U.S. Department of the Interior

U.S. Geological Survey

Open-File Report 2009–1142

Prepared in cooperation with the U.S. Fish and Wildlife Service, Region 1

Results of Laboratory Testing for Diphacinone in Seawater, 

Fish, Invertebrates, and Soil Following Aerial Application 

of Rodenticide on Lehua Island, Kauai County, Hawaii, 

January 2009
Cover. Black-footed albatross on Lehua Island, Kauai County, Hawaii (photograph courtesy of Eric VanderWerf, Pacific Rim 

Conservation). 
Results of Laboratory Testing for 

Diphacinone in Seawater, Fish, 

Invertebrates, and Soil Following Aerial 

Application of Rodenticide on Lehua Island, 

Kauai County, Hawaii, January 2009

By Carl E. Orazio, Michael J. Tanner, Chris Swenson, Jeffrey Herod, 

Peter Dunlevy, and Robert W. Gale

Prepared in cooperation with the U.S. Fish and Wildlife Service, Region 1

Open-File Report 2009–1142

U.S. Department of the Interior

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

KEN SALAZAR, Secretary

U.S. Geological Survey

Suzette M. Kimball, Acting Director

U.S. Geological Survey, Reston, Virginia: 2009

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Suggested citation:

Orazio, C.E., Tanner, M.J., Swenson, C., Herod, J., Dunlevy, P., Gale, R.W., 2009, Results of laboratory testing for 

diphacinone in seawater, fish, invertebrates, and soil following aerial application of rodenticide on Lehua Island, 

Kauai County, Hawaii, January 2009: U.S. Geological Survey Open-File Report 2009–1142, 15 p. with appendix.
iii

Contents

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

Introduction.....................................................................................................................................................1

Methods...........................................................................................................................................................3

Sample Collection and Handling ........................................................................................................3

Diphacinone Laboratory Analysis......................................................................................................4

Results .............................................................................................................................................................4

Seawater................................................................................................................................................4

Soil...........................................................................................................................................................4

Fish Fillets...............................................................................................................................................4

Whole Crabs ..........................................................................................................................................5

Limpets....................................................................................................................................................5

Conclusions.....................................................................................................................................................5

Acknowledgments.........................................................................................................................................5

References......................................................................................................................................................5

Tables...............................................................................................................................................................7

Appendix 1: Analytical Methods for Diphacinone ..............................................................................13

Materials ..............................................................................................................................................13

Sample Preparation and Quantification of Diphacinone in Seawater Samples ......................13

Sample Preparation and Quantification of Diphacinone in Biological Tissues and Soil ........13

Fish Samples...............................................................................................................................13

Limpet...........................................................................................................................................14

Whole Crab .................................................................................................................................14

Soil................................................................................................................................................14

Instrumental Analysis and Data Reduction....................................................................................14

HPLC-PDA Analysis...................................................................................................................14

Quality Assurance and Quality Control ...........................................................................................14

Criteria for Quantification of HPLC-PDA Data.......................................................................15

Acceptance or Rejection Criteria for Results .......................................................................15

Figures

1–2. Screen shots showing—

1. Lehua and Niihau Islands, Hawaii ....................................................................................2

2. Lehua Island, Niihau, Hawaii showing the three sampling sites on the 

south of the island ...............................................................................................................3
iv

Tables

1. Results of laboratory testing for diphacinone in Lehua Island, Hawaii, 

seawater samples ........................................................................................................................8

2. Results of laboratory testing for diphacinone in Lehua Island, Hawaii, 

soil samples ...................................................................................................................................9

3. Results of laboratory testing for diphacinone in Lehua Island, Hawaii, fish 

fillet samples................................................................................................................................10

4. Results of laboratory testing for diphacinone in Lehua Island, Hawaii, whole 

crab samples ...............................................................................................................................11

5. Results of laboratory testing for diphacinone in Lehua Island, Hawaii, soft 

tissue limpet samples.................................................................................................................12

Appendix Tables

A. Quality-control sample types for analysis of environmental samples...............................14

B. Quality-control sample types and levels selected for study ...............................................15
v

Conversion Factors

Multiply By To obtain

Length

meter (m) 0.3048 feet (ft)

centimeter (cm) 2.54 inch (in)

kilometer (km) 0.621388 mile (mi)

nanometer (nm) = 10-9 meter

Volume

microliter (μL) 0.00003382 ounce, fluid (fl. oz)

milliliter (mL) 0.03382 ounce, fluid (fl. oz)

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

Mass

kilograms (kg) 2.204 pounds (lbs)

gram (g) 0.03527 ounce, avoirdupois (oz)

microgram (μg) = 1 x 10-6 grams

nanogram (ng) = 1 x 10-9 grams

Concentration

molar (M) = moles per liter

millimolar (mM) = millimoles per liter (10-3 M)

nanograms per milliter (ng/mL) = part per billion (ppb; 10-9))

microgram per gram (μg/g) = parts per million (ppm: 10-6)

microgram per milliliter (μg/mL) = parts per million (ppm: 10-6)

nanogram per gram (ng/g) = parts per billion (ppb: 10-9)

microgram per liter (μg/L) = parts per billion (ppb: 10-9)

nanogram per liter (ng/L) = parts per trillion (ppb: 10-12)

kilograms per hectare (kg/ha) 0.8888 pounds per acre (lb/acre)

Area

hectare (ha) 2.47 acre

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

 °F= (1.8×°C) +32

Concentrations of chemical constituents in solid materials (tissues) are given in nanogram per 

gram (ng/g, or parts per billion, ppb) wet weight. Concentrations of chemical constituents in 

calibration standard solutions and in liquid samples (seawater) are given in nanograms per liter 

(ng/mL, or parts per billion, ppb).
vi
Results of Laboratory Testing for Diphacinone in 

Seawater, Fish, Invertebrates, and Soil Following Aerial 

Application of Rodenticide on Lehua Island, Kauai County, 

Hawaii, January 2009

By Carl E. Orazio1, Michael J. Tanner1, Chris Swenson2, Jeffrey Herod2, Peter Dunlevy3, and Robert W. Gale1

1

U.S. Geological Survey.

2

U.S. Fish and Wildlife Service.

3

U.S. Department of Agriculture.

