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Pesticide usage on the Southern High Plains and acute toxicity of four chemicals to the fairy shrimp Thamnocephalus platyurus (Crustacea: Anostraca).

Abstract -- Extensive pesticide use on crops grown on the Southern High Plains (SHP) represents a considerable anthropogenic stressor to ephemeral aquatic ecosystems. These short-lived aquatic ecosystems, known in the southwest as playas, are epicenters of biodiversity on the High Plains. Cotton is the major agricultural crop grown on the SHP, accounting for over half of all cotton produced in Texas. Currently there are 67 different chemicals used to control cotton pests, and when other crops such as grain sorghum are included the number approaches 100. Thamnocephalus platyurus is a fairy shrimp indigenous to the Southern High Plains that is also available commercially. In addition it is used as an invertebrate model for water quality and toxicity testing. Acute toxicity of four agricultural pesticides widely used on the SHP (Methyl Parathion 4E, Tempo[R] SC Ultra [active ingredient cyfluthrin], Roundup[R] [glyphosate], and Karmex[R] DF [diuron]) was determined using laboratory-derived T. platyurus. Twenty-four hour old nauplii experienced mortality (48 hour LC50) at concentrations ranging from 10.99 [micro]g/L for Tempo[R] SC Ultra and 1.248 mg/L for Roundup[R]. These results suggest that the current pesticide application rates have the potential to endanger the native playa invertebrate T. platyurus.


The Southern High Plains (SHP) is an 8.2 million hectare, 27 county region that starts at the Canadian River in west Texas and extends south past Lubbock, Texas, and west into eastern New Mexico (Fig. 1; Smith 2003). The major land use throughout this region is agriculture, with cotton being the most commonly grown crop species contributing 64% of Texas' crop (Crop Profile for Cotton in Texas 1999). Grain sorghum, soybean, corn, alfalfa, and sunflowers are relatively minor crops and although they may be important in local ecosystems, their limited areas of growth make them "islands" in a sea of cotton (Agricultural Statistics Board 2001). Therefore, the primary focus of this study is with the chemicals currently associated with the production of cotton in the SHP.

Cotton is grown throughout eleven different states in the southern United States, and geographic variation in agricultural practices results from differences in weed and insect pressures. This, in turn, results in major variation in the types and usage of pesticides (Gianessi & Puffer 1990).


Cotton typically is planted between May 5 and May 25 in Lubbock and the surrounding counties. Farther to the south, the planting window is slightly longer, lasting through early June. Blooms usually appear in mid July with heat unit accumulation ending around 5 October. Harvest begins around 15 October and lasts through November (Ritchie et al. 1994).

Cotton farming requires three to five times more kilograms of chemicals per hectare (ha) than corn or soybeans, and as much as 7 kg/ha of herbicides and 5 kg/ha of insecticides are applied annually to cotton fields throughout the United States. Cotton also requires more applications per year; 4.7 chemical applications as opposed to 1.2 applications for corn (Coupe et al. 1998).

Herbicides are one of the major chemical classes used in the SHP to control cotton weeds. Twenty-four different active ingredients in herbicide formulations are used on the SHP. Herbicides are applied at ten different times throughout the year starting with winter treatments and ending in preharvest applications. The most commonly used herbicide on the SHP was Roundup[R] and its other brand names used as a post-emergent herbicide, which can be applied at five different times throughout the growing season. It is used in high concentrations, with up to 4.5 kg active ingredient/hectare (a.i/ha) as the recommended application rate. Roundup[R] is very water soluble, therefore it can infiltrate into playa ecosystems through runoff or spray drift. The LC50 value for glyphosate (the active ingredient in Roundup[R]) (Relyea 2005) is relatively high (4,000 [micro]g/L for Daphnia magna), indicating that it poses minimal risk to aquatic invertebrates. However, the surfactant used in most Roundup formulations, polyethoxylated tallowamine [POEA], is exceedingly toxic to aquatic species (Relyea 2005). Another com-mon herbicide used is Karmex[R], with the active ingredient diuron. It is primarily used on both oranges and cotton as a pre-emergent herbicide. The recommended usage of diuron is as much as 1.36 kg a.i./ha/yr. Diuron has a long half life of 90 days in aquatic environments and a high solubility of 42 mg/L, allowing diuron to infiltrate into playas as well as remain for extended periods of time. Diuron is one of the more toxic herbicides with 48 h LC50 values of 1,400 [micro]g/L for D. magna (PAN Pesticides Database 2005, Muschal & Warne 2003).

