U.S. Department of the Interior

U.S. Geological Survey

Open-File Report 2008–1285

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

Determination of Diphacinone in Sea Water, Vertebrates, 

Invertebrates, and Bait Pellet Formulations Following 

Aerial Broadcast on Mokapu Island, Molokai, Hawai’i
Cover. Aerial photograph of Mokapu Island, Molokai, Hawai’i (Photo by C. Swenson, U.S. Fish and Wildlife Service).
Determination of Diphacinone in Sea Water, 

Vertebrates, Invertebrates, and Bait Pellet 

Formulations Following Aerial Broadcast 

on Mokapu Island, Molokai, Hawai’i

By Robert W. Gale, Michael Tanner, Carl E. Orazio

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

Open-File Report 2008–1285

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: 2008

For product and ordering information:

World Wide Web: http://www.usgs.gov/pubprod

Telephone: 1-888-ASK-USGS

For more information on the USGS--the Federal source for science about the Earth, its natural and living resources, 

natural hazards, and the environment:

World Wide Web: http://www.usgs.gov

Telephone: 1-888-ASK-USGS

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 materials contained within this report.

Suggested citation:

Gale, R.W., Tanner, M., and Orazio, C.E., 2008, Determination of diphacinone in sea water, 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. 
iii

Contents

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

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

Purpose and Scope ..............................................................................................................................3

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

Mokapu Sample History.......................................................................................................................3

Sample Preparation..............................................................................................................................4

Materials .......................................................................................................................................4

Sample Preparation for Diphacinone in Sea-Water Samples..............................................4

Sample Preparation and Quantification of Diphacinone in Tissues....................................6

Fish: Ta`ape, A`awa, and Hagi Fillets...............................................................................6

Limpet: Whole Opihi ...........................................................................................................7

Sample Preparation for Diphacinone in Ramik® Green Bait...............................................7

Instrumental Analysis and Data Reduction......................................................................................8

Final Sample Preparation ...........................................................................................................8

HPLC-PDA Analysis.....................................................................................................................8

Quality Assurance and Quality Control .............................................................................................8

Sea Water Holding Time.............................................................................................................8

Negative Control Bait Matrix .....................................................................................................8

Criteria for Quantification of HPLC-PDA Data.........................................................................8

Acceptance or Rejection Criteria for Results .........................................................................9

Results .............................................................................................................................................................9

Sea Waters..........................................................................................................................................10

Fish Fillets.............................................................................................................................................10

Limpets..................................................................................................................................................14

Ramik® Green Diphacinone Bait Pellets........................................................................................14

Conclusions...................................................................................................................................................14

Acknowledgements.....................................................................................................................................15

References....................................................................................................................................................15

Figures

1. Screen shot of Mokapu, Island, Molokai, Hawai’i ..................................................................2

2. Aerial photograph of Mokapu Island, Molokai, Hawai’i.........................................................2

3. Chart showing preparatory methods for diphacinone in the various matrices .................6

4–5. Graphs showing—

4. High performance liquid chromatography-photodiode array (HPLC-PDA)

chromatogram of diphacinone ..........................................................................................9

5. Photodiode array (PDA) spectrum of diphacinone......................................................10
iv

Tables

1. Sample collection information....................................................................................................5

2. Quality-control sample types for analysis of environmental samples...............................11

3. Quality-control sample types and levels selected for study ...............................................11

4. Diphacinone concentrations in sea water.............................................................................12

 5. Diphacinone concentrations in fish fillets..............................................................................13

 6. Diphacinone concentrations in whole Opihi limpets............................................................14

 7. Diphacinone concentrations in Ramik® Green bait lot composites .................................15

Conversion Factors

Multiply By To obtain

Length

meter (m) 0.3048 feet (ft)

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

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)

micromolar (μM) = micromoles per liter (10-6 M)

microgram per gram (μg/g) = parts per million (ppm: 106

)

nanogram per gram (ng/g) = part per billion (ppb; 109

)

microgram per milliliter (μg/mL) = parts per million (ppm: 106

)

microgram per liter (μg/L) = parts per billion (ppb: 109

)

nanogram per milliliter (ng/mL) = part per billion (ppb; 109

)

Application rate

pounds per acre (lb/acre) 1.125 kilograms per hectare (kg/ha)

Electrical resistance

mega-ohms (mΩ) = 106

 ohms

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). Concentrations of chemical constituents in calibration 

standard solutions and in liquid samples (sea water) are given in nanograms per milliliter (ng/

mL, or parts per billion, ppb).
Determination of Diphacinone in Sea Water, Vertebrates, 

Invertebrates, and Bait Pellet Formulations Following 

Aerial Broadcast on Mokapu Island, Molokai, Hawai’i

By Robert W. Gale, Michael Tanner, and Carl E. Orazio

Abstract

This report presents the results of a study to determine 

diphacinone concentrations in samples of sea water and in 

fillet samples of fish and in limpets from the ocean adjacent 

to Mokapu Island and from reference samples from Molokai, 

Hawai’i; concentrations of the active ingredient (diphacinone) 

were also determined in samples of the Ramik® Green bait 

pellets used for the broadcast study. After preparation, diphacinone concentrations were determined with high-performance 

liquid chromatography with photodiode array detection. No 

detectable concentrations of diphacinone were found in the 

fish, limpets, or sea-water samples from Mokapu Island or 

from the reference sites. The limit of detection for diphacinone in sea water was 18 nanograms per milliliter (parts per 

billion); the limit of detection in fish fillets was 10 nanograms 

per gram (parts per billion); and the limit of detection in 

limpets was 17 nanograms per gram. The average concentration of diphacinone in the Ramik® Green bait pellets was 45 

micrograms per gram (parts per million), which represents 90 

percent of the nominal concentration stated for the product by 

the manufacturer. 

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.