Abstract

In January 2009, rodenticide bait (Ramik Green® pellets) 

containing the active ingredient diphacinone was aerially 

applied to Lehua Island. Reported herein are the results of laboratory analyses to determine diphacinone concentrations in 

samples of seawater, fillet of fish, soft tissue of limpets (opihi), 

whole-body crabs, and soil collected from Lehua Island, Kauai 

County, Hawaii, after aerial application of the rodenticide 

bait. Diphacinone was specifically chosen because of its low 

toxicity to nontarget organisms. Its use on Lehua Island is the 

second time it has ever been used for an aerial application 

to eradicate rodents. Testing of the Lehua Island samples for 

diphacinone utilized high-performance liquid chromatography 

with photodiode array detection. No detectable concentrations 

of diphacinone were found in any of the samples from Lehua 

Island. The limits of detection for diphacinone were 0.4 nanograms per milliliter (parts per billion) seawater, 15 nanograms 

per gram (dry weight) soil, 20 nanograms per gram (parts per 

billion) fish fillet, 13 nanograms per gram whole crab, and 34 

nanograms per gram soft tissue limpet.

Introduction

Oceanic islands contain a disproportionate share of the 

world’s unique terrestrial species and are especially vulnerable 

to the impacts of invasions by nonnative species, including 

rats. More than 80 percent of all oceanic islands worldwide 

have been infested by some species of invasive rodent. The 

ecosystems on oceanic islands are extremely susceptible 

to disturbances caused by infestations of invasive species 

because of their limited habitat coverage and the close integration of niche species. Most species extinction events that have 

occurred or are occurring in these isolated ecosystems are 

caused by invasive species. Many island rodent eradication 

projects have been successfully conducted worldwide using 

anticoagulant rodenticides.

Lehua Island is an uninhabited 312-acre crescent-shaped 

volcanic cone located about three-fourths of a mile north of 

Niihau and 20 miles (mi) west of Kauai (fig. 1). Lehua Island 

is a State designated seabird sanctuary that supports colonies 

of seabirds such as Laysan (Phoebastria immutabilis) and 

black-footed albatross (Phoebastria nigripes), red-footed 

(Sula sula) and brown boobies (Sula leucogaster), black noddies (Anous minutus), Newell’s (Puffinus newelli) and wedgetailed shearwaters (Puffinus pacificus), red-tailed tropicbirds 

(Phaethon rubricauda), and band-rumped storm petrels 

(Oceanodroma castro). During the first biological surveys of 

Lehua in 1931, Polynesian rats (Rattus exulans) and European 

rabbits (Oryctolagus cuniculus) were found. Both of these species are alien to Hawaii and had been present on Lehua for an 

unknown period of time. Rabbits were eradicated from Lehua 

in 2006. Other restoration efforts include restoring native 

plant communities and managing the barn owl, an introduced 

seabird predator species. A coalition of conservation agencies 

have developed a comprehensive 10-year restoration plan that 

includes managing the owls, eradicating rats, and transplanting 

native birds, land snails, terrestrial arthropods and plants to the 

island.

For the Lehua Island rat eradication, the U.S. Fish and 

Wildlife Service (USFWS), the Hawaii Department of Fish 

and Wildlife (DOFAW), and the U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Wildlife 

Services (USDA–APHIS–WS) jointly proposed aerial application of diphacinone (0.005% active ingredient). Diphacinone 

is a chronic anticoagulant rodenticide that acts by disrupting 

the normal blood clotting mechanisms of vertebrates, competing at receptor sites in the liver with vitamin K, a necessary 

chemical for blood clotting. Diphacinone has been shown to 
2 Results of Lab Testing for Diphacinone Following Aerial Application of Rodenticide on Lehua Island, Kauai County, Hawaii

be an effective toxicant for rats in Hawaii and elsewhere. It is 

efficacious, yet has relatively low risk of impacts to nontarget 

species through consumption of prey that have consumed the 

bait pellets (secondary impacts). Diphacinone was specifically 

chosen for use because of its low toxicity to nontarget organisms, its relatively short half-life in the environment, and its 

low biomagnifications potential (U.S. Environmental Protection Agency, 1998). Its use on Lehua Island is the second time 

it has ever been used for an aerial application to eradicate 

rodents. The first such use was on Mokapu Island, Hawaii, 

near Molokai in winter 2008. Early wintertime was chosen 

for the bait application time when alternate rat foods and rat 

populations are lowest and migratory native nontarget species 

are not present or are only present in low numbers. 

Previous to the Lehua aerial applications, staff from 

various Federal and State agencies camped on the island and 

marked transects. Albatross (Laysan, black-footed) nests were 

mapped so pellets could be removed that fell near nests with 

chicks the day of application. Populations of desired nontarget 

species, including nesting seabirds and protected plants, were 

monitored actively for approximately 2 years prior to the 2009 

application to produce reliable population estimates of adverse 

impacts before and after rodenticide treatments.

On January 6 and 12, 2009, the USDA–APHIS–WS

Hawaii State office, in cooperation with USFWS Pacific 

Islands Wildlife Office and the State of Hawaii, applied a total 

of 7,800 pounds (lbs) of rodenticide bait (Green Ramik® pellets) containing the active ingredient diphacinone on Lehua 

Island. Bait pellet count within plots confirmed the targeted 

distribution of bait across the island. The January 12 application was conducted to ensure bait was available to every rat on 

the island for a lethal exposure period. During operations, four 

people worked on a 240-foot (ft) long barge staged near Lehua 

Island that served as a helicopter landing zone for loading bait, 

refueling, and a Geographical Information Station (GIS) workstation for downloading Global Positioning System (GPS) data 

during application to track and document bait distribution. As 

many as six people also worked on Lehua Island during bait 

applications to monitor bait distribution and to remove pellets 

around albatross chick nests. Two different helicopter companies were used for transport, one to ferry people to the island 

and another to transport gear and water to the Lehua Camp. 

A third helicopter and pilot were used for bait application. 

The bait was applied by a USDA–APHIS–WS and local pilot 

contractor team that had experience aerially applied rodenticide 11 months earlier on Mokapu Island. A fourth helicopter, 

operated by the U.S. Coast Guard, patrolled the area around 

Lehua Island and was on standby in the unlikely event of an 

emergency. Biologists were limited in their stay on Lehua 

Island during the project because of offshore military operations in the area.

