In Cooperation with the Maine Department of Environmental Protection 

Pilot Study of Sublethal Effects on Fish of Pesticides 

Currently Used and Proposed for Use on Maine 

Blueberries 

By Adria A. Elskus 

Open-File Report 2007–1110 

U.S. Department of the Interior 

U.S. Geological Survey 
U.S. Department of the Interior 

DIRK KEMPTHORNE, Secretary 

U.S. Geological Survey 

Mark D. Myers, Director 

U.S. Geological Survey, Reston, Virginia: 2007 

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Suggested citation: 

Elskus, A.A., 2007, Pilot study of sublethal effects on fish of pesticides currently used and proposed for use 

on Maine blueberries: U.S. Geological Survey Open-File Report 2007-1110, 10 p. 

Any use of trade, product, or firm names is for descriptive purposes only and does not imply 

endorsement by the U.S. Government. 

Although this report is in the public domain, permission must be secured from the individual 

copyright owners to reproduce any copyrighted material contained within this report. 

ii
Contents 

Contents ................................................................................................................................................................................i

Conversion Factors and Abbreviations ...........................................................................................................................i

Abstract................................................................................................................................................................................1

Background .........................................................................................................................................................................1

Objectives ............................................................................................................................................................................2

Materials and Methods .....................................................................................................................................................2

Materials ..........................................................................................................................................................................2

Zebrafish...........................................................................................................................................................................3

Dosing Solutions .............................................................................................................................................................3

Confirmation of Pesticide Dosing Concentrations....................................................................................................3

Pesticide Exposures.......................................................................................................................................................4

Mortality, Time to Hatch, Developmental Abnormalities ........................................................................................4

Innate Immune Function................................................................................................................................................4

Behavioral Assays..........................................................................................................................................................4

Results of Preliminary Studies on the Effects of Blueberry Pesticides on Fish......................................................5

Dosing Solution Concentrations ..................................................................................................................................5

Innate Immune Function................................................................................................................................................5

Mortality, Time to Hatch, Developmental Abnormalities ........................................................................................7

Spontaneous Swimming................................................................................................................................................8

Conclusions and Additional Research............................................................................................................................9

References Cited ................................................................................................................................................................9

Figures 

1-4. Graphs showing– 

1. Preliminary data on the effects of exposure to single pesticide formulations on the 

innate immune system of zebrafish embryo-larvae exposed from fertilization through 

swim-up larvae ..................................................................................................................................................6

2. Preliminary data on the effects of exposure to single pesticide formulations on the 

innate immune system of zebrafish embryo-larvae exposed from fertilization through 

the swim-up larval stage..................................................................................................................................6

3. Preliminary data on the effects of exposure to mixtures of pesticide formulations on 

the innate immune system of zebrafish embryo-larvae exposed from fertilization 

through the swim-up larval stage...................................................................................................................7

4. Preliminary data on single pesticide exposure effects on spontaneous swimming in 

zebrafish exposed from fertilization through swim-up ...............................................................................8

Tables 

1. Preliminary analyses demonstrating that nominal and actual dosing solutions concentrations 

are similar......................................................................................................................................................................5

2. Mean days to hatch for zebrafish exposed to pesticide formulations from fertilization through 

swim-up larvae.............................................................................................................................................................8
Conversion Factors and Abbreviations 

SI to Inch/Pound 

Multiply By To obtain 

Length 

centimeter (cm) 0.3937 inch (in.) 

millimeter (mm) 0.03937 inch (in.) 

Area 

square centimeter (cm2) 0.001076 square foot (ft2) 

square centimeter (cm2) 0.1550 square inch (ft2) 

Volume 

liter (L) 33.82 ounce, fluid (fl. oz) 

liter (L) 2.113 pint (pt) 

liter (L) 1.057 quart (qt) 

liter (L) 0.2642 gallon (gal) 

liter (L) 61.02 cubic inch (in3

) 

Flow rate 

milliliter per minute (mL/min) 0.0002642 gallon per minute (gal/min) 

Mass 

milligram (mg) 0.00003527 ounce, avoirdupois (oz) 

