permethrin were listed among the top six of 400 mL and there were three replicate beakers per

Environmental Toxicology DOI 10.1002/tox TOXICITY OF A. BAHIA AND P. PUGIO 3 treatment, with 10 animals per beaker. Larval grass shrimp Data Analysis and 7-day-old mysids were fed three to four drops of newly hatched Artemia daily. An airline was inserted into each Median lethal concentrations (24 h and 96 h LC50 values) test chamber through the lid using a 1-mL glass pipette. with 95% conﬁdence intervals (CIs) were determined using Experiments were conducted in an environmental chamber nominal chemical concentrations (SAS Probit Analysis, set at 258C, with a 16-h light:8-h dark photoperiod. Mortal- PROC PROBIT, SAS V.9.1.3, Cary, NC). Signiﬁcant dif- ity was assessed daily and any dead animals were removed, ferences (p \ 0.05) between LC50 values at each time point followed by a complete renewal of the test solution. Suc- for the different life stages/species were determined using cessful test criteria included \10% mortality in the controls the LC50 ratio test (Wheeler, 2006). and standard water quality parameter ranges (temperature 24.0–26.08C, dissolved oxygen 60% saturation, pH 7.6– 8.2, salinity 18–22 ppt). RESULTS

Chemical Analysis For lambda-cyhalothrin test concentrations of 3.125, 6.25, 12.5, 25, 50, 100, and 200 ng/L, the measured treatment con- Chemical analysis was conducted for two of the pyreth- centrations were: 3.1, 6.6, 14.4, 24.3, 49.6, 102.5, and 200.3 roids tested, lambda-cyhalothrin and permethrin (sum of ng/L, respectively. Lambda-cyhalothrin percent recovery cis and trans isomers), once at test initiation. Analytical ranged from 97.1% to 114.9% of the nominal values. For standards of lambda-cyhalothrin, permethrin and delta- permethrin test concentrations of 25, 50, 100, 200, 400, and hexachlorocyclohexane (d-HCH) were purchased from 800 ng/L, the measured permethrin treatment concentrations AccuStandard (New Haven, CT). Permethrin-phenoxy- were 27.0, 67.0, 91.9, 233.8, 398.2, and 680.6 ng/L, respec- 13 C6 used as an internal standard was from Cambridge tively. Permethrin percent recovery ranged from 85.1% to Isotope Laboratories (Andover, MA). Ethyl acetate, hex- 134.0% of the nominal values. Two lambda-cyhalothrin ane and HPLC water were purchased from Burdick & treatments, 12.5 and 25 ng/L, were resampled after 24 h. Jackson (Muskegon, MI). The percent losses from the initial measured concentrations Water samples were collected immediately after dosing were 1.7% and 17.5%, respectively. Measured control values from each treatment (one replicate only), spiked with an in- for both chemicals were below the detection limit. ternal standard and extracted using liquid/liquid extraction The 24 h and 96 h LC50 values determined for adult with ethyl acetate. For a reagent blank, HPLC grade water grass shrimp ranged from 23.20 to 385.81 ng/L and 5.80 to was used. The extracts were passed through anhydrous 161.00 ng/L, respectively, for the five pyrethroids tested, sodium sulfate, and reduced in volume to 0.5 mL using an with deltamethrin being the most toxic compound tested automated evaporation system (TurboVap II). The extracts followed by lambda-cyhalothrin, cypermethrin, permethrin, were transferred to autosampler vials with hexane rinse to and phenothrin (Table I). For larval grass shrimp the 24 h make a final volume of 1 mL. and 96 h LC50 values ranged from 20.80 to 284.23 ng/L Quantification of lambda-cyhalothrin and permethrin and 5.04 to 154.00 ng/L, respectively, with the same order was based on calibration curves with the internal standard of chemical toxicity (deltamethrin [lambda-cyhalothrin>- 13 permethrin-phenoxy- C6. The internal standard was added cypermethrin> permethrin> phenothrin) as adult grass to each sample prior to extraction and d-HCH was added as shrimp (Table I). The 24 h and 96 h LC50 values deter- a recovery standard before instrument analysis to enable mined for mysids ranged from 113.30 to 999.10 ng/L and the calculation of internal standard recoveries. Analysis 26.77 to 511.30 ng/L, respectively, and the order of chemi- was performed on a capillary gas chromatograph/mass cals from most toxic to least toxic after 96 h was deltameth- spectrometer (Agilent 6890N/5973) using electron impact rin>cypermethrin [lambda-cyhalothrin> permethrin> ionization operating in selective ion monitoring mode. A phenothrin (Table I). The order of chemicals toxicity after RH-5MS (Restek Corporation) capillary column (30 m 3 24 h was slightly different for mysids, with cypermethrin 0.25 mm 3 0.25 lm) was used. Calibration curves were being more toxic than lambda-cyhalothrin (Table I). linear for both pyrethroids from 0.5 to 200 ng/mL with r2 Most of the pyrethroid chemicals tested had very steep being greater than 0.99. toxicity curves (Fig. 1). For example, deltamethrin caused Quality assurance/quality control samples included one approximately 20% grass shrimp mortality at 4 ng/L and reagent blank, one matrix spike, one matrix spike duplicate approximately 100% mortality at 11 ng/L [Fig. 1(A)]. The sample, and one replicate sample for each batch of samples. increase in effect over time was also rapid. For example, Method detection limit was calculated based on weighted most of the toxicity observed occurred within 48 h for grass least-squares regression (Zorn et al., 1997) and was 1 ng/L shrimp exposed to deltamethrin, lambda-cyhalothrin, and for both analytes. Recoveries were estimated for fortified permethrin [Fig. 1(A,B)]. The increase in mortality over water samples (50 ng/L spike), and were 119% for lambda- time was more gradual for mysids exposed to deltamethrin, cyhalothrin and 101% for permethrin. lambda-cyhalothrin, and permethrin [Fig. 1(A,B)].