A second major class of chemicals used on cotton in the SHP is insecticides. The major pest species to cotton on the SHP is the boll weevil (Anthonomous grandis). Thirty-six different active ingredients are used as insecticides on crops in the SHP. The organophosphate Methyl Parathion 4E was determined to be the most widely used insecticide in this region, and third in the US, with over 3.3 million pounds of active ingredient applied annually (Thurman et al. 1998). It is used extensively on cotton and other crops in the SHP for control of fourteen pests, including the most common pest, boll weevil (Baugh et al. 2004). Based on crop consultant recommendations, Methyl Parathion 4E can be used at the highest application rate on the SHP with up to 2.25 kg a.i./ha (Baugh et al. 2004). Methyl parathion is one of the more toxic active ingredients used on the SHP, with a 48 hour LC50 value of 0.14 [micro]g/L for the aquatic invertebrate D. magna (Hazardous Substance Data Base 2005).

Cyfluthrin, the active ingredient in Baythroid[R] and Tempo[R], also is extremely toxic to aquatic invertebrates with a similar LC50 value of 0.14 [micro]g/L for D. magna. It is not used as heavily as Methyl Parathion 4E with only 28-56 g a.i./ha used ( 2005). However, it was deposited more often than Methyl Parathion 4E with over 40 million applications a year in the United States (Kiely et al. 2004). It was used on six cotton pests and many other sorghum and corn pests. Tempo[R] SC Ultra is composed of the active ingredient cyfluthrin as well as crystalline silica and the solvents, xylene, ethyl benzene, and trimethyl benzene which do not appear to affect acute toxicity (Cox 1994).

Harvest aids are used on the SHP but to a much lower extent than herbicides and insecticides. Also, their time of application, October through November, makes them irrelevant to this study.

In the cotton growing regions of the SHP there are over 20,000 playa lakes, which are small, shallow wetland depressions (Gustavson et al. 1994). These low-lying areas are fed solely by precipitation, thus playas are watersheds throughout the SHP. Playas are vital to maintaining the biodiversity and they harbor many species that could not otherwise survive in the arid environment of the SHP (Smith 2003). The actual number of species living in each playa is highly variable based on size of playa and relation to other playas, however all playas increase the diversity of species in the surrounding areas (Smith 2003). In addition to increasing biodiversity, playas in the SHP are important as the only recharge points for the Ogallala Aquifer, which underlies much of this region, inputting between 13 to 82 ml of water per year (Nativ & Riggio 1989, Stone 1990, Zartman et al. 1994).

Fairy shrimp are macroinvertebrates that are commonly found in playas of the SHP, as well as temporary water bodies throughout the world. Thamnocephalus platyurus (Crustacea: Anostraca) is the largest fairy shrimp species found in the SHP and can also be found throughout the southern United States living in ephemeral water bodies between 17[degrees]C and 32[degrees]C (Belk 1977). They typically emerge between May and October, which coincides with cotton growing season in the SHP and the application of most agricultural chemicals. Fairy shrimp are a major source of metabolizable energy to many vertebrates (Munuswamy & Subramoniam 1986) including mammals, amphibians, and waterfowl (Anderson & Hsu 1990, Mizutani et al. 1991, Fischer et al. 1982). Thamnocephalus platyurus are non-discriminate filter feeders that help maintain water quality in playa ecosystems (Eriksen & Belk 1999). Thamnocephalus platyurus eggs, known as cysts, are available commercially, therefore they are frequently used as a test species for water quality and toxicity testing (Thamnotoxkit F [Creasel Ltd., Belgium] and Thamnocephalus platyurus Toxkit [Vickers Laboratories Ltd., United Kingdom]).