Monitoring for primary and secondary adverse impacts 

of diphacinone on nontarget species was one of the foremost 

concerns during this rodent eradication project. Pre- and 

post-application samples of crabs, limpets (opihi), fish, and 

soil were collected from three sites on the south side of the 

Figure 1. Lehua and Niihau Islands, Hawaii (Created from GoogleEarth© image).
Methods 3

island (fig. 2). Pre-application specimens were collected 

on December 30 and 31, 2008, and January 2, 2009. Postapplication samples were collected on January 7, 12, and 19, 

2009, at 1-day and 1-week periods after each bait application. 

The location of the sampling sites was determined in part by 

human safety and logistical concerns. There was very high 

winter surf during most of this period coming primarily from 

the north and wrapping around the island. These three locations were the only places where the shore-based sampling 

crews could safely access the shoreline and where there 

would be potential use by people. In addition, the sites chosen 

represent the most conservative monitoring result and, thus, 

provide for the highest possible confidence that the results 

were indicative of actual circumstances for future analysis of 

risk of nontarget organism exposure. This is due to the fact 

that Lehua Island’s largest gulch drainages extend to the ocean 

along the south shore where runoff enters the sea near the 

three sampling sites. Thus, the biota and soils sampled in the 

area were a good representation of the higher end diphacinone 

exposure potential. However, it should be noted that the automated weather station on the island recorded no significant 

rainfall events following the Lehua bait application that would 

have washed pellets into the water. Plots across the island 

were monitored to measure the disappearance of diphacinone 

rodenticide and ensure enough bait pellets remained on the 

ground for all rats to consume a lethal amount. Transects were 

searched multiple times for rat carcasses and to document any 

nontarget mortality, if it occurred. More than 20 dead rats were 

found within transects on the island; no dead nontarget species 

were found.

The objective of this study was to assess the potential 

presence of diphacinone in seawater, biota, and soil as a 

consequence of aerial application of rodenticide to eradicate 

invasive, alien rats from Lehua Island. The study consisted of 

three sampling periods, each addressing the post-application 

diphacinone exposure levels in the immediate environment. 

Methods

Sample Collection and Handling

Personnel from the USFWS, DOFAW, and USDA–

APHIS–WS collected specimens from three sites for 

Figure 2. Lehua Island, Niihau, Hawaii (Created from GoogleEarth© image) showing the three sampling sites (yellow dots) on the 

south of the island. Indicated sample types were collected at each sampling location.
4 Results of Lab Testing for Diphacinone Following Aerial Application of Rodenticide on Lehua Island, Kauai County, Hawaii

determination of potential diphacinone exposure. Pre-application and post-application samples that were collected at each 

site include seawater, soil, limpet (Cellana exarata), aama 

crab (Grapsus tenuicrustatus), taape (Lutjanus kasmira), 

stocky hawkfish (Cirrhitus pinnulatus), nenue (Kyphosus biggibus), toau (Lutjanus fulvus), Christmas wrasse (Thalassoma 

trilobatum), and hogfish (Bodianus bilunulatus). Limpets 

are intertidal grazers and the crabs are omnivorous, intertidal 

scavengers, and were collected from the splash zone. For each 

site, samples were collected a distance of 25 to 50 meters 

(m) along the shoreline. The GPS locations of the sites were 

recorded. Site #1 was about 100 m from Site #2, and Site #2 

was 500 m from Site #3. Surface soils were collected 20 to 30 

m from the water’s edge at a depth of 2 to 5 inches (in.), at the 

base of gulches that drain into the ocean. Seawater and soil 

samples were contained in clean glass 250-milliliter (mL) jars 

with Teflon-lined lids. Limpets and aama crabs were collected 

in near-shore habitats and weighed whole. At each site, three 

to five fish were caught by hook and line; the six species of 

fish collected feed on a variety of organisms, including algae, 

invertebrates, and other fish. Weights of the fish fillet samples 

were recorded. The biological samples were wrapped in aluminum foil, placed in a plastic bag and labeled with the sample 

type, weight, site, date, and field number information. Tissues 

were stored frozen, whereas, soils and waters were stored cool.

Samples were shipped to the U.S. Geological Survey 

(USGS) Columbia Environmental Research Center (CERC) 

under chain of custody; a total of 18 fish samples, 9 soil 

composites, 9 seawaters, 9 composites of 5 to 8 whole limpet 

carcasses, and 7 composites of 2 whole crabs were shipped. (A 

similar set of samples was sent to the USDA–APHIS laboratory in Fort Collins, Colordao, for inter-laboratory comparison.) The pre-application samples arrived at CERC on January 

6 and the post-application samples arrived on January 23, 

2009. Upon receipt at CERC, the samples were logged into the 

sample database system and assigned a unique identification 

number. Fish fillets were analyzed as individuals; limpets were 

analyzed as composites of five to eight individuals; and crabs 

were analyzed as composites of two individuals, except for 

when only a single sample was available. Seawater and soil 

samples were refrigerated in the dark at 4 degrees Celsius (°C) 

and vertebrate and invertebrate tissue samples were stored in 

the dark at -20oC until analysis.

Diphacinone Laboratory Analysis

The analytical method consisted of extraction of diphacinone from the sample and subsequent concentration and 

purification by solid-phase extraction (SPE) or low pressure size-exclusion chromatography (LP-SEC). The detailed 

diphacinone laboratory analytical method is provided in 

appendix 1. Seawater (200 mL) was extracted with a solid 

phase cartridge; biological tissues (5 g aliquots) were column 

extracted; soil samples (15–20 g dry weight) were refluxed. 

The resulting extracts were then cleaned with C-18 SPE and 

size exclusion chromatography as needed. Sample preparation 

methods increased in their complexity as the richness of the 

sample matrices increased, generally in the order: seawater 

< soil < fish fillet < crab < limpet. Analytical separation of 

diphacinone was performed by high performance liquid chromatography (HPLC) followed by ultraviolet-visible photodiode array absorbance (PDA) detection and quantification following the methods of Yang and others (2001). Coumarin was 

the instrumental internal standard. Quality-control samples 

included diphacinone-fortified pre-application samples (matrix 

spikes) triplicate analysis, procedural blanks of sodium sulfate, 

and matrix blanks (pre-application samples). The Limits of 

Detection (LOD) for diphacinone were 0.4 nanograms per milliliter (ng/mL, parts per billion, ppb) for seawater, 15 nanograms per gram (ng/g, ppb) for dry weight soil, 20 ng/g for 

fish fillet, 13 ng/g for whole crab, and 34 ng/g for soft tissue of 

limpet. Detection limits for the diphacinone analysis method 

were calculated from the responses of the multiple matrix 

blanks (Keith and others, 1983, 1991).