Environmental Toxicology DOI 10.1002/tox 4 M. E. DELORENZO ET AL.

Phenothrin toxicity also increased more gradually with time [Fig. 1(B)]. Larval grass shrimp had the lowest 96 h LC50 values for all pyrethroids tested, and 7-day-old mysids had the highest 96 h LC50 values (Fig. 2). Larval grass shrimp were significantly more sensitive to lambda-cyhalothrin (at 96 h, but not at 24 h) and permethrin (both 24 h and 96 h) than adult grass shrimp (Table I). Larval grass shrimp were significantly more sensitive to all chemicals tested than mysids ng/L (95% CI) juvenile mysid (Table I). Adult grass shrimp were significantly more 50 sensitive than mysids to all chemicals tested except for permethrin (Table I). When the five pyrethroids tested are considered as ratios of their 96 h LC50 values, the estimated concentration that would cause 50% mortality in larval and adult grass shrimp is similar (ratios 1.05–2.25), whereas it is higher for 7-day-old mysids compared to adult grass shrimp (ratios 1.22–9.83) and higher still for 7-day- 0.05) [a] 511.30 (441.70–591.70) [b] a

> old mysids compared to larval grass shrimp (ratios p 2.66–18.03).

ratio test results for differences between larval grass DISCUSSION 50 ng/L (95% CI) adult grass shrimp LC

50 The ﬁve pyrethroid compounds tested were highly toxic to both larval and adult grass shrimp, and mysids. All the acute toxicity values determined were less than 1 lg/L. Some pyrethroids were more toxic than others. There was an approximately 20-fold difference in toxicity among the pyrethroids tested for adult grass shrimp, an approximately 30-fold difference for larval grass shrimp, and an approximately 20-fold difference for mysids. For all test organ- [a] 161.00 (128.00–203.00) a isms, deltamethrin was the most toxic pyrethroid tested and phenothrin was the least toxic pyrethroid tested. There were interactions among time, chemical, and spe-

not determined. Letters indicate LC cies/life stage on acute pyrethroid toxicity. Toxicity was 5 strongly time-dependent for some chemicals; deltamethrin for example, had 24 h toxicity values that were at least ng/L (95% CI) larval grass shrimp LC

