Proceedings of the International 

Cyanide Detection Testing 

Workshop

February 6-8, 2008 

Orlando, Florida 

Edited by

Andrew W. Bruckner 

Glynnis G. Roberts

National Oceanic and Atmospheric Administration 

National Marine Fisheries Service 

NOAA Technical Memorandum NMFS-OPR-40 

August 2008

U.S. Department of Commerce
This publication should be cited as: 

Bruckner, A.W. and G. Roberts (editors). 2008. Proceedings of the International Cyanide 

Detection Testing Workshop. NOAA Technical Memorandum NMFS-OPR-40, Silver Spring, 

MD 164 pp. 

Signifi cant support for the development of this document was provided by NOAA Fisheries, 

Offi ce of Habitat Conservation, and NOAA’s Coral Reef Conservation Program. The views 

expressed in this document are those of the authors and the participants of the workshop, 

and do not necessarily refl ect the offi cial views or policies of the U.S. Government, NOAA 

or DOS.

Additional copies of this publication may be requested from: 

John Foulks

National Oceanic and Atmospheric Administration

NOAA National Marine Fisheries Service

Offi ce of Habitat Conservation 

1315 East West Highway 

Silver Spring, MD 20910 

John.Foulks@noaa.gov

Front Cover Images: (Top) James Cervino – A fi sher uses cyanide to catch marine ornamental 

fi sh; (Bottom) Stephen Why – Pens used in Micronesia to hold live reef food fi sh.
Proceedings of the Cyanide 

Detection Testing Workshop 

Edited by Andrew W. Bruckner and Glynnis G. Roberts

Offi ce of Habitat Conservation

Ecosystem Assessment Division

NOAA National Marine Fisheries Service 

1315 East-West Highway 

Silver Spring, MD 20910-3282 

NOAA Technical Memorandum NMFS-OPR-40 

August 2008 

U.S. Department of Commerce 

Carlos Gutierrez, Secretary 

National Oceanic and Atmospheric Administration 

Vice Admiral Conrad C. Lautenbacher, Jr., USN (Ret.) 

Under Secretary for Oceans and Atmosphere 

National Marine Fisheries Service 

Jim Balsiger, Assistant Administrator for Fisheries 
Proceedings of the Cyanide Detection Testing 

Workshop 

Program Coordinator 

Andrew Bruckner, NOAA Fisheries

Organizing Committee

Andrew Bruckner, NOAA Fisheries, Headquarters 

Patty Debenham, SeaWeb 

Eric Punkay, SeaWeb/SeaStrategy Network

Kristine Johnson, Kingfi sher Foundation

Doug Thompson, Keystone Center

Workshop Group Leaders

Brian Logue, South Dakota State University

Robert Kobelski, Centers for Disease Control and Prevention

Patty Debenham, SeaWeb

Kristine Johnson, Kingfi sher Foundation

Peter Rubec, Florida Fish and Wildlife Conservation Commission

Sponsors

NOAA National Marine Fisheries Service

Offi ce of Habitat Conservation 

Ecosystem Assessment Division

Silver Spring, MD 

NOAA Coral Reef Conservation Program

Silver Spring, MD

Kingfi sher Foundation

San Francisco, CA

Workshop Facilitator

Doug Thompson, Keystone Center
ACKNOWLEDGEMENTS

The Proceedings of the International Cyanide Detection Testing Workshop summarizes the 

outcome of a meeting held in Orlando, Florida, funded by the NOAA Coral Reef Conservation 

Program and Kingfi sher, with additional travel support provided through a contract from 

NOAA Fisheries Offi ce of Habitat Conservation to the International Marinelife Alliance. The 

workshop was developed in response to a Resolution on enforcement adopted by the U.S. 

Coral Reef Task Force with the primary intent of mitigating the use of cyanide in the capture 

of reef fi sh for the marine aquarium trade (MAT) and the live reef food fi sh trade (LRFF). 