Mokapu is an approximately 10-acre island located 

approximately 1 kilometer (km) off the north coast of Molokai 

just east of the Kalaupapa Peninsula (figs. 1 and 2). The 

island is a Hawai’i State Seabird Sanctuary managed by the 

Hawai’i Department of Land and Natural Resources, Division 

of Forestry and Wildlife (DOFAW); the island supports native 

populations of white-tailed tropicbirds (Phaethon lepturus), 

red-tailed tropicbirds (P. rubricauda), black noddies (Anous 

minutus), and wedge-tailed shearwaters (Puffinus pacificus). 

Like the nearby islands of Okala and Huelo, Mokapu supports 

some of the most diverse native coastal plant communities in 

Hawai’i. For example, Mokapu contains 29 native plant species; several of these species are rare and vulnerable to extinction. The island is dominated by native shrubs, but retains 

small groves of native lama trees (Diospyros spp.), some 

native palm trees (Pritchardii hillebrandii), which dominate 

nearby Huelo, and 11 of the last 14 individuals of the shrub 

Pittosporum halophilum that is endemic to Molokai. Peucedanum sandwicense, a large perennial herbaceous plant, is listed 

as threatened under the Endangered Species Act of 1973, and 

Lepidium bidentatum var. o-waihiense, a succulent herbaceous 

plant, also is a species of concern on the island. In 2003, the 

U.S. Fish and Wildlife Service (USFWS) designated Mokapu 

Island as critical habitat for P. sandwicense and Tetramolopium 

rockii (perennial shrubs) and Brighamia rockii, a succulent 

perennial plant present on nearby adjacent islands. 

It is extremely likely that the presence of rats has terminated or slowed the recruitment of the threatened Peucedanum 

sandwicense as well as Pritchardia, Pittosporum, and Diospyros along with other native plant taxa on Mokapu. Rats are 

known to eat Pritchardia seeds, and their presence on Mokapu 

is believed to be contributing to the decline of this rare, 

endemic species. In 2006, only 12 mature Pritchardia palms 

and one seedling were present on Mokapu.

Likewise, only 11 individuals of Pittosporum, 20 individuals of Peucedanum, and two small groves of Diospyros

remain on Mokapu. In addition, observations from other 

Pacific islands document that rats depredate eggs, and sometimes prey upon the young and adults of three of the seabird 

species known to nest on Mokapu: red-tailed and white-tailed 
2 Determination of Diphacinone Following Aerial Broadcast on Mokapu Island, Molokai, Hawai’i

Figure 1. Screen shot of Mokapu, Island, Molokai, Hawai’i (courtesy of GoogleEarth©).

Figure 2. Aerial photograph of Mokapu Island, Molokai, Hawai’i. (Photo by C. Swenson, U.S. Fish and Wildlife 

Service).
Methods 3

tropicbirds and wedge-tailed shearwaters; therefore, rodent 

control is a critical management objective for maintaining and/

or restoring the ecological integrity of Mokapu Island.

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 be an effective toxicant for rats in Hawai’i 

and elsewhere. It is efficacious, yet has relatively low risk of 

impacts to nontarget species through consumption of bait pellets (direct impacts) and/or through consumption of prey that 

have consumed the bait pellets (secondary impacts).

The USFWS, the DOFAW, and the U.S. Department of 

Agriculture, Animal and Plant Health Inspection Service, 

Wildlife Services (USDA–APHIS–WS) proposed to eradicate 

Polynesian rats from Mokapu Island using the anticoagulant 

rodenticide diphacinone (0.005 percent active ingredient) 

applied by aerial broadcast. Operations were conducted only 

during the winter months (December through March) when 

alternate rat foods and rat populations are lowest and migratory native nontarget species were not present, or were present 

only in low numbers.

Toxic bait pellets containing the active ingredient diphacinone at 50 parts per million (ppm) were used to exterminate 

rats. The bait is dyed green by the manufacturer to reduce 

dietary exposure to birds. The rodenticide was broadcast 

uniformly across the emergent land area of the island at the 

approved application rate in an attempt to expose all rats to a 

lethal dose. Applications were completed by aerial broadcast 

across 100 percent of the land area of the island at a nominal 

rate of 10 pounds per acre (lb/acre) in two separate broadcast 

applications on February 6 and February 12, 2008. Coastlines 

and steep areas were treated with twice the rodenticide for 

each application.

Monitoring for primary and secondary adverse impacts 

of diphacinone on nontarget species was one of the foremost 

concerns for this rodent eradication project. Populations of 

desired nontarget species, including nesting seabirds and 

protected plants, were monitored actively for a sufficient 

period (approximately 2 years) to produce reliable population 

estimates of adverse impacts before and after rodenticide treatments. The preceding text was condensed from Swenson and 

Duvall, 2007.

This report presents the development of analytical chemistry methods for determining diphacinone concentrations in 

sea water, fish tissues, and limpets, and the quantification of 

diphacinone concentrations in these samples after application of the rodenticide to Mokapu Island. Sea water, fish, and 

limpets from Kalaupapa National Historical Park and from 

commercial sources were used as negative control (reference) 

samples. The results are intended to provide the program 

managers and other resource managers in the study area with 

reference data regarding the fate and effects of diphacinone 

on Mokapu Island. Additionally, the procedures developed for 

the various matrices could serve as prototypes for the development of similar methods on other matrices where diphacinone 

contamination may be an issue in the future.

Purpose and Scope

The objectives of this study were to assess the levels of 

diphacinone present in sea water and biota as a consequence of 

aerial broadcast of Ramik Green® rodenticide to exterminate 

Polynesian rats from Mokapu Island. These results will serve 

as a demonstration of the actual risk of exposure of non-target 

organisms to the use of diphacinone in the proposed rodent 

eradication strategy. The study consisted of several analytical 

sample sets, each addressing the post-broadcast diphacinone 

exposure levels in the immediate environment.

Methods

Mokapu Sample History

Personnel from the USFWS, DOFAW, and/or USDA–

APHIS–WS collected grab samples of reference sea water on 

January 23, 2008, at Kalaupapa National Historical Park (Ka 

Laea Point) for diphacinone analysis. Aerial broadcasts of 

Ramik® Green rodenticide bait were conducted on February 6 

and February 12, 2008. On February 17, after aerial broadcasts 

were completed, sea-water samples were collected from several 

points surrounding Mokapu Island for diphacinone analysis. 