Results

Seawater

No diphacinone was detected in the Lehua Island seawater (table 1, at the back of the report). The LOD for diphacinone in the Lehua Island seawater samples was 0.4 ng/mL. 

The quality-control data indicated that the analysis of the 

seawater is of high quality. Extraction efficiency from diphacinone fortified seawater ranged from 80 to100 percent; the 

seawater spike analyzed with this study resulted in 87 percent 

recovery. The negative control seawater sample did not have 

any detectable concentrations of diphacinone.

Soil

No diphacinone was detected in the soil samples (table 

2, at the back of the report). The LOD for diphacinone in 

the Lehua Island soil samples was 15 ng/g (dry weight). The 

quality-control data indicated that the analysis of the soil was 

of high quality. Extraction efficiency of diphacinone from 

fortified pre-application soil sample was 101 percent and the 

negative control soil sample did not have any detectable concentrations of diphacinone. 

Fish Fillets

No diphacinone was detected in the fish samples (table 

3, at the back of the report). The LOD for diphacinone in the 

Lehua Island fish samples was 20 ng/g (wet weight). The 

quality-control data indicated that the analysis of the fish fillets 

was of high quality. For biological samples including fish, 
References 5

extraction efficiency of diphacinone for this method ranged 

from 80 to 100 percent; recoveries from two fortified preapplication fish samples analyzed with this study were 85 and 

94 percent. The negative control fish sample did not have any 

detectable concentrations of diphacinone. 

Whole Crabs

No diphacinone was detected in the crab samples (table 4, 

at the back of the report). The LOD for diphacinone in the 

Lehua Island crab samples was 13 ng/g (wet weight). The 

quality-control data indicated that the analysis of the crabs 

was of high quality. Extraction efficiency of diphacinone from 

fortified pre-application crab samples was 101 percent and 

the negative control crab sample did not have any detectable 

concentrations of diphacinone. 

Limpets

No diphacinone was detected in the limpets (table 5, 

at the back of the report). The LOD for diphacinone in the 

Lehua Island limpet samples was 34 ng/g (wet weight). The 

quality-control data indicated that the analysis of the limpets 

was of high quality. For biological samples including limpets, 

extraction efficiency is 80 to 100 percent; recovery of diphacinone from the fortified pre-application limpet sample was 

99 percent. The negative control limpet samples (pre-application samples) did not have any detectable concentrations of 

diphacinone.

Conclusions

Diphacinone was not detected in any of the Lehua Island 

environmental samples that were collected after the aerial 

application of the rodenticide bait. The lack of detectable 

diphacinone residues in Lehua Island marine habitats mirrors 

the test results from Mokapu Island eradication in 2008 (Gale 

and others, 2008). Inter-laboratory analysis of duplicate Lehua 

Island samples corroborates the finding that diphacinone 

was not detected in the Lehua samples (T.M. Primus, written 

commun., 2009). No live rats have been detected on Lehua 

Island since the January 2009 bait application. Monitoring 

will continue for 2 years before the eradication is declared a 

success. No nontarget mortalities were documented on Lehua 

Island. The Lehua monitoring represents the largest marine 

sampling effort to date associated with any aerial rodenticide 

application.

Acknowledgments

The authors thank staff of USDA–APHIS–WS, Catherine 

Swift and others of USFWS for their contributions in this 

effort. They made the primary selection of the study area and 

continue to work on follow-up monitoring and other aspects of 

this study.

References

Gale, R.W., Tanner, M., and Orazio, C.E., 2008, Determination of diphacinone in seawater, vertebrates, invertebrates, 

and bait pellet formulations following aerial broadcast on 

Mokapu Island, Molokai, Hawai’i: U.S. Geological Survey 

Open-File Report 2008–1285, 16 p.

Hunter, K., and Sharp, E.A., 1988, Modification to procedures 

for the determination of chlorophacinone and for multiresidue analysis of rodenticides in animal tissue: Journal of 

Chromatography A, v. 437, p. 301–305. 

Keith, L.H., Crummet, W., Deegan, J., Jr., Libby, R.A., Taylor, 

J.K., and Wentler, G., 1983, Principles of environmental 

analysis: Analytical Chemistry, v. 55, p. 2,210–2,218.

Keith, L.H., 1991, Environmental sampling and analysis—A 

practical guide: Boca Raton, Fla., Lewis Publishers, Inc., 

143 p.

Mesmer, M.Z., and Flurer, R.A., 2000, Determination of chlorophacinone and diphacinone in commercial rodenticides by 

liquid chromatography-UV detection and liquid chromatography-electrospray ionization mass spectrometry: Journal of 

Chromatography A, v. 89, p. 249–55.

Primus, T.M., Griffin, D.L., Volz, S.A., and Johnston, J.J., 

1998, Reversed-phase ion-pair liquid chromatographic 

determination of chlorophacinone and diphacinone in 

steam-rolled oat baits and steam-rolled oat/wax baits: 

Journal of the Association of Official Analytical Chemists 

International, v. 81, p. 349–57.

U.S. Fish and Wildlife Service and Hawaii Department of 

Land and Natural Resources, 2008, Final supplemental 

environmental assessment Lehua Island ecosystem restoration project October 2008: Honolulu, Hawaii, 172 p.

U.S. Environmental Protection Agency, 1998, Reregistration 

eligibility decision (RED)—Rodenticide cluster, EPA–

738–R–98–007.

Yang, S.Y., Pan, G.M., Meng, G.F., Zhang, D.M., 2001, Study 

of diphacinone in biological samples by high performance 

liquid chromatography/diode array detection: Separations 

and Purifications, v. 19, p. 245–247.
6 Results of Lab Testing for Diphacinone Following Aerial Application of Rodenticide on Lehua Island, Kauai County, Hawaii
Tables 

7

Tables
8 Results of Lab Testing for Diphacinone Following Aerial Application of Rodenticide on Lehua Island, Kauai County, Hawaii

Table 1. Results of laboratory testing for diphacinone in Lehua Island, Hawaii, 

seawater samples.

