Joint Toxicity and Risk of Bifenthrin and Zeta-cypermethrin to a Non-target Insect

Principle Investigator: Gadi V.P. Reddy Project personnel: Frank Antwi

Western Triangle Agricultural Research Center, Montana State University, 9546 Old Shelby Rd.,

P.O. Box 656, Conrad, MT 59425

Aim of the Study

The purpose of this paper was to test the (1) hypotheses of independent, uncorrelated and similar (additive) joint actions, (2) assess whether the interaction was synergistic, antagonistic, or additive, and (3) determine the risks of bifenthrin and zeta-cypermethrin mixture using T. molitor as a surrogate species.

Materials and Methods

Insects

Tenebrio molitor (mealworm larvae) third instars were purchased from Petco (Petco Company, Joliet, IL). T. molitor was chosen because of ease of handling and rearing, and immature life stage. A sample of 100 T. molitor were weighed to obtain the average weight per larva (mean wet weight = 134.9 mg; SD = 30.1 mg).

Bioassay

The bioassay experiments were conducted between June 30 and July 12, 2015 in a laboratory at Western Triangle Agricultural Research Center, Conrad, Montana. The prevailing laboratory conditions were 25 ºC ± 1 temperature, 66 ± 5% relative humidity, and 16:8 h light: dark photoperiod. Ten grams (10 g) of potting mix soil (Scotts Miracle-Gro Company, Marysville, OH) was placed in 2.25 oz. (66.55 ml) Gelatin shot glasses (NorthWest Enterprises, Elk Grove Village, IL 60007). Two T. molitor third instar larvae were placed in each cup and 1 ml of tap water was sprayed with a 473 ml capacity plant and garden atomizer sprayer (Sprayco, Livonia, MI) on each soil in the cups. A small slice of potato (1 cm3) was placed in the cup as a food source. Hero (a.i. zeta-cypermethrin 3.75% and bifenthrin 11.25%; Mustang Max (a.i. zeta- cypermethrin 9.15%), Brigade 2EC (a.i. bifenthrin 25.1%) from FMC Corporation, Philadelphia, PA were used as treatments. A stock solution (10X) of the insecticidal treatment was prepared out of which serial dilutions were made (0, 0.25X, 0.50X, 1X, 2X field fold). One ml of each dilution was sprayed on the soil, food source, and on the larvae of the surrogate organism (T. molitor). The lids of the cups were perforated and the perforated lids were used to cover the cups. The covered cups were then placed in trays on a wheel shelve in the laboratory. Counting for insect mortalities were recorded for 3 days. Insect that did not move when prodded with forceps were considered dead.

Data analysis

Abbott method (Abbott 1925) was used to account for control or natural mortalities. Water was used as the control. The data were analyzed with SAS. The LC50 values were determined with PROC PROBIT. Mortalities were regressed on concentrations using PROC REG. Graphs of

percentage mortality against log10 concentration were plotted with Sigma Plot. Risk Quotients (RQ) were determined by integrating estimated environmental concentration (EEC) and the toxic endpoint LC50 to characterize the risk. The Risk Quotient values were then compared with an RQ Level of Concern (LOC) of ≥ 0.5 for terrestrial animals used by USEPA.

Independent and Similar actions

Equivalent concentrations of z-cypermethrin and bifenthrin were obtained by multiplying the total active concentration in Hero EC by 3.75 and 11.25% for z-cypermethrin and bifenthrin, respectively. The hypothesis of independent action was determined using the equation below:

P = P1 + P2 - P1P2 (1)

Where amounts of the two constituents of the mixture resulted in kills of proportions P1, P2 respectively of the tested organism (Bliss 1934). These proportions were then multiplied by 100 to obtain the expected percentage of mortalities for independent action in Table 5. For similar action the equivalent concentrations of zeta-cypermethrin and bifenthrin were substituted in the regression equations for Hero EC treatment in Table 3 for the respective percentage mortalities. These were then summed up to obtain the expected percentage mortalities for similar action in Table 5. Based on the observed and expected percentage mortalities Chi Square Analysis was done to test the hypotheses of independent and similar actions for the constituents of the mixture Hero EC (Bliss 1934).

A chi-square (χ2) test was used to determine synergistic, additive, or antagonistic interactions between Brigade (bifenthrin) and Mustang Max (zeta-cypermethrin) in the treatments. Abbott method [30] was used to correct for control mortalities. The expected additive proportional percentage mortality Me for the zeta-cypermethrin - bifenthrin mixture was determined as Me = Mz + Mb (1 - Mz), where Mz and Mb are the observed proportional percentage mortality by zeta-cypermethrin (Mustang Max) and Brigade (bifenthrin) alone, respectively. A χ2 test determined as (χ2 = (Mzb - Me)2/Me) whereby Mzb = the observed percentage mortality for the zeta-cypermethrin - bifenthrin mixture was compared with the χ2 table value for 4 df. A non- additive effect between two actives was suspected when the calculated χ2 value exceeded the table value (Finney 1964). A significant interaction was considered as synergistic when the difference Mzb - Me had a positive value. When the difference Mzb - Me had a negative value, a significant interaction was considered antagonistic.