Following aerial broadcast of Ramik® Green, surface 

water grab samples were collected within 30 feet (ft) of the 

eastern and western sides of Mokapu Island. Six 250 milliliter (mL) samples were collected at each of the six stations. 

Three stations were located off the eastern shore and three off 

the western shore. Parts of each sample were shipped cold [4 

°Celsius (°C)] to the U.S. Geological Survey (USGS) and the 

other participating laboratories using chain of custody protocols. Two samples from each site were provided to the USGS 

for analysis. An additional experiment was incorporated to 

validate the effect of holding time for the analysis of diphacinone in sea-water samples (stored at 4 °C for 53 days).

Diphacinone was determined in invertebrates (limpets) 

and vertebrates (fish) from reference sites and from Mokapu 

Island following aerial broadcast of Ramik® Green to quantify 

reference and post-broadcast diphacinone levels. Personnel 

from the USFWS, DOFAW, and/or USDA–APHIS–WS collected one species of mollusk (Opihi, the Hawai’ian Limpet) 

from a reference location (Kalaupapa) and from Mokapu sites 

after aerial broadcast on February 17, 2008. Six individual 

fish samples (duplicate samples of three separate species, i.e. 

Ta`ape, A`awa, and Hagi) were collected from within 50 ft of 

the western shore of Mokapu on February 17, 2008, following 

aerial broadcast. Reference samples of Ta`ape were purchased 

from a commercial Oahu market. Fish and limpet samples 

were packaged in aluminum foil packets sealed in zip-lock 

bags and stored frozen. Whole limpet carcasses and fillet 
4 Determination of Diphacinone Following Aerial Broadcast on Mokapu Island, Molokai, Hawai’i

portions of each fish were shipped frozen to the participating 

laboratories under chain of custody on March 17, 2008 (table 

1).

The diphacinone content of Ramik® Green rodenticide 

bait samples stored at the Maui DOFAW base yard and subsequently used in the rat eradication efforts on Mokapu Island, 

Hawai’i, was verified. Personnel from the USFWS, DOFAW 

and/or USDA–APHIS–WS collected 10 random Ramik® 

Green rodenticide bait samples from the Maui DOFAW base 

yard on February 12, 2008. Parts [~30 2-gram (g) pellets] of 

each sample were shipped frozen to the participating laboratories under chain of custody, and were received by March 17, 

2008 (table 1). 

Upon receipt at the USGS, the sea-water samples were 

logged in to the sample data-base system, assigned a unique 

identification number, and stored refrigerated at 4 °C until 

analysis. The tissue and bait samples were logged and stored 

in the dark at -20 oC until analysis.

Sample Preparation

Sample preparation methods for diphacinone are matrixdependent and were developed for each target matrix: sea water, fillet, whole limpet, and bait. The general method consisted of extraction of diphacinone from the matrix of interest, 

and subsequent concentration and purification of the extract 

by solid-phase extraction (SPE) or low performance sizeexclusion chromatography (LP-SEC). The general schemes 

developed are presented in figure 3.

Analytical separation of diphacinone was performed by 

high performance liquid chromatography (HPLC) followed 

by ultraviolet-visible photodiode array absorbance (PDA) 

detection and quantification after the methods of Yang and 

others (2001). Coumarin was used as the instrumental internal 

standard.

Materials

Solid-phase extraction cartridges [Oasis-HLB (hydrophilic/lipophilic balance) 6 mL x 500 milligrams (mg)] 

were purchased from Waters Corp., Milford, Massachusetts. 

Octadecyl SPE cartridges [Isolute C18(EC) 6 mL x 1 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), acetic acid (HPLC-grade), 

anhydrous sodium sulfate, ascorbic acid (reagent grade), and 

2 N o-phosphoric acid 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. Tetrabutylammonium 

hydroxide (TBAH) was purchased from Sigma-Aldrich, St. 

Louis, Missouri. Tetrabutylammonium phosphate (TBAP) was 

purchased from ACROS Organics, Somerville, New Jersey. 

Diphacinone and coumarin standard solutions (in methanol) 

were purchased from AccuStandard, New Haven, Connecticut. 

Milli-Q water [18 mega-ohms (mΩ), Millipore Synergy UV, 

Millipore Corp., Bedford, Massachusetts] was used throughout 

the analytical process.

Aqueous tetrabutylammonium hydroxide ion pair (TBAHIP) solutions used for HPLC and for SPE were prepared at 0.1 

molar (M) and 0.03 M in water and pH adjusted to 6.0 with 

o-phosphoric acid. Solid TBAP (not pH adjusted) was used to 

prepare the methanolic 5 millimolar (mM) tetrabutylammonium phosphate solution used for the bait reflux-extractions. 

Sample Preparation for Diphacinone in Sea-Water 

Samples

Diphacinone has a water solubility of 30 ppm and is 

subject to hydrolysis at pH 5 or less; however, it is stable to 

hydrolysis from pH 7 to 9 (U.S. Environmental Protection 

Agency, 1998). The sea-water samples collected for this study 

were stored refrigerated at 4 °C and in the dark for 53 days 

before analysis. The samples were collected on February 17, 

2008, shipped to USGS on March 17, 2008, and extracted on 

April 10, 2008, following method development and validation. 

The storage stability of diphacinone in sea water for a similar 

period was investigated to ensure that the holding time for 

diphacinone was not exceeded. A 200 mL aliquot of Kalaupapa reference sea water (pH 8.1) was fortified with about 5 

micrograms (μg) of diphacinone and was returned to refrigerated storage to simulate the holding times for the samples. 

Another 200 mL volume of Kalaupapa reference sea water (pH 

8.1) was fortified with about 5 μg diphacinone at the time of 

analysis to determine recovery efficiency. The storage stability 

sample was analyzed on May 19, 2008 (53 days post fortification), using the methods described for sea-water samples.