[USGS, U.S. Geological Survey; ID, identification; ng/mL, nanograms per milliliter (parts per billion, ppb); <, less than; LOD, limits of detection; hr, hour; --, not applicable; %, percent]

USGS

identification Sample identification Field ID pH Diphacinone(ng/mL)

45045 Site 1: baseline (1/2/09) JJH09-ST001 8.10 <LOD [0.4]

45046 Site 2: baseline (1/2/09) JJH09-ST002 8.13 <LOD [0.4]

45047 Site 3: baseline (1/2/09) JJH09-ST003 8.20 <LOD [0.4]

45176 Site 1: 24hr post (1/7/09) JJH09-006 8.10 <LOD [0.4]

45177 Site 2: 24hr post (1/7/09) JJH09-005 8.14 <LOD [0.4]

45178 Site 3: 24hr post (1/7/09) JJH09-004 8.01 <LOD [0.4]

45179 Site 1: 24hr/7 day (1/13/09) JJH09-009 8.22 <LOD [0.4]

45180 Site 2: 24hr/7 day (1/13/09) JJH09-008 8.23 <LOD [0.4]

45181 Site 3: 24hr/7 day (1/13/09) JJH09-007 8.10 <LOD [0.4]

45182 Site 1: post 7 day (1/19/09) JJH09-12 8.10 <LOD [0.4]

45183 Site 2: post 7 day (1/19/09) JJH09-11 8.12 <LOD [0.4]

45184 Site 3: post 7 day (1/19/09) JJH09-10 8.18 <LOD [0.4]

Quality-control samples

Expressed as 200 mL equivalent sample volume basis

Mock (3/16/09) Diphacinone standard -- -- 0.76

PB031609 Procedural blank -- -- <LOD [0.4]

MB031609 Matrix blank (composite) -- -- <LOD [0.4]

MS031609 Matrix spike (composite) -- -- 0.66 (87%)
Tables 9

Table 2. Results of laboratory testing for diphacinone in Lehua Island, Hawaii, 

soil samples.

[USGS, U.S. Geological Survey; ID, identification; ng/g, nanograms per gram (parts per billion, ppb); values reported on a dry-sediment weight (dw) basis; <, less than; LOD, limits of 

detection; hr, hour; --, not applicable; %, percent]

USGS 

identification Sample identification Field ID Diphacinone(ng/g) dw

45048 Site 1: baseline (1/2/09) JJH09-ST001 <LOD [15]

45049 Site 2: baseline (1/2/09) JJH09-ST002 <LOD [15]

45050 Site 3: baseline (1/2/09) JJH09-ST003 <LOD [15]

45185 Site 1: 24hr post (1/7/09) JJH09-006 <LOD [15]

45186 Site 2: 24hr post (1/7/09) JJH09-005 <LOD [15]

45187 Site 3: 24hr post (1/7/09) JJH09-004 <LOD [15]

45188 Site 1: 24hr/7 day (1/13/09) JJH09-009 <LOD [15]

45189 Site 2: 24hr/7 day (1/13/09) JJH09-008 <LOD [15]

45190 Site 3: 24hr/7 day (1/13/09) JJH09-007 <LOD [15]

45191-1 Site 1: post 7 day (1/19/09) JJH09-12 <LOD [15]

45191-2 Replicate #2 JJH09-12 <LOD [15]

45191-3 Replicate #3 JJH09-12 <LOD [15]

45192 Site 2: post 7 day (1/19/09) JJH09-11 <LOD [15]

45193 Site 3: post 7 day (1/19/09) JJH09-10 <LOD [15]

Quality-control samples

Expressed as 20-g equivalent sample dry mass basis

Mock (3/18/09) Diphacinone standard -- 34

PB031809 Procedural blank -- <LOD [15]

MB031809 Matrix blank (45048) -- <LOD [15]

MS031809 Matrix spike (45048) -- 34.5 (101%)

SPE mock 33

SPE recovery: soil 31.5 (97%)
10 Results of Lab Testing for Diphacinone Following Aerial Application of Rodenticide on Lehua Island, Kauai County, Hawaii

Table 3. Results of laboratory testing for diphacinone in Lehua Island, Hawaii, fish fillet samples.

[Concentrations given in micrograms of diphacinone per gram (ug/g) wet weight (ww) of fish sample, USGS, U.S. Geological Survey; ID, identification; ng/g, nanograms per gram (parts per billion, ppb); values reported on a wet-tissue weight basis; <, less than; LOD, limits of detection; --, 

not applicable; %, percent; QAQC, quality assurance/quality control]

CERC 

identification Sample identification Fish Field ID Diphacinone(ng/g fish) ww

45054 Site 1: baseline (1/2/09) Not identified JJH09-ST001 <LOD [20]

45055 Site 2: baseline (12/30/08) Hawiian hogfish JJH08-ST002 <LOD [20]

45056 Site 3: baseline (12/30/08) Blueline snapper JJH08-ST003 <LOD [20]

45201-1 Site 1: 24hr post (1/7/09) Toau JJH09-006 St001 <LOD [20]

45201-2 Replicate #2 Toau JJH09-006 St001 <LOD [20]

45201-3 Replicate #3 Toau JJH09-006 St001 <LOD [20]

45202 Site 1: 24hr post (1/7/09) Hogfish JJH09-006 St001 <LOD [20]

45203 Site 1: 24hr post (1/7/09) Stocky hawkfish JJH009-06 St001 <LOD [20]

45204 Site 2: 24hr post (1/7/09) Stocky hawkfish JJH09-005 St002 <LOD [20]

45205 Site 2: 24hr post (1/7/09) Stocky hawkfish JJH09-005 St002 <LOD [20]

45206 Site 3: 24hr post (1/7/09) Hinalea JJH09-004 St003 <LOD [20]

45207 Site 3: 24hr post (1/7/09) Taape JJH09-004 St003 <LOD [20]

45208 Site 3: 24hr post (1/7/09) Stocky hawkfish JJH09-004 St003 <LOD [20]

45209 Site 3: 24hr post (1/7/09) Stocky hawkfish JJH09-004 St003 <LOD [20]

45210 Site 1: 24hr/7 day (1/12/09) Stocky hawkfish JJH09-009 St001 <LOD [20]

45211 Site 1: 24hr/7 day (1/12/09) Hawkfish #2 JJH09-009 St001 <LOD [20]

45212 Site 2: 24hr/7 day (1/12/09) Nenue JJH09-008 St002 <LOD [20]

45213-1 Site 3: 24hr/7 day (1/12/09) Nenue JJH09-007 St003 <LOD [20]