Initial consultations with the U.S. Coral Reef Task Force (USCRTF) Coral Trade Enforcement 

Working Group, including representatives from the U.S. Fish and Wildlife Service (Sheila 

Einsweiller), U.S. Department of State (Christine Dawson), NOAA (Andrew Bruckner; Beth 

Lumsden), U.S. Department of Justice (Karen Wardzinski) and U.S. Agency for International 

Development (Barbara Best) assisted in identifying workshop objectives and in developing a 

work plan.

The workshop would not have been possible without the dedicated efforts of the steering 

committee, Andy Bruckner, Patty Debenham, Eric Punkay and Kristine Johnson, who 

were responsible for developing the agenda, objectives, work plan and working group tasks, 

selecting participants, and pulling together background documents. We are grateful to SeaWeb, 

especially Eric Punkay, who helped with all logistical aspects of the workshop, including 

planning and travel arrangements and conference support. Dr. Peter Rubec also provided 

hours of dedicated time and assistance in the workshop, including organization and logistical 

arrangement for workshop participants from Vietnam, Indonesia, and the Philippines, and 

compilation of extensive background information on cyanide detection. We are also grateful 

to Doug Thompson for his assistance in the fi nal development of the agenda and working 

group tasks and expertise as a facilitator. Without Doug’s involvement, we are unlikely to have 

accomplished anywhere near as much, and are especially appreciative of his efforts at keeping 

us on track.

We are grateful to Brain Logue for his comprehensive white paper summarizing cyanide testing 

options and his patience in helping those of us that are not chemists in understanding some 

of the diffi culties in detecting cyanide exposure. We would like to recognize the dedicated 

efforts of Bob Kobeleski, Patty Debenham and Kristine Johnson as chairs of the working 

group sessions and for compiling the working group recommendations. All of the workshop 

participants need to be recognized for their excellent background presentations and white 

papers, and their cooperative efforts in addressing the terms of reference and developing 

recommendations within their respective working groups. Finally, special thanks go to Glynnis 

Roberts, a Knauss Sea Grant Fellow who was thrown into the middle of this on her fi rst week 

in the offi ce. Without Glynnis’ dedicated efforts, these proceedings would not be possible. 

I would also like to thank everyone for their cooperation and congenial interactions with other 

participants, given the complex and somewhat controversial nature of the problem. We had 

a very productive discussion of the issues surrounding the use of cyanide and options for 

iii
testing, as well as other holistic strategies to begin tackling this complex issue. I feel we made 

excellent progress in identifying possible next steps and look forward to working with all of 

you to implement the recommendations, and getting to the point where we can implement a 

network of CDT labs. I recognize the importance of the on-the-ground efforts geared toward 

communities dependent on coral reef resources, and commend all of the ongoing efforts in 

the Philippines, Indonesia and Vietnam. I am also happy to hear about the renewed interest 

of the government agencies in this issue, the progress that has been made, and the recognition 

of the importance of partnerships between exporting and importing countries to address this 

complex issue.

Andy Bruckner, May 1, 2008

iv
PREFACE

The International Cyanide Detection Testing Workshop was conceived to identify possible 

options for reducing the use of cyanide in the capture of coral reef fi shes for the marine 

aquarium trade and the live reef food fi sh trade. Because the emphasis was on identifying 

options for cyanide testing in exporting and importing countries, as well as management and 

enforcement opportunities, participants included forensic chemists with expertise in cyanide 

testing, as well as government and non-government representatives from the United States 

and three major exporting countries — Philippines, Indonesia, and Vietnam. Attempts were 

made to involve key experts who either had developed a possible test for cyanide or had 

implemented cyanide testing on marine fi shes, as well as those involved in conservation and 

education initiatives directed at major stakeholders, including fi shermen and other industry 

representatives and non-government conservation groups.