A solid phase extraction method was developed for the 

isolation of diphacinone from sea water. Although the isolation 

of diphacinone from coconut crab (Birgus latro) tissues is well 

understood (Tanner and Orazio, written commun., 2008), no 

work has been reported to assess the recovery of diphacinone 

from sea water. Generally, the water samples were extracted by 

an appropriate SPE cartridge to adsorb diphacinone that was 

then recovered by elution of the SPE and quantified by HPLC 

with PDA detection. Potential matrix effects from salts on 

recovery of diphacinone from the SPE sorbent or the retention of diphacinone were investigated and determined to be 

negligible.

The pH of the Mokapu Island water samples was determined using a Mettler-Toledo Seven Easy pH meter (Schwerzenbach, Inc., Switzerland). The pH meter was calibrated with 

4.00 and 7.00 buffer solutions (Fisher Scientific, Fair Lawn, 

New Jersey) before pH determinations. Fortified samples were 

prepared using 200 mL of Kalaupapa reference sea water or 

100 mL of aqueous Oceanic Natural Sea Salt solution and 

adding 2.5 μg diphacinone. The final concentrations of the 

fortified reference sea-water samples and the Oceanic Natural 
Table 1. Sample collection information.[USGS, U.S. Geological Survey; ID, identification; mL, milliliters; Al, aluminum; g, grams]

Collection

date

USGS

ID Site Sample description Sample type Sample container Am

1/23/2008 42033 Kalaupapa National Historical 

Park (at Ka Laea Point), Molokai

Sea water Water 250-mL widemouth jars 3x250 mL 

1/23/2008 42034 Kalaupapa National Historical 

Park (at Ka Laea Point), Molokai

Limpet, Opihi (Cellata exarata) Whole organism (incl. shell) Al-foil/Ziploc 3 individuals—a

3/17/2008 42035 Oahu Commercial Fish Market Blue-lined Snapper, Ta`ape (Lutjanus kasmira) Skin-on fillet Al-foil/Ziploc 4 individuals—a

2/17/2008 42036 Mokapu Station 1 Sea water Water 250-mL widemouth jars 2x250 mL 

2/17/2008 42037 Mokapu Station 2 Sea water Water 250–mL widemouth jars 2x250 mL 

2/17/2008 42038 Mokapu Station 3 Sea water Water 250–mL widemouth jars 2x250 mL 

2/17/2008 42039 Mokapu Station 4 Sea water Water 250–mL widemouth jars 2x250 mL 

2/17/2008 42040 Mokapu Station 5 Sea water Water 250–mL widemouth jars 2x250 mL 

2/17/2008 42041 Mokapu Station 6 Sea water Water 250–mL widemouth jars 2x250 mL 

2/17/2008 42042 Mokapu Station 1 Limpet, Opihi (Cellata exarata) Whole organism (without shell) Al-foil/Ziploc ~4 individuals—

2/17/2008 42043 Mokapu Station 2 Limpet, Opihi (Cellata exarata) Whole organism (without shell) Al-foil/Ziploc ~4 individuals—

2/17/2008 42044 Mokapu Station 3 Limpet, Opihi (Cellata exarata) Whole organism (without shell) Al-foil/Ziploc ~4 individuals—

2/17/2008 42045 Mokapu Station 1-A Blue-lined Snapper, Ta`ape (Lutjanus kasmira) Skinless fillet Al-foil/Ziploc 1 individual—ab

2/17/2008 42046 Mokapu Station 1-B Blue-lined Snapper, Ta`ape (Lutjanus kasmira) Skinless fillet Al-foil/Ziploc 1 individual—ab

2/17/2008 42047 Mokapu Station 1-C Blue-lined Snapper, Ta`ape (Lutjanus kasmira) Skinless fillet Al-foil/Ziploc 1 individual—ab

2/17/2008 42048 Mokapu Station 1-D Blue-lined Snapper, Ta`ape (Lutjanus kasmira) Skinless fillet Al-foil/Ziploc 1 individual—ab

2/17/2008 42049 Mokapu Station -E Hogfish, A`awa (Bodianus bilunulatus) Skinless fillet Al-foil/Ziploc 1 individual—ab

2/17/2008 42050 Mokapu Station 1-F Bridled triggerfish, Hagi (Sufflamen fraenatus) Skinless fillet Al-foil/Ziploc 1 individual—ab

2/17/2008 42051 Maui-1 Ramik® Green (diphacinone bait pellets) Individual box of pellets #1 Ziploc ~25–30 pellets 3

2/17/2008 42052 Maui-2 Ramik® Green (diphacinone bait pellets) Individual box of pellets #2 Ziploc ~25–30 pellets 3

2/17/2008 42053 Maui-3 Ramik® Green (diphacinone bait pellets) Individual box of pellets #3 Ziploc ~25–30 pellets 3

2/17/2008 42054 Maui-4 Ramik® Green (diphacinone bait pellets) Individual box of pellets #$ Ziploc ~25–30 pellets 3

2/17/2008 42055 Maui-5 Ramik® Green (diphacinone bait pellets) Individual box of pellets #5 Ziploc ~25–30 pellets 3

2/17/2008 42056 Maui-6 Ramik® Green (diphacinone bait pellets) Individual box of pellets #6 Ziploc ~25–30 pellets 3

2/17/2008 42057 Maui-7 Ramik® Green (diphacinone bait pellets) Individual box of pellets #7 Ziploc ~25–30 pellets 3

2/17/2008 42058 Maui-8 Ramik® Green (diphacinone bait pellets) Individual box of pellets #8 Ziploc ~25–30 pellets 3

2/17/2008 42059 Maui-9 Ramik® Green (diphacinone bait pellets) Individual box of pellets #9 Ziploc ~25–30 pellets 3

2/17/2008 42060 Maui-10 Ramik® Green (diphacinone bait pellets) Individual box of pellets #10 Ziploc ~25–30 pellets 3
6 Determination of Diphacinone Following Aerial Broadcast on Mokapu Island, Molokai, Hawai’i

Sea Salt water samples were 12.5 nanograms per milliliter 

(ng/mL) and 25 ng/mL, respectively.