45213-2 Replicate #2 Nenue JJH09-007 St003 <LOD [20]

45213-3 Replicate #3 Nenue JJH09-007 St003 <LOD [20]

45214 Site 3: 24hr/7 day (1/12/09) Stocky hawkfish JJH09-007 St003 <LOD [20]

45215 Site 3: 24hr/7 day (1/12/09) Stocky hawkfish #2 JJH09-007 St003 <LOD [20]

45216 Site 1: post 7 day (1/19/09) Surge wrasse JJH09-012 St001 <LOD [20]

45217 Site 2: post 7 day (1/19/09) Stocky hawkfish JJH09-011 St002 <LOD [20]

45218 Site 2: post 7 day (1/19/09) Stocky hawkfish JJH09-011 St002 <LOD [20]

Quality-control samples

Expressed as 5-g equivalent sample mass basis

Mock (2/19/09) Mock: diphacinone standard -- -- 873

PB021909 Procedural blank -- -- <LOD [20]

MB021909 Matrix blank (45055) -- -- <LOD [20]

MS021909 Matrix diphacinone spike (45055) -- -- 738 (85%)

QAQC SPE mock (2/25/09) Mock SPE: diphacinone standard -- -- 975

QAQC SPE recovery (2/25/09) Recovery SPE: diphacinone standard -- -- 970 (100%)

Mock (2/23/09) Mock: diphacinone standard -- -- 455

PB022309 Procedural blank -- -- <LOD [20]

MB022309 Matrix blank (45055) -- -- <LOD [20]

MS022309 Matrix diphacinone spike (45055) -- -- 429 (94%)

QAQC SPE mock (2/26/09) Mock SPE: diphacinone standard -- -- 1,000

QAQC SPE recovery (2/26/09) Recovery SPE: diphacinone standard -- -- 1,000
Tables 11

Table 4. Results of laboratory testing for diphacinone in Lehua Island, Hawaii, whole 

crab samples.

[USGS, U.S. Geological Survey; ID, identification; ng/g, nanograms per gram (parts per billion, ppb); 

values reported on a wet-tissue weight basis; <, less than; LOD, limits of detection; --, not applicable; 

%, percent]

USGS 

identification Sample identification Field ID Diphacinone(ng/g)

45051 Site 1: baseline (12/31/08) JJH08-St001 (n=2) <LOD [13]

45052 Site 2: baseline (12/31/08) JJH08-St002 (n=2) <LOD [13]

45053 Site 3: baseline (12/31/08) JJH08-St003 (n=1) <LOD [13]

45194 Site 1: 24hr post (1/7/09) JJH09-006 (n=2) <LOD [13]

45195 Site 2: 24hr post (1/7/09) JJH09-005 (n=2) <LOD [13]

45196 Site 3: 24hr post (1/7/09) JJH09-004 (n=2) <LOD [13]

45197 Site 1: 24hr/7 day (1/12/09) JJH09-009 (n=1) <LOD [13]

45198 Site 1: post 7 day (1/20/09) JJH09-12 (n=2) <LOD [13]

45199 Site 2: post 7 day (1/19/09) JJH09-11 (n=2) <LOD [13]

45200-1 Site 3: post 7 day (1/19/09) JJH09-10 (n=2) <LOD [13]

45200-2 Replicate #2 JJH09-10 (n=2) <LOD [13]

45200-3 Replicate #3 JJH09-10 (n=2) <LOD [13]

Quality-control samples

Expressed as 5-g equivalent sample mass basis

Mock (4/1/09) Diphacinone standard -- 107

PB040109 Procedural blank -- <LOD [13]

MB040109 Matrix blank (45057) -- <LOD [13]

MS040109 Matrix spike (45058) -- 108 (101%)

SPE mock -- -- 105

SPE recovery: crabs Recovery SPE -- 102 (99%)
12 Results of Lab Testing for Diphacinone Following Aerial Application of Rodenticide on Lehua Island, Kauai County, Hawaii

Table 5. Results of laboratory testing for diphacinone in Lehua Island, Hawaii, soft 

tissue limpet samples.

[USGS, U.S. Geological Survey; ID, identification; ng/g, nanograms per gram (parts per billion, ppb); 

values reported on a wet-tissue weight basis; <, less than; LOD, limits of detection; --, not applicable; 

%, percent]

USGS identification Sample identification Field ID Diphacinone

(ng/g)

45057 Site 1: baseline (12/30/08) JJH08-001 (n=5) <LOD [34]

45058 Site 2: baseline (12/30/08) JJH08-002 (n=8) <LOD [34]

45059 Site 3: baseline (12/30/08) JJH08-003 (n=6) <LOD [34]

45219-1 Site 1: 24hr post (1/7/09) JJH09-006 (n=5) <LOD [34]

45219-2 Replicate #2 JJH09-006 (n=5) <LOD [34]

45219-3 Replicate #3 JJH09-006 (n=5) <LOD [34]

45220 Site 2: 24hr post (1/7/09) JJH09-005 (n=5) <LOD [34]

45221 Site 3: 24hr post (1/7/09) JJH09-004 (n=5) <LOD [34]

45222 Site 1: 24hr/7 day (1/13/09) JJH09-009 (n=5) <LOD [34]

45223 Site 2: 24hr/7 day (1/13/09) JJH09-008 (n=5) <LOD [34]

45224 Site 3: 24hr/7 day (1/13/09) JJH09-007 (n=6) <LOD [34]

45225 Site 1: post 7 day (1/19/09) JJH09-12 (n=5) <LOD [34]

45226 Site 2: post 7 day (1/19/09) JJH09-11 (n=6) <LOD [34]

45227 Site 3: post 7 day (1/19/09) JJH09-10 (n=5) <LOD [34]

Quality-control samples

Expressed as 5-g equivalent sample mass basis

SPE mock/5-g equiv Diphacinone standard -- 125

SPE recovery: limpet Recovery SPE -- 124 (99%)