Results

Time-concentration-mortality response

The mortality responses of T. molitor with time is represented in Table 2, and Fig. 1. Mortality responses ranged from 0 to 80% at day 1 for all treatments (Hero, Mustang Max and Brigade) across the concentrations (Table 2, Fig. 1). At day 2, mortalities were 0 to 86.2% for Hero, 0 to 57.7% for Mustang Max, and 0 to 45.1% for Brigade across the concentrations (Table 2, Fig. 1). Across the concentrations at day 3 percentage mortalities ranged from 0 to 46.2% for Hero, 0 to 94.7% for Mustang Max, and 0 to 100% for Brigade (Table 2, Fig. 1).

Mortality and concentration relationships

The relationships between T. molitor mortalities and log concentrations are as shown in Table 3 and Fig. 1 for days 1 to 3. The regression models were significant (Table 3). The regression models explained 66.3 to 85.29%, 28.0 to 72.63%, 35.27 to 92.77% of the total response variation for T. molitor, at days 1 to 3 for the treatments Hero, Mustang Max, and Brigade, respectively (Table 3). The standard deviation of T. molitor sensitivity to the treatments varied from 0.21 - 0.28%, 0.23 - 1.02%, and 0.21 - 0.69% at days 1 to 3 on treatment with Hero, Mustang Max, and Brigade, respectively (Table 3). For a unit change in concentration, mortalities of T. molitor varied from 3.59 to 4.78%, 0.98 to 4.34%, and 1.45 to 4.71% at days 1 to 3 for Hero, Mustang Max, and Brigade, respectively (Table 3).

Lethal concentrations and risk quotients

The lethal concentrations, relative potencies, and risk quotients on treatment of T. molitor larvae with Hero, Mustang Maxx, and Brigade insecticides are presented in Table 4. The lethal concentrations generally decreased with time for Hero and Mustang Maxx, and increased for Brigade at days 1 to 3 (Table 4). The lethal concentrations at days 1 to 3 were for Hero (0.0000102 - 1.11371 g a.i./L), for Mustang Maxx (0.01117 - 0.01207 g a.i./L), and for Brigade (8.84121 × 10-7 – 0.0001239 g a.i./L) (Table 4).

Except at day 3 for Hero, none of the treatments exceeded the relative potency of Brigade insecticide (Table 4). The relative potencies were for Hero (7.94 × 10-7 – 1.21 × 101), for Mustang Maxx (7.32 × 10-5 - 1.11 × 10-2), and for Brigade (1.00) (Table 4).

Also, except at days 1 and 2 for Hero, risk quotients (RQs) were exceedingly high (> 1) for the treatments (Table 4). The risk quotients were for Hero (0.13 - 13747.35), for Mustang Maxx (2.16 - 2.97), and for Brigade (7.35 ×102 - 1.63 × 102) (Table 4).

Joint action and interaction

The joint action or toxicity of the insecticide mixture Hero (zeta-cypermethrin + bifenthrin) at various time points and concentrations are shown in Table 5. The interaction between Brigade (bifenthrin) and Mustang Maxx (zeta-cypermethrin) against T. molitor at various concentration and at days 1 to 3 are presented in Table 6.

The expected or predicted mortalities were always less than the observed mortalities for both independent and similar action hypotheses tested (Table 5, Fig. 1). The observed mortalities ranged from 56.7 - 80%, 27.6 - 86.2%, and 33.3 - 46.2% at days 1 to 3 (Table 5, Fig. 1). For independent action, expected mortalities ranged from 12.56 - 20.44%, 10.42 -16.46%, and 10.72- 17.45% at days 1 to 3 (Table 5, Fig. 1). For similar action, expected mortalities varied from 12.96 - 21.60%, 10.70 - 17.19%, and 11.01 - 18.28% at days 1 to 3 (Table 5, Fig. 1). The calculated χ2 values at days 1 to 3 were always greater than the Table value (χ2 0.050, df = 4 = 9.488), for both independent and similar actions (Table 5). Therefore, the hypotheses of independent and similar actions were rejected (Table 5). Except at day 2 for Mustang Max, and Brigade at 1X and 2X, across the concentrations (0.25X to 2X) at days 1 to 3 for the treatments (Hero, Mustang Maxx, and Brigade) the interactions were antagonistic (Table 6).

Acknowledgement

This material is based upon work that is supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, Hatch project under Accession# 1009746.

References

Abbott, W.S. 1925. A method for computing the effectiveness of an insecticide, J. Econ. Entomol. 18: 265-267.