The pesticide registration process requires acute toxicity testing to nontarget organisms such as birds, mammals, fish, plants, and terrestrial and aquatic invertebrates. However, toxicity tests used in the pesticide registration process often are conducted on species that are not present in the ecosystems impacted by pesticide application. Generally, aquatic inverterate toxicity testing is done on five species, Daphnia magna, D. pulex, Gammarus fasciatus, G. pseudolimnaeus, and G. lacustris (cf. Walker 1995). Currently, the EPA only requires that the active ingredient be tested for toxicity, not the pesticide formulation (EPA document 40 CFR 158.145, Touart 1995). Pesticide formulations consist of both active ingredients and inert ingredients. The active ingredient destroys or mitigates the pest while inert ingredients, or other ingredients, are not intended to affect the target pest. Inert ingredients consist of surfactants, wetting agents, dispersing agents, emulsifiers, solubilizers, and bioenhancers (Tominack 2000). The US Environmental Protection Agency currently acknowledges almost 1,700 inert ingredients being used on crops in the United States. Twelve-hundred of these are classified as unknown toxicity and of the 500 tested, only eight are classified as non-toxic (United States Environmental Protection Agency 1987). This study was conducted to determine if pesticide application of formulations used on the SHP would be injurious of a native macroinvertebrate (T. platyurus).

This study evaluated the acute toxicity (48 hour LC50) of four commonly used pesticides: Methyl Parathion 4E, Tempo[R] SC Ultra, Roundup[R], and Karmex[R] DF in order to determine their potential effects on T. platyurus. Chemicals were selected based on their potential to occur in playas in the SHP due to the amount used, number of applications per year, and the time when they are applied.


Chemicals were purchased from a local pesticide distributor. The chemicals purchased were Methyl Parathion 4E [methyl parathion; O,O-dimethyl O-4-nitrophenyl phosphorothioate, CAS 298-00-0] (Cheminova Inc., Wayne, NJ), Tempo[R] SC Ultra [cy-fluthrin; cyano(4-fluro-3-phenoxyphenyl)methyl 3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropane carboxylate, CAS 68359-37-5] (Bayer Corporation, Kansas City, MO), Roundup[R] [glyphosate; N-(phosphonomethyl) glycine, CAS 1071-83-6] (Monsanto Company, St. Louis, MO), and Karmex[R] DF [diuron; N'-(3,4-dichloro-phenyl)-N,N-dimethylurea, CAS 330-54-1] (Griffin Chemical, Valdosta, GA).

Laboratory derived T. platyurus cysts were obtained from Thamnotoxkit F and hatched in moderately hard synthetic freshwater as defined by EPA document EPA/600/4-90/027F. The water contained 60 mg/L magnesium sulfate, 96 mg/L sodium bicarbonate, 4 mg/L potassium chloride, and 60 mg/L calcium sulfate dehydrate (CaS[O.sub.4]x2[H.sub.2]O) (pH = 7.53, alkalinity = 92 CaC[O.sub.3]/L, and hardness = 66 CaC[O.sub.3]/L) (United States Environmental Protection Agency 1993). Cysts were placed in 250 ml beakers and hatched in a Plant Growth Chamber (Altair Refrigeration, Stafford, Texas) on a 14:10 light:dark schedule with light intensity of 1,575 lux and temperature regulated at 25[degrees]C. Twenty-four hours after cysts were placed in water, free-swimming nauplii were randomly selected for toxicity testing. Toxicity tests were conducted in 250 ml beakers using twenty, 24-hr-old nauplii. Each experiment was carried out using seven different chemical concentrations (eight for Karmex[R] DF) and one control group. Each treatment group was replicated three times. All beakers were filled with 175 ml of EPA water plus chemical. Each concentration was made using a serial dilution with final concentrations being 0.01, 0.1, 4, 50, 500, 10,000, 20,000 [micro]g/L for Methyl Parathion 4E, 0.008, 0.08, 2.2, 10.1, 84.7, 8,474, 84,745 for Tempo[R] SC Ultra, 19.9, 199, 747, 1,595, 4,175, 19,920, 199,200 [micro]g/L for Roundup[R], and 0.006, 0.06, 6, 37.5, 187.5, 625, 6,250, 12,500 [micro]g/L for Karmex[R] DF as nominal concentrations. At 4, 8, 12, 24, and 48 h after addition of the chemical, dead nauplii were counted and recorded with total mortality at 48 hours used to generate a dose response curve. Death was defined by lack of phyllopod movement.