Oasis-HLB SPE cartridges were cleaned and preconditioned with 10 mL acetonitrile followed by 10 mL methanol at 

a flow rate of about 1 mL/minute and then dried under vacuum 

for about 3 minutes. Immediately before extraction, the SPE 

cartridge was conditioned with 10 mL aqueous TBAH-IP 

reagent followed by 20 mL of Milli-Q water, at about 1 

mL/minute (cartridge remained wet). A 200 mL water sample 

(100 mL for replicates, laboratory blanks, and laboratory-fortified samples) was applied to the cartridge at about 3 mL/minute. The sample container was rinsed quantitatively with about 

20 mL Milli-Q water, which then was applied to the cartridge. 

Next, the cartridge was washed with 3 mL Milli-Q water and 

dried under vacuum for about 3 minutes. 

Diphacinone was recovered from the cartridge with 

13 mL acetonitrile; the eluant was collected in a 15-mL amber 

culture tube (fraction 1). Any more strongly bound diphacinone was recovered from the cartridge with 5 mL 70:30 

(methanol:aqueous TBAH-IP reagent; volume:volume); this 

eluant was collected in a separate 15-mL amber culture tube 

(fraction 2).

The first diphacinone eluants (fraction 1) were evaporated 

to dryness using nitrogen with a water bath temperature of 

< 50 oC (N-EVAP, Organomation, Inc., Berlin, Massachusetts). 

The residues were dissolved in 700 microliters (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 residual 

diphacinone eluants (fraction 2) were fortified with 5 μg coumarin and analyzed directly.

Sample Preparation and Quantification of 

Diphacinone in Tissues

Hunter and Sharp (1988) described the addition of 

ascorbic acid to the extraction solvent, as well as to the matrix 

before dehydration for extractions from vertebrate liver 

samples. This increased the recovery of indandione-group 

anticoagulant rodenticides in all of the liver samples tested. 

Before analysis of Mokapu field samples, it was established 

that ascorbic acid treatment was not required to achieve 

adequate recoveries of any diphacinone from the fish and limpet matrices. Further method development efforts established 

that a C18 SPE cleanup was necessary for fish fillet samples, 

whereas both C18 SPE and LP-SEC cleanup steps were necessary to remove residual lipid materials from the limpet matrix.

Fish: Ta`ape, A`awa, and Hagi Fillets

Fortified fish fillet samples were prepared in about 5 g

 of reference Ta`ape matrix or sodium sulfate (procedural 

spikes) by adding 2.5 μg diphacinone to the dehydrated reference material. The final concentration of the fortified matrix 

samples was 500 nanograms per gram (ng/g) wet-weight.

Homogenized Ta`ape (Lutjanus kasmira), A`awa (Bodianus bilunulatus), or Hagi (Sufflamen fraenatus) fillet tissue 

samples (5 g fish skinless fillet) were dehydrated with 25 g 

anhydrous sodium sulfate. The mixture was allowed to dehydrate at least 2 hours, and then was blended with stainless steel 

blades and a commercial blender until a free flowing powder 

was obtained. 

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 

Column

Extraction

C18 SPE

(lipid removal)

HPLC-PDA

Fillet

Sonication (DCM)

Reflux (MethanolicTBAP)

Sonication (DCM/Acetone 

/Acetic Acid)

HPLC-PDA

Bait

HLB SPE

F1 

Unbound

F2

Bound

Sea water

HPLC-PDA HPLC-PDA

Column 

Extraction

Limpet

C18 SPE

(lipid removal)

LP-SEC (biogenic removal)

HPLC-PDA

Figure 3. Preparatory methods for diphacinone in the various matrices.
Methods 7

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

at a flow rate of approximately 2 mL/minute 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 °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 co-extracted interferences (mainly lipid material) 

were removed by C18 SPE. The cartridges were conditioned 

with 10 mL methanol followed by 10 mL 0.03 M TBAH-IP, 

dried under vacuum for about 1 minute, and finally by rinsing 

with 10 mL acetonitrile (the cartridge remained wet). The 

2 mL sample extracts were applied to the cartridge at about 

2 mL/minute (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 diphacinone eluants were evaporated 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.

Limpet: Whole Opihi

Fortified samples were prepared with about 3 g of reference limpet matrix or sodium sulfate (procedural spikes) by 

adding 2.5 μg diphacinone to the dehydrated reference material. The final concentration of the fortified matrix samples 

was 830 ng/g.

Whole Opihi (Cellata exarata) limpet sample homogenates (about 3 g) were dehydrated with 30 g anhydrous sodium 

sulfate. The mixture was allowed to dehydrate at least 2 hours, 

and then was blended with stainless steel blades and a commercial blender until a free flowing powder was obtained.

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 mL/minute 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 °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 co-extracted interferences (mainly lipid material) 

were removed by C18 SPE. The cartridges were conditioned 

with 10 mL methanol followed by 10 mL 0.03 M TBAH-IP, 

dried under vacuum for about 1 minute, and finally by rinsing 

with 10 mL acetonitrile (the cartridge remained wet). The 

2 mL sample extracts were applied to the cartridge at about 

2 mL/minute (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 diphacinone eluants were evaporated to dryness 

with nitrogen. The residues were dissolved in 3 mL dichloromethane. 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/minute). The diphacinone fractions (LP-SEC collect 

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 dichloromethane 

sample extracts were evaporated 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.

Sample Preparation for Diphacinone in Ramik® 

Green Bait

Primus and others (1998) described the extraction of 

diphacinone from steam rolled oat baits using 5 mM methanolic tetrabutylammonium phosphate ion pairing solution. Mesmer and Flurer (2000) described the extraction of diphacinone 

by sonication of commercial indanedione rodenticides with 

methanol containing 2 percent formic acid. The extraction 

recovery of diphacinone from the Ramik® Green formulation 

was established in this study before analysis of field samples. 

The removal of co-extracted inert ingredients (green dye, 

waxes, etc.) using suitable SPE and other techniques also was 

investigated.

No reference material without active ingredient was 

available; therefore, no method quality-control samples were 

analyzed with the sample set. Instead, steps were taken to optimize extraction efficiency based on the nominal active ingredient concentration of diphacinone reported by the manufacturer 

in Ramik® Green bait (50 ppm).