50 4-fold higher than their corresponding 96 h values for both

value (ng/L) and 95% conﬁdence interval (CI), of each pyrethroid determined for adult grass shrimp, 1 day old grass shrimp life stages and mysids. Phenothrin toxicity,

50 however, increased more gradually with time; 24 h toxicity values approximately 2-fold higher than 96 h values. There were also species differences, with mysids generally being

9696 5.04 (4.11–6.18) [a]96 6.24 (5.23–7.44) [a]96 19.08 (17.27–21.09) [a]96 50.00 (40.00–50.00) [a] 154.00 (139.00–170.00) 5.80 (4.70–7.15) [a]slower 11.44 (9.28–14.09) [b] 21.20 (16.84–26.74) [a] to 112.70 (93.66–135.70)exhibit [b] mortality 26.77 (20.91–34.27) [b] 112.50 (96.47–131.10) [c] 50.72 (41.29–62.29) [b] (e.g. 137.40 (128.30–147.10) [b] moribund individuals were apparent for several days), whereas grass shrimp mortality was more immediate. These differences could be attributed to physiological differences, such as lower sodium channel activity or greater reversibility of the pyrethroid effects on the mysid axons. For the ﬁve pyrethroids tested, toxicity was not related to log Kow value. Differences in pyrethroid toxicity to the estuarine crustaceans tested may be explained, in part, by structural differences. Type II pyrethroids are characterized by an alpha cyano group and have been shown to have Published in (Key et al., 2011).

a increased biological activity (greater nerve membrane TABLE I. Median lethal concentrations, LC larval grass shrimp, and 7-day-old juvenile mysids. ND shrimp, adult grass shrimp, and mysids. Values sharing the samePyrethroid letter were not signiﬁcantly different ( Time (h) LC Deltamethrin (Type 1I)Lambda–cyhalothrin (Type I1)Cypermethrin (Type I1) 24Permethrin 24 (Type 1)Phenothrin (Type 1) 43.68 (29.74–59.56) [a] 20.80 (12.24–29.59) [a] 24 24 72.30 (62.42–80.86) [a]depolarization 24 175.73 (156.26–198.85) [a] 37.82 (31.31–44.54) [a] 23.20 (19.80–26.84) [a] 284.23 (253.49–376.71) [a] and 59.92 (50.42–72.23) [a] block 273.78 (241.37–305.71) [b] 229.78 (ND) [b] of 113.3 385.83 (80.06–190.37) (333.42–456.57) [b] [b] sensory 174.50 (124.88–410.23) [b] 268.35 and (235.91–308.63) [b] motor 999.10 (896.80–1117.00) [c] axons)

Environmental Toxicology DOI 10.1002/tox TOXICITY OF A. BAHIA AND P. PUGIO 5

Fig. 1. Concentration-response curves generated for each pyrethroid and each species/life stage tested. (A) Type II pyrethroids (deltamethrin, lambda-cyhalothrin, and cypermethrin); (B) Type I pyrethroids (permethrin and phenothrin). Percent mortality was assessed at each time point during the exposure (average and standard error). [Color ﬁgure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