Pressure 

kilopascal (kPa) 0.009869 atmosphere, standard (atm) 

kilopascal (kPa) 0.01 bar 

kilopascal (kPa) 0.1450 pound-force per inch (lbf/in) 

kilopascal (kPa) 20.88 pound per square foot (lb/ft2

) 

kilopascal (kPa) 0.1450 pound per square inch (lb/ft2

) 

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows: 

°F=(1.8×°C)+32 

Temperature in degrees Fahrenheit (°F) may be converted to degrees Celsius (°C) as follows: 

°C=(°F-32)/1.8 

Abbreviations: 

ng, nanogram 

nm, nanometer 

mL, milliliter 

μL, microliter 

ppb, parts per billion 

uv, ultraviolet 
Pilot Study of Sublethal Effects on Fish of Pesticides 

Currently Used and Proposed for Use on Maine 

Blueberries 

By Adria A. Elskus 

Abstract 

Blueberry pesticides have been detected consistently in some Down East Maine rivers, yet 

little is known about the sublethal effects of these pesticides on fish early life stages. The Maine 

blueberry industry is proposing to replace the insecticide ImidanTM (active ingredient phosmet) and 

the herbicide VelparTM (active ingredient hexazinone), two of the pesticides found in these rivers, 

with candidate alternatives SpinTor TM (active ingredient spinosad) and Callistso TM (active 

ingredient mesotrione). Our objective is to evaluate potential sublethal effects of these four 

formulations before the industry adopts the two candidate alternatives. We exposed zebrafish 

(Danio rerio) early life stages, from fertilization through larval swim-up, to a range of pesticide 

concentrations and evaluated their response relative to untreated controls. In this report we provide 

preliminary data on immune function as well as on parameters in addition to those originally 

proposed: development and performance fitness. We also provide information on our progress 

towards optimizing chemical protocols for analyzing the concentration of active ingredient in each 

of our formulation dosing solutions, another new parameter we added to those originally proposed. 

Preliminary results indicate that at environmentally realistic concentrations, these pesticides may 

have no significant effect on innate immunity, development rate or behavior (spontaneous 

swimming), however further replication is needed to confirm these initial findings. We have also 

observed some degree of developmental abnormalities in both pesticide-treated and control 

zebrafish embryos; however, additional replication is underway to determine if these groups differ 

significantly. 

Background 

Dramatic declines in Atlantic salmon (Salmo salar) populations in the northeastern U.S. 

have led to the complete loss of wild salmon in all New England states except for eight rivers in 

Maine where Atlantic salmon are now listed as an endangered distinct population segment (DPS) 

(National Research Council, 2004). Despite intensive efforts to restore Maine populations through 

juvenile stocking programs, adult returns continue to be well below the estimated carrying 

capacities of these rivers (Maine Atlantic Salmon Task Force, 1997). 

The Maine Board of Pesticide Control has consistently detected blueberry pesticides, 

including phosmet and hexazinone, in certain DPS rivers (Jackson, 2003). With little to no data on 

the relative risk to salmon health posed by blueberry pesticides, restoration managers cannot rule 

out the possibility that these pesticides are hampering recovery and restoration efforts for 

endangered Maine Atlantic salmon. 

Early life stages are considered the most sensitive to stressors, and because biochemical, 

hormonal and morphological changes that occur during early development are irreversible, toxicant 

1
exposure during early life is almost certain to have permanent effects on populations (Lawrence 

and Hemingway, 2003). Indeed, developmental exposure to atrazine, a triazine related to 

hexazinone, produced altered immune function in adulthood (Rooney and others, 2003). Moreover, 

a broad range of species may be affected since pesticides, including organophosphates and 

triazines, deleteriously affect immune function in a wide range of invertebrate and vertebrate 

species (Dunier, 1996; Galloway and others, 2003). 

Many pesticides exert their toxicity during metabolism and do not accumulate in biological 

tissues, including the pesticides being evaluated in the current study. Consequently, body burden 

analysis, as is done in many government-funded monitoring programs to evaluate the effect of 

persistent organic contaminants, will not reflect pesticide exposure or effect. Instead what is 

necessary is to characterize the exposure regime and define effect levels in carefully controlled 

exposure studies. 