GPC mock Diphacinone standard -- 116

GPC recovery: limpet Recovery LP-GPC -- 114 (98%)

Mock (3/23/09) Mock -- 140

PB032309 Procedural blank -- <LOD [34]

MB032309 Matrix blank (45057) -- <LOD [34]

MS032309 Matrix spike (45058) -- 138 (99%)
Tables 13

Appendix 1: Analytical Methods for Diphacinone

Materials

Solid-phase extraction cartridges (Oasis-HLB (hydrophilic/lipophilic balance) 6 milliliters (mL) x 500 milligrams (mg)) were purchased from Waters Corp., Milford, 

Massachusetts. Octadecyl SPE cartridges (Isolute C18 

(EC) 6 mL x 1 gram (g)) were purchased from International Sorbent Technology, Mid Glamorgan, United 

Kingdom. The size exclusion material (SX-3 biobeads 

200–400 mesh) was purchased from Bio-Rad Co., Richmond, California. Acetone, acetonitrile, dichloromethane, 

methanol (OPTIMA grade), anhydrous sodium sulfate, 

and 2 N o-phosphoric acid (ACS grade) were purchased 

from Fisher Scientific, Fair Lawn, New Jersey. Whatman 

0.45-micrometer (μm) polytetrafluoroethylene (PTFE) 

syringe filters were purchased from Whatman, Inc., 

Sanford, Maine. A 1-molar (M) solution of tetrabutylammonium hydroxide (TBAH) was purchased from SigmaAldrich, St. Louis, Missouri. Diphacinone and coumarin 

standard solutions (in methanol) were purchased from 

AccuStandard, New Haven, Connecticut. Milli-Q water 

(18mΩ, Millipore Synergy UV, Millipore Corp., Bedford, 

Massachusetts) was used throughout the analytical process. 

Aqueous tetrabutylammonium hydroxide ion pair (TBAHIP) reagents used for high performance liquid chromatography (HPLC) and for SPE were prepared at 0.03 M in 

water and pH adjusted to 6.0 with 2N o-phosphoric acid. 

Sample Preparation and Quantification of 

Diphacinone in Seawater Samples

The seawater samples (200 mL) were extracted by 

HLB-SPE cartridge to adsorb diphacinone that was then 

recovered by acetonitrile elution of the SPE and quantified 

by HPLC with PDA detection; the pH of the water was 

measured using a Mettler-Toledo Seven Easy pH meter 

(Schwerzenbach, Inc., Switzerland) (Gale and others, 

2008). The final extract was prepared in 700 microliters 

(μL) methanol with 300 μL of the aqueous TBAH-IP 

reagent added to match the liquid chromatography mobile 

phase, and 1 microgram (μg) coumarin (instrumental internal standard) added. 

Sample Preparation and Quantification of 

Diphacinone in Biological Tissues and Soil

Fish Samples

Homogenized fillet tissue samples (5 g of skinless fillets) were dehydrated for 2 hours with 25 g anhydrous sodium 

sulfate. The mixture was then blended into a free-flowing 

powder with a stainless steel-bladed commercial blender. It 

has been previously established that ascorbic acid treatment, 

needed for some types of vertebrate samples, was not required 

for fish, crab, and limpet matrices (Hunter and Sharp, 1988). 

The dehydrated sample was loaded into an extraction column 

with a total of about 20 mL of acetonitrile rinses of the sample 

container and then saturated with acetonitrile. The acetonitrile saturated dehydrated matrix was allowed to interact 

approximately 1 hour before extraction. Additional acetonitrile 

(150 mL) was added to the column, and the sample extracted 

at a flow rate of approximately 2 milliliters per minute 

(mL/min) until flow ceased; additional acetonitrile (100 mL) 

was added to the column, the extraction continued, and the 

eluant collected with the original extract. The extracts were 

rotary evaporated under vacuum (<50 degrees Celsius (°C)) to 

approximately 3 mL, and quantitatively transferred to 15-mL 

culture tubes with three sequential 2 mL rinses with acetonitrile. The sample extracts were evaporated by nitrogen stream 

to 2 mL and mixed thoroughly. Some coextracted interferences 

(mainly lipid material) were removed by C18 SPE-(EC). The 

cartridges were conditioned with 10 mL methanol followed by 

10 mL 0.03 M TBAH-IP, dried under vacuum for about 1 minute with 10 mL acetonitrile (the cartridge remained wet). The 

2 mL sample extracts were applied to the cartridge at about 

2 mL/min (with three 1 mL acetonitrile rinses). Diphacinone 

was recovered from the cartridge with 7 mL acetonitrile (total 

acetonitrile 10 mL: three 1 mL rinses plus 7 mL for elution). 

The eluant was collected in a 15-mL amber culture tube. 

The final sample extracts were prepared for analysis by first 

evaporating the solvent to dryness with nitrogen. The residues 

were dissolved in 700 μL methanol, 300 μL of the aqueous 

TBAH-IP reagent was added to match the liquid chromatography mobile phase, and 1 μg coumarin (instrumental internal 

standard) was added. The final samples were filtered through 

0.45-μm PTFE syringe filters directly into 1-mL amber autosampler vials.
14 Results of Lab Testing for Diphacinone Following Aerial Application of Rodenticide on Lehua Island, Kauai County, Hawaii

Limpet

Limpet (opihi) (Cellana exarata) (soft tissues) sample 

homogenates (5 g) were accurately weighed and then dehydrated with 25 g anhydrous sodium sulfate and prepared with 

the similar procedure used for the fish samples with the addition of a couple of steps: Additional lipids and other biogenic 

material were removed by low-performance size-exclusion 

chromatography (SX-3 biobeads, dichloromethane mobile 

phase at a flow of 3.5 mL/min). The diphacinone fractions 

(LP-SEC collection window from 40 to 60 minutes) were 

rotary evaporated under vacuum (<50°C) to approximately 

3 mL, and quantitatively transferred to 15-mL culture tubes 

with three sequential 2 mL rinses with dichloromethane. The 

extract was then run through C18 SPE-(EC). The final 1 mL 

sample extracts were prepared for analysis in methanol and 

aqueous TBAH-IP in the same manner described above.