Water was not changed during the testing period because water replacement would have added an additional external stressor and possibly confounded results of toxicity tests. Water quality parameters were measured three times throughout each test for the highest concentration.

Mortality data was modeled using logit analysis. The concentration that killed 50% of test organisms (i.e. LC50) was determined using the statistical program R version 2.0.1 (R Development Core Team, Boston, MA, USA) and recorded.


Tempo[R] SC Ultra was the most lethal of the compounds tested (Table 1) with an LC50 value for T. platyurus of 10.99 [+ or -] 1.24 [micro]g/L (mean [+ or -] SE). Methyl Parathion 4E had a 48 hour static LC50 value of 31.30 [+ or -] 7.52 [micro]g/L. Interestingly, the herbicides were much more toxic than expected based on previous studies on the active ingredient alone. The herbicide Karmex[R] DF had LC50 values of 75.97 [+ or -] 7.41 [micro]g/L. Roundup[R] had an LC50 of 1,243.38 [+ or -] 47.75 [micro]g/L (Table 5). Acute lethality curves are given for Methyl Parathion 4E (Fig. 2), Tempo[R] SC Ultra (Fig. 3), Roundup[R] (Fig. 4), and Karmex[R] DF (Fig. 5).

Overall, the insecticides were more toxic to T. platyurus than the herbicides, as expected. This study found that T. platyurus is more tolerant of Methyl Parathion 4E and Tempo[R] SC Ultra than Daphnia magna, but less tolerant of Karmex[R] DF and Roundup[R].





The use of pesticide formulations rather than active ingredient alone can substantially alter the toxicity of pesticides. In this study, a 4-fold higher toxicity (as compared to D. magna) was determined for Roundup[R] compared to glyphosate alone. In three frog species (Rana sylvatica, Rana pipiens, Hyla versicolor), the addition of POEA surfactant can lower the acute lethal concentration (48 h LC50) by 10 fold (3.9-15.5 mg/L with POEA versus 108-161 mg/L for technical grade glyphosate) (Relyea, 2005). POEA is an ethoxylated long-chain alkyl amine that is derived from animal fatty acids and functions by enhancing uptake of glyphosate by decreasing water surface tension and breakdown of the cuticle (Giesy et al, 2000). It is hypothesized that POEA also can affect the structural integrity and function of the exoskeleton of aquatic invertebrates. In this study, a 20-fold increase in toxicity was seen for Karmex[R] DF (tested on T. platyurus) over diuron (D. magna) alone. It's suspected that this is also a result of inert ingredients found in Karmex[R] DF although no research exists on this. Also, T. platyurus could potentially be more sensitive to Karmex[R] DF than other previously studied tested species as a result of interspecies variation. This result for herbicides opposes the finding of Centenu et al. (1995) suggesting that acute toxicity values are typically higher for T. platyurus than for D. magna.

Typically, herbicides as a whole are only minimally toxic to aquatic invertebrates, and are virtually nontoxic at ecologically relevant concentrations (Thurman et al. 1998). However, alterations in metabolism causing decreased fecundity and retarded growth are possible when macroinvertebrates are exposed to herbicides (Sibly & Calow 1989, Thurman et al. 1998). T. platyurus becomes sexually mature based on size (Ali & Dumont 1995). If growth is significantly retarded, reproduction will not occur before the playa dries eventually resulting in the elimination of T. platyurus in the playa. However, from this study it is the consensus of the authors that there is a need to reevaluate the toxicity of herbicides based on the pesticide formulations, not the active ingredient alone due to the increased toxicity seen in this study.

The insecticide with the greatest potential for exposure among the aquatic invertebrates in the SHP based on usage and time of application was determined to be Methyl Parathion 4E. However, Methyl Parathion 4E is relatively insoluble in water, thus potentially limiting exposure among aquatic species (Thurman et al. 1998). Other insecticides which have the potential to infiltrate into playas include Baythroid[R] and Tempo[R] (cyfluthrin), Asana[R] XL (esfenvalerate), Decis[R] 1.5E (deltamethrin), and Scout[R] X-tra 0.9E (tralomethrin), based on high concentrations used and high toxicity values. Of the top five insecticides used on the SHP, four are synthetic pyrethroids.