For this pilot study, the U.S. Geological Survey, in cooperation with the Maine Department 

of Environmental Protection, used zebrafish, a well-characterized aquatic toxicology model for 

which a sensitive assay of immune system function has recently been developed (Hermann and 

others, 2004), whose development is well-documented (Westerfield, 1993) and for which 

behavioral assays have been published (Samson and others, 2001). This paper presents the 

preliminary results of this study. 

Objectives 

The original objectives of this study were to: 

1. Evaluate the immunotoxic effects on fish early life stages of two currently used blueberry 

pesticides, phosmet and hexazinone, consistently detected in Maine Down East rivers. 

2. Evaluate the immunotoxic effects of the proposed alternative pesticides, spinosad and 

mesotrione. 

3. Determine whether pesticide mixtures reflective of those observed for currently used pesticides 

in Maine Down East rivers have additive, synergistic, or antagonistic effects on immune 

function. 

4. Provide preliminary information to the Maine Board of Pesticide Control regarding the 

sublethal effects of candidate pesticides on fish before they are adopted by Maine blueberry 

growers.

Additional objectives subsequently incorporated into this study were to: 

5. Evaluate developmental and behavioral effects on fish early life stages of two currently used 

pesticides, phosmet and hexazinone, and their proposed alternatives, spinosad and mesotrione. 

6. Develop and optimize analytical protocols for measuring aqueous concentrations of the 

currently used pesticides, phosmet and hexazinone, and their proposed alternatives, spinosad 

and mesotrione. 

Materials and Methods 

The materials and methods used during the course of this pilot study are presented below. 

Materials 

Pesticide formulations ImidanTM (Gowan), Velpar-LTM (DuPont), CallistoTM (Syngenta), and 

SpinTorTM (Dow AgroSciences) were obtained from the University of Maine Blueberry Extension 

Office, courtesy of Dr. Frank Drummond. Styrene-divinylbenzene (SDB-L) solid phase extraction 

2
(SPE) columns (500 mg/6 mL) were obtained from Phenomenex (Torrance, Cal.). Solvents were 

high purity pesticide grade from Fisher Scientific, Inc Pittsburgh, PA. Dihydrodichlorofluorescein 

diacetate (H2DCFDA) was from Invitrogen (Carlsbad, Cal.), phorbol 12-myristate 13-acetate 

(PMA) and black 96 well plates were from Fisher Scientific, Inc. (Pittsburgh, Pa.). Pesticide 

standards were obtained from the U. S. Environmental Protection Agency's (USEPA) National 

Pesticide Standard Repository (Fort Meade, Md.). 

Zebrafish 

Zebrafish embryos (AB strain) were obtained from the University of Maine’s Zebrafish 

Core (University of Maine, 2007). Tanks of 14 females and 12 males were spawned as needed to 

provide these fish. 

Dosing Solutions 

Dosing concentrations were based on pesticide levels reported in the Pleasant River by the 

Maine Board of Pesticide Control for hexazinone and phosmet (Jackson, 2003). VelparTM, 

ImidanTM, CallistoTM, and SpinTorTM stock solutions were prepared in egg water and diluted to the 

desired concentrations. Solution concentrations were based on the concentration of active 

ingredient in the formulation. To determine whether pesticide mixtures measured in the Down East 

rivers of Maine have additive, synergistic, or antagonistic effects, we exposed embryos to single 

formulations and to mixtures (VelparTM+ImidanTM, CallistoTM + SpinTorTM), in combinations and 

concentrations measured in the Down East rivers of Maine for phosmet+hexazinone (0.2, 0.75, 2.0, 

3.0 ppb, (Jackson, 2003) and doses 10 times as high (7.5, 30 ppb). Pesticide solutions were 

prepared within 2 to 3 h (hours) of the start of the experiment and then held at 28 °C in the 

incubators with the fish on a 14-h/10-h light/dark cycle to mimic conditions under which singleapplication pesticide exposures 'age' in the field. To prevent the buildup of ammonia in the 

exposure plates, treatment water was replaced daily using the incubator-held dosing solutions. 