Whole Crab

Whole crabs (Grapsus tenuicrustatus) were rinsed with 

distilled water and blended (shell included). Sample homogenates (5 g) were accurately weighed and then dehydrated 

with 25 g anhydrous sodium sulfate and extracted with the 

procedure used for the fish samples. The dichloromethane 

sample extracts were prepared for analysis in the same manner 

as the fish and limpets. The deep red color of the crab extract 

required dual C18 SPE-(EC). The final 1 mL sample extracts 

were prepared for analysis in methanol and aqueous TBAH-IP 

in the same manner described above.

Soil

Moistures of the soil samples were determined by oven 

drying on a heating block at 120°C and measuring moisture 

loss. Wet portions of soil samples equivalent to 20 g dry mass 

were extracted by refluxing with methanol for 8 hours. The 

extracts were cleaned up and prepared for analysis using the 

C18 SPE-(EC) procedure described above. The final 1 mL 

sample extracts were prepared 

for analysis in the same manner 

described above.

Instrumental Analysis and 

Data Reduction

HPLC-PDA Analysis

Quantification of diphacinone 

was performed with a Surveyor® 

HPLC system (Thermo-Fisher, Inc., 

San Jose, California) consisting 

of an autosampler, gradient pump, 

photodiode array (PDA) detector, and XCalibur® chromatography data collection and processing software. A Luna® 

C18(2) 100 Å, 150 x 2 millimeter (mm) x 3-μm analytical 

column with a SecurityGuard® C18 guard column cartridge 

(Phenomenex, Torrance, California) was used for the separation of diphacinone. The ion-pair reagent for the mobile phase 

was 0.03 M TBAH-IP in water adjusted to a pH of 6.0 using 

2 N o-phosphoric acid. The mobile phase, methanol/0.03 M 

TBAH-IP (70:30 volume:volume) was delivered isocratically 

at 0.8 mL/min. The sample was applied onto the column via 

20 μL full-loop injections. Diphacinone was detected by wavelength scanning from 230 to 400 nanometers (nm) with quantification at the primary wavelength (286 nm) and confirmation 

at the two secondary wavelengths (314 and 326 nm). The crab 

samples were quantified using the secondary wavelengths 

because of primary wavelength interference from the extract’s 

color. The primary wavelength for the instrumental internal 

standard coumarin was 276 nm with a secondary wavelength 

of 312 nm. Calibration of the instrument was achieved through 

a range of standards from 20 to 5,000 ng/mL diphacinone 

with coumarin used as the instrumental internal standard. The 

instrumental limit of detection was 3 ng/mL, which equates 

to 0.6 ng/g for a 5 g sample analysis. The method LOD were 

based upon repeat analysis of each of the different matrices.

Quality Assurance and Quality Control

The quality-assurance plan for this study was to ensure that 

the analyses provided accurate and precise measurements 

of the samples collected in this study. The general scheme 

included replication of various stages (table A), comparison 

and calibration against known standards, proper maintenance 

and calibration of equipment, accurate sample tracking and 

custody, proper documentation at all steps of sample processing, and other considerations of good laboratory practice. 

The accuracy and precision of analytical methods for 

this study were assessed by the following monitors of sample 

preparation and instrumental analysis: individual method stage 

recoveries, replicated sample or reference matrices, procedural blanks, negative control (reference) matrix blanks, and 

Table A. Quality-control sample types for analysis of environmental samples.

Sample type Quality-control function

Procedural blank Determines analyte laboratory background levels or background interferences with analyte signal.

Negative control material (matrix 

blank)

Determines background interferences with analyte signal 

related to a representative and controllable sample matrix.

Fortified negative control material 

(matrix spike)

Determines analyte recovery and assesses potential signal 

enhancement or suppression from a representative and 

controllable sample matrix.

Replicate sample

 within set

 between sets

Determines repeatability analyte signal associated with a 

specific environmental matrix.

Determines the reproducibility of analyte signal associated 

with a specific environmental matrix.
Tables 15

fortified negative control matrix samples. The fortified matrix 

samples were amended with native analyte during sample 

preparation. The numbers of quality-control samples of each 

type are presented in table B. 

Criteria for Quantification of HPLC-PDA Data

Method limits of detection and limits of quantification 

(LOQ) were estimated from low-level standards and determined by the signal-to-noise ratio generated by a matrix blank 

sample. Keith and others (1983, 1991) established the LOD 

as 3 times the matrix blank background signal, and the LOQ 

as 10 times background signal. For the positive identification 

and quantification of each analyte, the following criteria were 

established:

1. The analyte peak area must be greater than 10 times 

matrix blank background signal (LOQ) for quantification, or 3 times background signal (LOD) to be 

considered detected, but <LOQ. If a peak is not present, or is less than three times background signal, it 

will be considered “Not Detected”.

2. The analyte elution must occur at retention times that 

are equivalent to those for the corresponding calibration standards (within ± 3 seconds or < 1 percent 

difference, as established by the method validation).

3. The spectrum of an unknown analyte must be comparable to the spectrum of a corresponding calibration 

standard (within purity factors established by the 

method validation and the expertise of the analyst).

Acceptance or Rejection Criteria for Results

Background responses from procedural and matrix blanks 

were quantified and used to estimate method limits of detection and quantification. 

Table B. Quality-control sample types and levels selected for study. 

Sample type Quality-control level (number of samples)

Seawater Fish fillet Whole limpet Crab Soil

Mock fortification solutions 1 2 1 1 1

Procedural blanks 1 2 1 1 1

Fortified procedural blanks 0 2 1 1 1

Negative control (reference) 

matrices

1 2 1 1 1

Fortified negative control 

(reference) matrices

1 2 1 1 1

Replicates within set 0 1 1 1 1
16 Results of Lab Testing for Diphacinone Following Aerial Application of Rodenticide on Lehua Island, Kauai County, Hawaii

Publishing support provided by: 

Rolla Publishing Service Center 

For more information concerning this publication, contact:

Director, USGS Columbia Environmental Research Center

4200 New Haven Road

Columbia, MO 65201

(573) 875–5399

Or visit the Columbia Environmental Research Center Web site at:

http://www.cerc.usgs.gov
Tables 17
Orazio and others— Results of Lab Testing for Diphacinone Following Aerial Application of Rodenticide on Lehua Island, Kauai County, Hawaii—OFR 2009–1142