The pesticides least likely to affect playa ecosystems are insect growth regulators due to extremely low concentrations used and limited numbers of applications. Insect growth regulators counteract Juvenile hormone in insects and therefore do not allow the larva to morph into a pupa and inhibit activation of ovarian follicles and development of accessory sex glands in adults (Muegge et al. 2003). Each of the insect growth regulators are specific to the group of insects it is designed to eliminate. All of these insecticides are used in low concentrations and have very high LC50 values for aquatic invertebrates.

The LC50 value for methyl parathion for D. magna is 0.14 [micro]g/L whereas the formulation found in Methyl Parathion 4E had a 48 hour toxicity value of 31 [micro]g/L in fairy shrimp. Similarly for cyfluthrin the LC50 for D. magna is 0.14 [micro]g/L and found a value of 1.1 [micro]g/L for Tempo[R] SC Ultra. These results coincide with the findings of Centenu et al. (1995).

The results of this study demonstrate that pesticide formulations can potentially be much more toxic than what is typically reported for the active ingredient alone. This is especially true for herbicides where many inert ingredients are added. Both herbicides tested in this study, Roundup[R] and Karmex[R] DF, were toxic to T. platyurus in concentrations less than what was seen for the active ingredient in the test species D. magna. This indicates that these herbicides may have the potential to cause mortality and metabolic alterations of a native playa species like fairy shrimp. Methyl Parathion 4E and Tempo[R] SC Ultra were less toxic to T. platyurus than in tests using their active ingredients seen in the test species D. magna. However, results of this study show added ingredients used in herbicides to increase weed killing capabilities can also increase toxicity of the herbicide to aquatic invertebrates possibly through common mechanisms. Therefore, herbicide toxicity needs to be reevaluated for herbicide formulations when surfactants and other inert ingredients are incorporated into the mixture.

Pesticides are not typically found individually in the environment, but are usually found as mixtures, thus altering toxicity, which needs to be considered further. Also, the actual concentration of chemicals in playas needs to be examined for a wide range of time periods to determine the extent of which chemicals get into the playa ecosystem. Future work needs to be done in order to determine non-lethal effects at lower concentrations as well as determine the acute toxic concentrations of native shrimp and other species living in the playas of the SHP.


We thank D. Shroyer of grounds maintenance at Texas Tech University for assisting us in purchasing these pesticides.


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JMB at:

John M. Brausch, Stephen Cox and Philip N. Smith

The Institute of Environmental and Human Health Department of Environmental Toxicology Texas Tech University Lubbock, Texas 79409
Table 1. Acute toxicity values of four common pesticides on
Thamnocephalus platyurus (this study) and Daphnia magna (literature) in
the Southern High Plains.

 Water Quality Parameters
Pesticide Temperature pH Dissolved Oxygen

Methyl Parathion 24.6 [+ or -] 7.67 [+ or -] 8.32 [+ or -]
 4E 0.2[degrees]C 0.05 0.02
Tempo SC Ultra 24.6 [+ or -] 7.26 [+ or -] 8.19 [+ or -]
 0.2[degrees]C 0.05 0.01
Roundup Super 24.6 [+ or -] 5.05 [+ or -] 8.27 [+ or -]
 Concentrate 0.2[degrees]C 0.05 0.04
Karmex DF 24.6 [+ or -] 7.70 [+ or -] 8.30 [+ or -]
 0.2[degrees]C 0.05 0.05

 Acute Toxicity Literature Value
 ([micro]g/L) ([micro]g/L)
Pesticide Mean [+ or -] SE Daphnia magna

Methyl Parathion 31.30 [+ or -] 7.52 0.14
Tempo SC Ultra 10.99 [+ or -] 1.24 0.14
Roundup Super 1243.38 [+ or -] 47.75 4000
Karmex DF 75.97 [+ or -] 7.41 1400
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Author:Brausch, John M.; Cox, Stephen; Smith, Philip N.
Publication:The Texas Journal of Science
Date:Nov 1, 2006
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