Three composites of the bait samples were prepared 

containing two baits from each sample-lot submitted as follows: Lots 1, 2, and 3; Lots 4, 5, and 6; Lots 7, 8, 9, and 10. 

Two bait pellets were removed from each of the zip-lock bags, 

individual weights recorded, and the pellets were transferred 

to a ceramic mortar. The individual pellets were chopped into 

smaller pieces using a stainless steel knife, and the composite 

samples were ground to a fine powder using a ceramic pestle.

Approximately 2 g portions of each composite were 

weighed into 250-mL boiling flasks, approximately 15 mL 

dichloromethane added, and the composites sonicated for 

1 hour. Next, the dichloromethane extracted samples were 

extracted by reflux with 5 mM methanolic tetrabutylammonium phosphate (TBAP; 55 mL) for 8 hours. The extracts were 
8 Determination of Diphacinone Following Aerial Broadcast on Mokapu Island, Molokai, Hawai’i

filtered through Whatman 41 ashless filter paper with repeated 

rinses of the boiling flask with extraction solvent. 

A second extraction was performed by soncating the 

post-refluxed composite bait samples with 15 mL 

dichloromethane:acetone:acetic acid (1:1:2%; 

volume:volume:volume) for 2 hours to recover any residual 

diphacinone. This second extract was filtered with repeated 

rinses with the extraction solvent. Final volumes were adjusted 

to 100 mL (for the reflux extract) or 25 mL (for the sonicated 

extract) using the appropriate extraction solvent and the solutions were thoroughly mixed. Sub-samples of each of the first 

extracts (2 mL) and each of the second extracts (10 mL) were 

transferred to 15-mL amber culture tubes.

The first and second extracts of the composite bait samples were evaporated individually 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. 

Instrumental Analysis and Data Reduction

Final Sample Preparation

As per previous discussion, all extracts were evaporated to 

approximately 3 mL by rotary evaporation and transferred to 

15-mL screw capped amber culture tubes with three sequential 

2-mL acetonitrile rinses. The concentrated extracts were evaporated to dryness by nitrogen evaporation and reconstituted in 

700 μL of methanol. Once the residues were dissolved, 300 

μL of 0.03 M tetrabutylammonium hydroxide ion pair reagent 

(TBAP-IP) in water was added and mixed thoroughly. The 

samples were filtered through 0.45 μm PTFE syringe filters 

directly in to 1-mL amber autosampler vials. 

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, 

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 TBAHIP 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/minute. 

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 primary 

wavelength for the instrumental internal standard, coumarin 

was 276 nm with a secondary wavelength of 312 nm. A diphacinone standard chromatogram is shown in figure 4; a photodiode array detector spectrum is shown in figure 5. Calibration 

of the instrument was achieved through a range of standards 

from just above the limit of quantification of about 10 ng/mL 

to 5,000 ng/mL diphacinone [and coumarin].

Quality Assurance and Quality Control

All research was conducted in accordance with the USGS 

Quality Assurance Plan, a system of checks managed by a 

Quality-Assurance system that assures that defined standards 

of quality are being met (at stated levels of confidence). The 

objective of the quality assurance plan for this study was to 

assure that the analytical and/or biochemical analyses provided 

accurate and precise measurements of the samples collected in 

this study. The general scheme included replication of various 

stages (table 2), 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 was assessed by the following checks of sample preparation and instrumental analysis: replicated sample or reference 

matrices, procedural blanks, fortified procedural samples, 

negative control (reference) matrix blanks, and fortified negative control matrix samples. The fortified matrix samples 

were amended with native analyte(s) during sample preparation. The numbers of quality control samples of each type are 

presented in table 3. 

Sea Water Holding Time

Because of questions related to the storage of sea-water samples (dark, refrigerated at 4 °C), a stability check 

experiment was conducted to determine the feasibility of the 

approximately 50 day holding time from sample collection 

(February 17, 2008), shipping to the USGS (March 17, 2008), 

and subsequent analysis (April 10, 2008). 

Negative Control Bait Matrix

The lack of a reference bait material without active ingredient limited the quality control samples to be analyzed with 

the bait sample set. Steps were taken to maximize extraction 

efficiency, based on the manufacturer’s nominal active ingredient concentration of 50 ppm diphacinone in Ramik® Green 

bait.

Criteria for Quantification of HPLC-PDA Data

Method limits of detection (LOD) and limits of quantification (LOQ) were estimated from low-level standards and 
Results 9

determined by the signal-to-noise ratio of the peak. Keith 

and others (1983; 1991) established the LOD as 3 times the 

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 

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. Acceptable recoveries of spiked 

samples were determined by diphacinone methods development studies as 50 to150 percent.

Results

Final analytical results were adjusted using the response 

of the instrumental internal standard and then adjusted for any 

Results 

0 2 4 6 8 10 12 14

0

20,000

40,000

60,000

80,000

100,000

120,000

140,000

160,000

180,000

200,000

220,000

240,000

260,000

2.60

4.97

Coumarin (internal standard)

Diphacinone (5,000 nanograms per milliliter) 

TIME, IN MINUTES

Figure 4. High performance liquid chromatography-photodiode array (HPLC-PDA) chromatogram of 

diphacinone (wavelength range 230 to 400 nanometers).
10 Determination of Diphacinone Following Aerial Broadcast on Mokapu Island, Molokai, Hawai’i

background levels of analytes by subtraction of mass-weighted 

procedural blank amounts. Similarly, matrix spike recoveries 

were estimated after any necessary corrections.

The sample preparation methods increased in their complexity as the complexity of the sample matrices increased, 

generally in the order: sea water < fillet < limpet. Preparation 

of bait composite samples required strong and specific reaction conditions to completely release diphacinone bound in the 

bait matrix and the wax-like coating materials. The efficiency 

and selectivity of the preparatory methods that were developed 

for this study was reflected in the achievement of consistently 

low detection limits without background interferences for all 

matrices.