Confirmation of Pesticide Dosing Concentrations 

To determine if nominal dosing concentrations reflect actual dosing concentrations, we are 

optimizing protocols for analyzing the concentration of active ingredients in our dosing solutions at 

the start and conclusion of the 5-d (day) exposure periods. While protocols for extracting phosmet 

and hexazinone from river water have been established (L.B. Perkins, University of Maine, oral 

comm.), there are few protocols for the candidate pesticides, mesotrione and spinosad. With 

guidance from Dr. L. Brian Perkins (University of Maine) and Dr. Larry LeBlanc (University of 

Maine) we have conducted preliminary experiments evaluating the utility of styrene 

divinylbenzene-coated silica gel (SDB-L) solid phase extraction (SPE) cartridges to capture 

mesotrione and spinosad from aqueous solutions of CallistoTM and SpinTorTM, respectively, and 

confirmed and optimized the ability of this cartridge packing to capture hexazinone and phosmet 

from aqueous solutions of VelparTM and ImidanTM, respectively. Dr. L. Brian Perkins (University of 

Maine) is working with us to optimize analysis conditions for spinosad and mesotrione by high 

performance liquid chromatography (HPLC). Briefly, 500- to 1,000-mL pesticide dosing solutions 

(VelparTM, ImidanTM, CallistoTM, SpinTorTM) were pulled through SBD-L SPE cartridges under 

vacuum (25 kPa), the cartridges eluted with acetonitrile (CallistoTM, SpinTorTM for meotrione, 

spinosad) or ethyl acetate (VelparTM, ImidanTM for hexazinone, phosmet) and two fractions collected 

(1 mL, F1; 5 mL, F2). Eluates for VelparTM and ImidanTM were dried over sodium sulfate (baked at 

600 °C for 12 h), volume reduced to 900 μL, spiked with 100 μL of chlorpyrifos (10 ng/μL) as an 

internal standard, and injected onto a Hewlett Packard 5890/5870 gas chromatograph-mass 

spectrometry (GC/MS) system (2 μL, splitless), with helium as the carrier gas, under the following 

conditions: 275°C injection port, ramping from 80 °C – 250 °C at 20 °C/min (minute), 1.2 mL/min 

3
flow rate. Full scan mode was used to identify the quantification ions and qualifying ions for 

hexazinone, phosmet, chlorpyrifos (a surrogate for phosmet), and metribuzin (a surrogate for 

hexazinone). Eluates for CallistoTM and SpinTorTM were dried over sodium sulfate, volume reduced 

under high purity nitrogen to 1 mL, and injected (20 μL) onto a Hewlett Packard High Performance 

Liquid Chromatograph Series 1050 fitted with a C-l8 column (100 x 4.6 mm). We used a mobile 

phase of 75 percent ACN, 25 percent water, a flow rate of 1 mL/min, and monitored the analytes 

spinodad and mestrione at a wavelength of 271 nm. This wavelength was determined to provide the 

maximum signal by performing a full-UV scan of each analyte using diode-array UV detection. 

Pesticide Exposures 

Zebrafish embryos were exposed to pesticides or egg water (0.6-percent Instant Ocean 

prepared in nanopure water) for 5 days in 100-mm diameter plastic petri dishes (40-50 

embryos/dish) at 28 °C on a 14-h/10-h light/dark cycle from 2 to 3 h post-fertilization (Day 0) to 

120 h post-fertilization (Day 4), an age where they display immunologic competence (Hermann 

and others, 2004). Treatment water in the petri dishes was renewed daily. For the immune system 

assays, experiments were terminated on Day 4 (120 h post-fertilization). For developmental and 

behavioral studies, zebrafish were transferred on Day 4 from the petri dish pesticide exposures to 

clean egg water in 250-mL beakers suspended in flow-through tanks at 28 °C. Zebrafish larvae 

were held in the flow-through system through the end of the experiment (Day 7) with daily 

feedings of rotifers or dry food once per day on Days 5 to 7. Each replicate experiment used 

embryos from a different spawn. 

Mortality, Time to Hatch, Developmental Abnormalities 

Zebrafish embryos were monitored daily from fertilization through the end of each 

experiment for mortality, days to hatch, and evidence of developmental abnormalities. 