Bliss C. I. 1939. The toxicity of poisons applied jointly, Ann. Appl. Biol. 26: 585-615. Finney, D.J. 1964. Probit Analysis, Cambridge University Press, London, 1964.

Table 1. Insecticide treatments and concentrations used.

a Insecticide concentration: 0X, control (water); 0.25X, 0.5X, 1X, and 2X the lowest field application rate.

b Hero: 0X = water; 0.25X = 0.09076 ml/L (0.013614 g a.i./L); 0.5X = 0.18152 ml/L (0.027228 g a. i./L); 1X = 0.36304 ml/L (0.054456 g a.i./L); 2X = 0.72608 ml/L (0.108912 g a.i./L).

c Mustang Maxx: : 0X = water; 0.25X = 0.0726075 ml/L (0.0066425 g a.i./L); 0.5X = 0.145215 ml/L (0.013285 g a. i./L); 1X = 0.29043 ml/L (0.02657434 g a.i./L); 2X = 0.58086 ml/L (0.0531487 g a.i./L).

d Brigade 2EC: : 0X = water; 0.25X = 0.06807 ml/L (0.0170856 g a.i./L); 0.5X = 0.13614 ml/L (0.03417115 g a.i./L); 1X = 0.27228 ml/L (0.06834228 g a.i./L); 2X = 0.54456 ml/L (0.1366846 g a.i./L).

Table 2. Time-concentration-mortality response of Tenebrio molitor larvae to Hero, Mustang, and Brigade insecticides.

a DAT, Days after treatment.

b Insecticide concentration: 1X the lowest label application rate equals Hero, 0.36304 ml/L; Mustang Maxx, 0.29043 ml/L; Brigade 2 EC, 0.27228 ml/L.

c Mortalities were adjusted for using the Abbot method (Abbott 1925).

d Water, control.

Table 3. Relationship between mortality and log concentrations of Hero, Mustang and Brigade insecticides to mealworm larvae

a Regression model: Y = Mortality (%); X = Concentration (g a.i./L).

b SD = Standard deviation of sensitivity to the treatments

Table 4. Lethal concentrations, relative potencies, and risk quotients for Tenebrio molitor larvae on treatment with Hero, Mustang Max, and Brigade insecticides

a Ratios of the lethal concentrations (LC50) of standard insecticide Brigade 2EC) to the treatments at 50% mortality for days 1 to 3.

b EEC = maximum commercial field rate

c Hero EC maximum commercial field rate = 0.93483 ml/L (10.3 fl. oz/acre) (0.140223 g a.i./L).

d ND, no data as confidence interval could not be determined by statistical analysis.

e Mustang Max maximum commercial field rate = 0.36304 ml/L (4 fl. oz/acre) (0.0332183 g a.i./L). f Brigade 2EC maximum commercial field rate = 0.36304 ml/L (4 fl. oz/acre) (0.0911225 g a.i./L). g 8.84121E-7 = 8.84121 × 10-7

Table 5. Joint action of Hero (zeta-cypermethrin + bifenthrin) insecticide to Tenebrio molitor larvae at three time points and four concentration levels

a Hero EC concentration: 0.013614 g a.i./L = 0.25X; 0.027228 g a.i./L = 0.5X; 0.054456 g a.i./L = 1X; 0.108912 g a.i./L = 2X.

b Concentration: equivalent zeta-cypermethrin (g a.i./L; 3.75%).

c Concentration: equivalent bifenthrin (g a.i./L; 11.25%).

d (Observed % Mortality – Expected % Mortality)2/Expected % Mortality.

Table 6. Interaction between Brigade (bifenthrin) and zeta-cypermethrin (Mustang Max) against Tenebrio molitor larvae at three time points and four concentration levels

a Insecticide concentration: 0.25X, 0.5X, 1X, and 2X the lowest field application rate.

b Observed proportional percentage mortality caused by Mustang Maxx (zeta-cypermethrin) alone.

c Observed proportional percentage mortality caused by Brigade (bifenthrin) alone.

d Expected additive proportional mortality for Hero (zeta-cypermethrin - bifenthrin) mixture.

e Observed proportional mortality for Hero (zeta-cypermethrin - bifenthrin) mixture.

f Interaction between treatments: Antagonistic (Mzb - Me = a negative value), Non-additive effect (synergistic or antagonistic; χ > 3.841), (Synergistic (Mzb - Me = a positive value).

Fig. 1. Pecentage mortality of third instar larvae of Tenebrio molitor on treatment with different concentrations of insecticides: Hero EC (bifenthrin + zeta-cypermethrin); Brigade 2EC (bifenthrin); Mustang Maxx (zeta-cypermethrin); independent action predictions for Hero HeroIA; similar action predictions for Hero (HeroSA).