Sea Waters

The concentrations of diphacinone in Mokapu Island seawater samples were below the LOD determined for this matrix 

and method (table 4). The LOD was 18 ng/mL (or parts per 

billion), and the LOQ was 61 ng/mL according to the methods 

of Keith et al. (1983; 1991). Fraction 1 contained greater than 

98 percent of the diphacinone in fortified sea-water samples. 

The instrumental internal standard (coumarin) response ranged 

from 96 to 108 percent of standards. Instrumental blanks, 

procedural blanks, and negative control samples for the seawater analyses did not have any detectable concentrations of 

diphacinone.

Fish Fillets

The concentrations of diphacinone in Mokapu Island 

Ta`ape, A’awa, and Hagi skinless fillet samples were below 

the LOD [table 5; 10 nanograms per gram (ng/g), or ppb]. The 

instrumental internal standard (coumarin) responses ranged 

from 96 to 104 percent. Instrumental blanks, procedural 

blanks, and negative control (reference) samples for the fish 

240 260 280 300 320 340 360 380 400

WAVELENGTH, IN NANOMETERS

0

50,000

100,000

150,000

200,000

250,000

300,000

350,000

400,000

450,000

500,000

550,000

600,000

650,000

700,000

750,000

800,000

276.00

310.00

Diphacinone (retention time 5.2 

minutes, from figure 4)

Figure 5. Photodiode array (PDA) spectrum of diphacinone.
Results 11

Table 2. 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

Replicate sample—between sets

Determines repeatability analyte signal associated with a specific 

environmental matrix.

Determines the reproducibility of analyte signal associated with a 

specific environmental matrix.

Positive control material Determines analyte recovery and assesses potential signal enhancement or suppression from a representative and controllable sample 

matrix on an ongoing basis within or between laboratories.

Table 3. Quality-control sample types and levels selected for study. 

[--, not applicable]

Quality-control level (number of samples)

Sample type Sea water Fish fillet Whole limpet Ramik® Green Bait

Mock fortification solutions 1 1 1 1

Procedural blanks 1 1 1 1

Fortified procedural blanks -- 1 1 1

Negative control (reference) 

matrices 1 1 1 --

Fortified negative control 

(reference) matrices 1 1 1 --

Replicate samples within 

set

3 3 3 3

Total 7 8 8 6
12 Determination of Diphacinone Following Aerial Broadcast on Mokapu Island, Molokai, Hawai’i

Table 4. Diphacinone concentrations in sea water.

[USGS, U.S. Geological Survey; ID, identification; ng/mL, nanograms per milliliter (parts per billion, ppb); <, less than; ND, not 

detected; --, not applicable]

USGS

ID Field label pH Diphacinone(ng/mL)

Recovery

(percent)

42033 Kalaupapa National Historical Park Ka Laea Point, 

Molokai

8.10 < 18 (ND) --

42036 Mokapu Station 1 8.20 < 18 (ND) --

42037 Mokapu Station 2 8.17 < 18 (ND) --

42038 Mokapu Station 3 8.10 < 18 (ND) --

42039 Mokapu Station 4 8.21 < 18 (ND) --

42040 Mokapu Station 5 8.16 < 18 (ND) --

42041-1 Mokapu Station 6—replicate 1 8.17 < 18 (ND) --

42041-2 Mokapu Station 6—replicate 2 8.17 < 18 (ND) --

42041-3 Mokapu Station 6—replicate 3 8.17 < 18 (ND) --

HPLC blank -- < 18 (ND) --

SPE blank -- < 18 (ND) --

Procedural blank -- < 18 (ND) --

42033 Negative control (reference) sea water blank

Kalaupapa National Historical Park

7.96 < 18 (ND) --

Fortified procedural Ocean Sea Salt solution 7.96 -- 88

42033-fortified Fortified negative control (reference) sea water

Kalaupapa National Historical Park

8.11 -- 86
Results 13

Table 5. Diphacinone concentrations in fish fillets.

[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; 

ND, not detected; --, not applicable]

USGS

ID

Field

label Sample type Diphacinone(ng/g)

Recovery

(percent)

42035 Oahu Commercial Fish Market Blue-lined Snapper, Ta`ape (Lutjanus kasmira) < 10 (ND) --

42045 Mokapu Station 1-A Blue-lined Snapper, Ta`ape (Lutjanus kasmira) < 10 (ND) --

42046-1 Mokapu Station 1-B—replicate 1 Blue-lined Snapper, Ta`ape (Lutjanus kasmira) < 10 (ND) --

42046-2 Mokapu Station 1-B—replicate 2 Blue-lined Snapper, Ta`ape (Lutjanus kasmira) < 10 (ND) --

42046-3 Mokapu Station 1-B—replicate 3 Blue-lined Snapper, Ta`ape (Lutjanus kasmira) < 10 (ND) --

42047 Mokapu Station 1-C Blue-lined Snapper, Ta`ape (Lutjanus kasmira) < 10 (ND) --

42048 Mokapu Station 1-D Blue-lined Snapper, Ta`ape (Lutjanus kasmira) < 10 (ND) --

42049 Mokapu Station E Hogfish, A`awa (Bodianus bilunulatus) < 10 (ND) --

42050 Mokapu Station 1-F Bridled Triggerfish, Hagi (Sufflamen fraenatus) < 10 (ND) --

Procedural blank < 10 (ND) --

42035 Negative control Ta`ape (reference) Blue-lined Snapper, Ta`ape (Lutjanus kasmira) < 10 (ND) --

Fortified procedural sample -- 102

42035-fortified Fortified negative control Ta`ape 

(reference)

Blue-lined Snapper, Ta`ape (Lutjanus kasmira) -- 100
14 Determination of Diphacinone Following Aerial Broadcast on Mokapu Island, Molokai, Hawai’i

fillet analyses did not have any detectable concentrations of 

diphacinone. 

The performance throughout the method was monitored 

by fortified procedural samples and fortified reference Ta`ape 

matrix samples. No diphacinone residues were detected in the 

procedural blanks or reference Ta`ape samples. Method recoveries ranged from 100 to 102 percent.