Innate Immune Function 

We evaluated respiratory burst, a simple immune system assay, as described by Herman 

and others (2004)). Briefly, on Day 4 zebrafish larvae were transferred from exposure dishes to 

black 96 well plates, one larva per well, and exposed to either substrate alone (H2DCFDA, 6 wells) 

or substrate plus phorbol 12-myristate 13-acetate (PMA) (6 wells). PMA provokes the production 

of superoxide. In turn, superoxide oxidizes the substrate H2DCFDA (a non-fluorescent dye) to 

dichlorofluorescein (DCF, a fluorescent product). In fish with a healthy immune system, PMA 

exposure in the presence of H2DCFDA will provoke substantial production of DCF. PMA thus 

serves both as the stimulant and as a positive control to confirm the assay is working properly. 

Evolution of DCF was monitored for up to 3.5 h in a Perkin Elmer FusionTM fluorescence plate 

reader at an excitation/emission of 485nm/530nm. 

Behavioral Assays 

We evaluated spontaneous swimming using the protocol described by Samson and others 

(2001). Briefly, larvae were placed individually into 100 mL of egg water in an 8-cm-diameter 

finger bowl placed over a 1-cm2

 grid. After a 2-min acclimation period, the number of lines 

crossed in 30 sec (seconds) by the larvae was recorded. Five random larvae were tested from each 

replicate for each treatment on Days 4 and 7 post-fertilization. A second behavioral assay, prey 

capture, a well-established measure of performance fitness in fish (Samson and others, 2001) is 

currently underway. 

4
Results of Preliminary Studies on the Effects of Blueberry Pesticides on 

Fish 

A discussion of preliminary study results follows and includes a presentation of dosing 

solution concentrations, innate immune function, mortality, time to hatch, developmental 

abnormalities, and spontaneous swimming. 

Dosing Solution Concentrations 

Pesticide standards were readily detectable by GC/MS (hexazinone and phosmet), and by 

HPLC (spinosad, mesotrione). Surrogates for phosmet (chlorpyrifos) and hexazinone (metribuzin) 

were also easily detected by GC/MS. We are currently in the process of obtaining surrogates for 

spinosad (spinetoram) and mesotrione (sulcotrione) from the manufacturers as they are not 

available from the USEPA National Pesticide Repository. These surrogates will be used to spike 

the dosing solutions prior to extraction through SPEs and, with internal standard spikes, will be 

used to correct for analyte recovery. 

Preliminary analyses of the pesticide dosing solutions indicate that the actual (measured) 

concentrations are close to nominal concentrations for ImidanTM and VelparTM (table 1). 

Table 1. Preliminary analyses demonstrating that nominal and actual dosing solutions 

concentrations are similar. 

[ppb, parts per billion] 

Dosing Formulation Dose Active Ingredients Nominal (ppb) Actual (ppb) 

VelparTM plus ImidanTM Low hexazinone 0.75 0.35 

phosmet 0.75 0.21 

High hexazinone 7.50 5.6 

phosmet 7.50 5.0 

Innate Immune Function

We found no consistent effects of the blueberry pesticide formulations on the innate 

immune function of developing zebrafish. The results of initial experiments with exposure to 

single pesticide formulations over a wide range of doses, from environmentally relevant (0.2-3.0 

ppb) to 10 times as high (7.5, 30 ppb), indicated there were no effects of any of the formulations on 

the respiratory burst response of embryo-larval zebrafish (fig. 1). In these initial experiments, the 

zebrafish response was monitored for 2 h. 

5
Figure 1. Preliminary data on the effects of exposure to single pesticide formulations on the innate 

immune system of zebrafish embryo-larvae exposed from fertilization through swim-up larvae. Bars 

represent means ± SD for n=2-4 replicates of 6 larvae per replicate. EW=Egg Water, V=VelparTM, I = 

ImidanTM, C = CallistoTM, S = SpinTorTM. Doses were 0.2, 0.75, 2.0, 3.0, 7.5, and 30 ppb. Respiratory 

burst was measured for 2 hours. 

To improve and optimize the sensitivity of the assay, we extended the monitoring time to 

3.5 h. However, even with the improved signal strength afforded by the longer monitoring 

duration, there appear to be no effects of these pesticide formulations on zebrafish respiratory burst 

(fig. 2). 