relative to their Type I analogs (Ecobichon, 1996). In this developed chemical metabolism pathways. Toxicity does study, Type II pyrethroids (cypermethrin, deltamethrin, and not always follow this trend; conversely, adults and newly lambda-cyhalothrin) were more toxic to estuarine crusta- hatched larvae were similar in sensitivity to deltamethrin, ceans than the Type I compounds (permethrin and pheno- cypermethrin, and phenothrin (Key et al., 2011). Further- thrin). Pyrethroid ether compounds are considered to be more, adults and older larvae (18 days) were similar in less toxic than the Type I or Type II esters of carboxylic sensitivity to the organophosphate compound chlorpyrifos acids. The pyrethroid ether, etofenprox, had an LC50 value (Key and Fulton, 1993), and adults were more sensitive of 1260 ng/L for adult grass shrimp (DeLorenzo and De than newly hatched larvae to ﬁpronil and endosulfan Leon 2010), which is 2.4–63 times less toxic than the Type (Key et al., 2003a). I pyrethroids, and 60–210 times less toxic than the Type II Mysids were less sensitive than adult and larval grass pyrethroids determined in this study. shrimp to the ﬁve pyrethroids tested. The difference in Larval grass shrimp were similar in sensitivity compared sensitivity varied, with mysids exhibiting approximately to adult grass shrimp for most pyrethroids tested, but the 18-fold lower sensitivity to lambda-cyhalothrin than the larvae were more than twice as sensitive to permethrin as grass shrimp, whereas mysids and adult grass shrimp were adult shrimp. Previous research has often found larval similar in sensitivity to permethrin. This may also relate to (1–2-day-old) grass shrimp to be generally more sensitive the aforementioned mechanistic differences between the than adult grass shrimp to various pesticides [e.g. chloro- Type I and Type II pyrethroids. The 7-day-old mysids thalonil (Key et al., 2003b), malathion (Key et al., 1998), tested in this study were less sensitive compared to 24- resmethrin (Key et al., 2005), bifenthrin (Harper et al., h-old mysids tested with cypermethrin (96 h LC50 5 27 2008), and etofenprox (DeLorenzo and De Leon 2010)]. ng/L) (Cripe, 1994) and permethrin (96 h LC50 5 20 ng/L) This may be due to higher metabolism and chemical uptake (Schimmel, 1983); thus life stage of the estuarine test spe- (larger surface to volume ratio) in the larvae, and less cies is clearly an important consideration. Cripe (1994) also

Environmental Toxicology DOI 10.1002/tox 6 M. E. DELORENZO ET AL.

Fig. 1. Continued

found mysids were four times more sensitive to cypermeth- Pyrethroids have been detected in a variety of environ- rin than another estuarine crustacean, 3- to 5-day-old pink mental samples, including surface waters and sediments, but shrimp, but 2.6 times less sensitive than pink shrimp to the concentrations are typically not available for marine and es- pyrethroid fenvalerate. tuarine waters. Lambda-cyhalothrin was detected from 110 Due to their sensitivity to insecticides, ease of collec- to 140 ng/L in surface water from agricultural watersheds in tion and handling, and high environmental relevance, Stanislaus County, California (He et al., 2008). The maxi- grass shrimp may be a preferred estuarine toxicity test mum expected environmental concentration (EEC) modeled species for assessment of pyrethroid risk in coastal eco- for lambda-cyhalothrin application to cotton was 100 ng/L systems. In this study, using 96-h static renewal bioas- (USEPA, 1993). Cypermethrin was found at 490 ng/L in an says, we found 7-day-old mysids to be less sensitive to Argentinian stream with nearby soybean ﬁelds (Jergentz pyrethroids than grass shrimp. It may not be appropriate et al., 2005). The maximum EEC for cypermethrin applica- to base acute toxicity thresholds on mysids alone tion to cotton was 2200 ng/L (USEPA, 2006). In a Canadian because they are not always the most sensitive crusta- pond, deltamethrin was measured at 24 000 ng/L after an cean species. However, mysids are more suitable for full agricultural application (CCME, 1999). An EEC is not life-cycle toxicity testing, particularly endocrine disrup- reported for deltamethrin, which is currently under USEPA tor testing (Verslycke et al., 2007), than grass shrimp registration review. Deltamethrin is also a metabolite of the due to their shorter life-span. pyrethroid tralomethrin. Permethrin was detected in

Environmental Toxicology DOI 10.1002/tox TOXICITY OF A. BAHIA AND P. PUGIO 7

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Environmental Toxicology DOI 10.1002/tox 8 M. E. DELORENZO ET AL.

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Environmental Toxicology DOI 10.1002/tox