Limpets

The concentrations of diphacinone in Mokapu Island limpet (whole body Opihi) samples were below the LOD (table 

6; 17 ng/g). The instrumental internal standard (coumarin) 

responses ranged from 99 to 101 percent. Instrumental blanks, 

procedural blanks, and negative control (reference) samples 

for the whole Opihi limpet analyses did not have any detectable concentrations of diphacinone. 

The performance throughout the method was monitored 

by fortified procedural samples and fortified field Opihi limpet 

matrix. No diphacinone residues were detected in the procedural or reference Opihi blanks. Method recovery was 102 

percent for the fortified procedural samples and the fortified 

Opihi sample.

Ramik® Green Diphacinone Bait Pellets

The concentrations of diphacinone in the three composited lots of Ramik® Green baits used for the Mokapu Island 

rat eradication study were determined to contain from 44 to 

46 μg/g (parts per million) diphacinone as determined by this 

methodology (table 7). The LOD and LOQ were not established because negative control bait (without diphacinone) 

was unavailable. The nominal concentration for the baits as 

reported by the manufacturer was 50 μg/g (parts per million); 

thus, the concentrations of diphacinone in composited lots of 

baits ranged from 88 to 92 percent of the nominal concentration. The amount of diphacinone recovered by reflux extraction averaged 99.4 percent of the total extractable diphacinone 

determined by combined reflux and sonication extraction 

steps. The instrumental internal standard (coumarin) responses 

ranged from 97 to 99 percent.

Conclusions

The analytical part of this study demonstrated that there 

were no matrices that contained diphacinone at or above the 

Table 6. Diphacinone concentrations in whole Opihi limpets.

[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; ND, not detected; --, not applicable]

USGS

ID Field label Sample description Diphacinone(ng/g)

Recovery

(percent)

42034

42042

42043

42044-1

42044-2

42044-3

42034-fortified

Kalaupapa National Historical 

Park Ka Laea Point, Molokai

Mokapu Station 1

Mokapu Station 2

Mokapu Station 3—replicate 1

Mokapu Station 3—replicate 2

Mokapu Station 3—replicate 3

Fortified C18 SPE procedural 

sample

Fortified LP-SEC procedural 

sample

Procedural blank

Fortified procedural sample

Fortified field Opihi (reference)

Limpet, Opihi (Cellata 

exarata)

Limpet, Opihi (Cellata 

exarata)

Limpet, Opihi (Cellata 

exarata)

Limpet, Opihi (Cellata 

exarata)

Limpet, Opihi (Cellata 

exarata)

Limpet, Opihi (Cellata 

exarata)

Limpet, Opihi (Cellata 

exarata)

< 17 (ND)

< 17 (ND)

< 17 (ND)

< 17 (ND)

< 17 (ND)

< 17 (ND)

< 17 (ND)

--

--

--

--

--

--

--

--

99

100

--

102

102
References 15

Table 7. Diphacinone concentrations in Ramik® Green bait lot composites.

[USGS, U.S. Geological Survey; ID, identification; μg/g, micrograms per gram (parts per million, ppm); values 

reported on an as-received weight basis]

USGS

composite ID Field label Sample type Diphacinone(μg/g)

42051/42052/42053 Maui-1/-2/-3 Ramik® Green (diphacinone bait 44

pellets)

42054/42055/42056 Maui-4/-5/-6 Ramik® Green (diphacinone bait 45

pellets)

42057/42058/42059/42060 Maui-7/-8/-9/-10 Ramik® Green (diphacinone bait 46

pellets)

method limit of detection, which ranged from about 10 to 

18 parts per billion (nanograms per milliliter for sea-water 

samples, or nanograms per gram for tissue samples). The 

methods developed were satisfactory, with negligible background interferences being encountered and efficient recoveries of diphacinone, which ranged from about 86 to 102 percent 

in fortified matrix samples. 

Concentrations of diphacinone, the active ingredient, in 

Ramik® Green bait samples, averaged 45 micrograms per 

gram (parts per million), which was 90 percent of the nominal 

concentration. Refluxing for 8 hours with methanolic solutions 

of the tetrabutylammonium phosphate (TBAP) ion-pairing 

reagent was required for efficient recovery of diphacinone 

from the bait samples.

For quality control, confirmation of peak identity and 

purity was made by comparing the retention times and peak 

spectra to diphacinone standards. All samples with diphacinone concentrations less than the limit of detection did not 

have peaks that matched the retention times or spectra of 

diphacinone standards. Additionally, the area ratios of the 

primary and secondary quantitation wavelengths did not correspond to known diphacinone standards, with the exception 

of diphacinone fortified quality-control samples

The isolation, concentration, and high performance liquid 

chromatography-photodiode array (HPLC-PDA) method 

performed well throughout the analyses of all sample matrices 

(sea water, fish fillet, whole limpet, and Ramik® Green bait 

samples). No instances of interfering compounds co-eluting 

with diphacinone (or with coumarin) were noted. The co-elution of compounds that interfere with analyte response (quantitation) or analyte spectra (identification) typically results in 

reporting an analyte as not quantifiable at an increased limit of 

quantification.

Acknowledgements

The authors thank Peter Dunlevy (USDA–APHIS–WS) 

and Cathrine Swift (USFWS) for their contributions in this 

effort. They have made the primary selection of the study area, 

and continued to work on logistics, sampling, site assessment, 

and other aspects of this study.

References

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. 249–255.

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. 349–357.

Swenson, C., and Duvall, F.P., II, 2007, Draft environmental assessment—Eradication of Polynesian rats (Rattus 

exulans) from Mokapu and Alau Islands, Hawai’i. accessed 

April 18, 2008, at URL: www.fws.gov/pacificislands/

FinalEAMokapuRatEradication.pdf

U.S. Environmental Protection Agency, 1998, Reregistration 

Eligibility Decision (RED)—Rodenticide Cluster: EPA 

738–R–98–007.
16 Determination of Diphacinone Following Aerial Broadcast on Mokapu Island, Molokai, Hawai’i

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. 

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
References 17
Gale and others—Determination of Diphacinone Following Aerial Broadcast on Mokapu Island, Molokai, Hawai’i—Open-File Report 2008–1285