Figure 2. Preliminary data on the effects of exposure to single pesticide formulations on the innate 

immune system of zebrafish embryo-larvae exposed from fertilization through the swim-up larval 

stage. Bars represent means ± SD for n=2-4 replicates of 6 larvae per replicate. EW=Egg Water, 

V=VelparTM, I = ImidanTM, C = CallistoTM, S = SpinTorTM. Doses were 0.75 and 7.5 ppb. Respiratory 

burst was measured for 3.5 hours. 

6
Due to ground-water contamination in the watershed, hexazinone is present year-round in 

the Pleasant River, one of the salmon rivers in Down East Maine, and thus is present in July when 

the insecticide ImidanTM (phosmet) is applied. To determine if pesticide mixtures have additive, 

synergistic, or antagonistic effects, we looked at pesticide mixtures at a variety of doses. 

Preliminary results (for one replicate per mixture) indicate mixtures do not affect the respiratory 

burst response of developing zebrafish (fig. 3); however, further replication is needed before firm 

conclusions can be made. 

Figure 3. Preliminary data on the effects of exposure to mixtures of pesticide formulations on the 

innate immune system of zebrafish embryo-larvae exposed from fertilization through the swim-up 

larval stage. Bars represent means for one replicate, 6 larvae per replicate. EW=Egg Water, 

V=VelparTM, I = ImidanTM, C = CallistoTM, S = SpinTorTM. Doses were combinations of 0.2, 0.75, 2.0, 3.0, 

7.5, and 30 ppb. Respiratory burst was measured for 3.5 hours. 

Mortality, Time to Hatch, Developmental Abnormalities 

Zebrafish embryo mortality is typically 20 to 50 percent within the first 24 h postfertilization (M. Nilan, University of Maine, oral commun., 2007). We found similar rates of 

mortality in our exposures, with no difference among treatments (data not shown). 

We observed evidence of developmental abnormalities in the pesticide treated groups, with 

very few occurrences in the controls. These abnormalities included reduced growth, small head 

with small or no eyes, pericardial edema, and scoliosis. A few individuals were moribund. All 

individuals displaying these abnormalities failed to hatch. These data will be recorded 

quantitatively in future exposures. 

Despite evidence of abnormal development, preliminary data indicate no significant 

difference in developmental rate (P<0.05, two-tailed Student's t-test) as measured by the mean 

number of days it took the embryos to hatch (table 2). 

7
Table 2. Mean days to hatch for zebrafish exposed to pesticide formulations from fertilization 

through swim-up larvae. Mean ± SD for n=3 replicates of 50 embryos per replicate. 

[n, number; ppb, parts per billion; SD, Standard Deviation] 

Treatment Dose (ppb) Mean days to hatch SD 

Egg water control 0 2.90 0.33 

VelparTM 0.75 2.95 0.56 

7.5 3.08 0.42 

ImidanTM 0.75 3.42 0.11 

7.5 3.18 0.20 

CallistoTM 0.75 3.36 0.71 

7.5 3.18 0.54 

SpinTorTM 0.75 2.89 0.26 

7.5 3.11 0.22 

Spontaneous Swimming 

Preliminary data on the effects of pesticide exposure on spontaneous swimming show no 

definitive trend (fig. 4). The large variability in these data reflect the small sample size (n=2). 

Further experiments replicating this endpoint are underway to reduce this variability. Swimming 

activity and the ability to capture prey are related behaviors, which together provide a measure of 

performance fitness. Ongoing experiments evaluating prey capture in dosed zebrafish will be 

related to our results with spontaneous swimming to determine if alterations in prey capture may be 

related to alterations in swimming activity. 

Figure 4. Preliminary data on single pesticide exposure effects on spontaneous swimming in 

zebrafish exposed from fertilization through swim-up. Bars represent means ± SD for n=2 replicates 

of 5 larvae per replicate. Swimming measured on Days 3 and 6 post-fertilization. EW=Egg Water, 

V=VelparTM, I = ImidanTM, C = CallistoTM, S = SpinTorTM. Doses were 0.75 and 7.5 ppb for Low and High, 

respectively. 

8
Conclusions and Additional Research

The long-term goal of our work is to provide risk assessors, blueberry growers, and 

restoration managers with data to aid in making science-based decisions regarding blueberry 

pesticide Best Management Practices in Maine (University of Maine Cooperative Extension 2007). 

We have little to no information to determine whether pesticides that are commonly in use now and 

pesticides that generally have fewer environmental effects and might be available in the near future 

(1-2 years) have detrimental effects on fish early life stages.

Preliminary data from the physiological and behavioral assays indicate there are no effects 

of environmentally realistic, and 10 times as high, pesticide exposures, done singly or in binary 

mixtures, on developing zebrafish. These data are preliminary, and further replication of the assays 

described above is underway to assess their statistical significance. 

Replication of these assays will increase the statistical power required to detect differences 

among treatments. These assay replications will be coupled with additional behavioral studies to 

assess the effects of these pesticides on the ability of larval zebrafish to capture prey, a sensitive 

assessment of performance fitness. We will continue to develop chemical protocols for analyzing 

aqueous solutions of the four pesticide formulations. These protocols will be used to measure the 

dosing concentrations at the start and end of the 5-d exposure periods. The results of these studies 

will be compared to published findings and the significance of these findings evaluated for the use 

of these pesticides in river systems that support developing fish. 

The results of this research will provide the basis for more extensive studies on the 

sublethal effects of blueberry pesticides, alone and in environmentally relevant combinations, on 

resident fish species in the Down East rivers of Maine. The results of such studies could influence 

which pesticides are used in Maine, could indicate whether Best Management Practices for 

currently-used and proposed for use pesticides need to be refined to further reduce potential aquatic 

contamination, and would provide data regarding the potential effects on resident fish of candidate 

pesticides before these pesticides come into use. 

References Cited 

Dunier, M., 1996, Water pollution and immunosuppression of freshwater fish: Italian Journal of 

Zoology, v. 63, p. 303-309. 

Galloway, B.J., Munkittrick, K.R., Currie, S., Gray, M.A., Curry, R.A., and Wood, C.S., 2003, 

Examination of the responses of slimy sculpin (Cottus cognatus) and white sucker (Catostomus 

commersoni) collected on the Saint John River (Canada) downstream of pulp mill, paper mill, 

and sewage discharges: Environmental Toxicology and Chemistry, v. 22, p. 2898-2907. 

Hermann, A.C., Millard, P.J., Blake, S.L., and Kim, C.H., 2004, Development of a respiratory burst 

assay using zebrafish kidneys and embryos: Journal of Immunological Methods, v. 292, p. 119-

129. 

Jackson, H.P., 2003, 2003 Drift study of two aerially applied blueberry pesticides: Augusta, Maine, 

Maine Board of Pesticide Control, 19 p. 

Jackson, H.P., 2003, 2003 Drift study of two aerially applied blueberry pesticides: Augusta, Maine, 

Maine Board of Pesticide Control, 19 p. 

Lawrence, A.J., and Hemingway, K.L., 2003, Effects of Pollution on Fish: Molecular Effects and 

Population Responses: U.K., Blackwell Publishing, 368 p. 

Maine Atlantic Salmon Task Force, 1997, Atlantic Salmon conservation plan for seven Maine 

rivers: Maine Atlantic Salmon Task Force, March 1997, 434 p. 

9
National Research Council, 2004, Atlantic salmon in Maine: Washington D.C., National 

Academies Press, 275 p. 

Rooney, A.A., Matulka, R.A., and Luebke, R.W., 2003, Developmental atrazine exposure 

suppresses immune function in male, but not female Sprague-Dawley rats: Toxicological 

Sciences, v. 76, p. 366-375. 

Samson, J.C., Goodridge, R., Olobatuyi, F., and Weis, J.S., 2001, Delayed effects of embryonic 

exposure of zebrafish (Danio rerio) to methylmercury (MeHg): Aquatic Toxicology, v. 51, p. 

369-376. 

University of Maine, 2007, Zebrafish Facility: Accessed April 10, 2007, at URL 

http://www.umaine.edu/bmmb/zebrafish.htm

University of Maine Cooperative Extension, 2007, Wild blueberry: Accessed April 10, 2007, at 

URL http://www.wildblueberries.maine.edu/ 

Westerfield, M., 1993, The zebrafish book: Eugene, Oregon, University of Oregon Press: 

Accessed on April 11, 2007, at URL http://zfin.org/zf_info/zfbook/zfbk.html 

10