This workshop represents one component of a series of initiatives being implemented by the 

NOAA Coral Reef Conservation Program to address unsustainable and destructive trade in 

coral reef species. The mandates for this work include the Executive Order on Coral Reef 

Protection (13089) issued by President Clinton in 1998, the U.S. Coral Reef Task Force Coral 

Reef Action Strategy, and the Coral Reef Conservation Act of 2000. The Executive Order 

called for the creation of the U.S. Coral Reef Task Force (USCRTF), a multi-agency federal 

body chaired by NOAA and the Department of the Interior, with involvement by state and 

territorial governments. It also specifi cally identifi ed unsustainable coral trade as an item the 

United States needed to address. Between 1998 and 2000, USCRTF members developed a 

road map for coral reef conservation — the National Action Plan to Conserve Reefs. One 

component of the Plan outlines seven key actions the United States should take to ensure the 

trade in coral reef species is sustainable, one of which targets efforts to improve law enforcement 

both domestically and internationally. At the 14th Meeting of the USCRTF in Palau, the Task 

Force called on its members to increase efforts to build enforcement capacity. The Steering 

Committee was charged with developing an enforcement “toolbox” in cooperation with 

the International Coral Reef Initiative (ICRI), to help coral reef management communities 

build enforcement capacity. A decision was also adopted by the USCRTF to address the use 

of poisons in the capture of reef fi shes. The decision called for the creation of a working 

group on enforcement to 1) identify and recommend specifi c experts in law enforcement, 

fi eld forensics, and toxicology/biomarkers; and 2) utilize their expertise to identify existing 

or potential cyanide detection methods or tests which could be used to determine if fi sh had 

been exposed to cyanide. They also asked the group to explore the usefulness of convening a 

broader expert panel to resolve the issues associated with cyanide detection tests.

Through funding from the NOAA Coral Reef Conservation Program, a workshop of 

experts was convened in February 2008. This document summarizes the outcomes of that 

meeting. Included are summary recommendations, working group reports, abstracts and 

white papers from speakers, and background information on cyanide fi sheries. The Executive 

Summary highlights the major outcomes and conclusions from the workshop, including nine 

specifi c recommendations. These proceedings provide the framework for moving forward in 

implementing networks of cyanide detection laboratories. 

v

TABLE OF CONTENTS

Acknowledgements ................................................................................................................... iii

Preface .......................................................................................................................................... v

Executive Summary .................................................................................................................... 1

Introduction ................................................................................................................................. 9

Terms of Reference for Working Groups ............................................................................ 12

Report of Working Group 1 ................................................................................................... 16

Report of Working Group 2 ................................................................................................... 26

Report of Working Group 3 ................................................................................................... 33

White Papers .............................................................................................................................. 41

Country Reports ..................................................................................................................... 117

 Vietnam ...................................................................................................................... 119

 Philippines .................................................................................................................. 135

 Indonesia .................................................................................................................... 141

Workshop Abstracts ............................................................................................................... 145

Appendix I: Workshop Agenda ............................................................................................ 159

Appendix II: Workshop Participants .................................................................................... 161

Appendix III: U.S. Coral Reef Task Force Resolution 15-1 ............................................ 163

vii

EXECUTIVE SUMMARY

The International Cyanide Detection Testing Workshop (February 6-8, 2008, in Orlando, 

Florida) brought together participants from Indonesia, the Philippines, the United States, 

and Vietnam, with representatives from fi sheries and law enforcement agencies, forensic 

laboratories, CITES Parties, non-governmental organizations, industry, and academia. The 

primary objective of the workshop was to review the state of testing methods for cyanide 

and identify simple, cost-effective, rapid, and internationally accepted tests to detect cyanide 

or its metabolites in reef fi shes at different points along the supply chain, including collection 

sites, export facilities, and ports of import. The workshop was organized by the United States 

(NOAA Coral Reef Conservation Program) with logistical support provided by SeaWeb, 

Kingfi sher Foundation, and the International Marinelife Alliance (IMA).

The workshop opened with a series of presentations on the extent of trade in live reef food 

fi shes (LRFF) and marine aquarium fi shes (MAF), patterns of cyanide use in these fi sheries, 

and the impacts of cyanide on target and non-target species and coral reef ecosystems. A 

discussion followed on possible conservation, management, and enforcement strategies that 

have been implemented or are under consideration in the major exporting and importing 

countries to address the use of cyanide to capture reef fi shes. 

The participants noted that cyanide fi shing is a widespread practice that has been reported 

in at least 15 countries or island territories. The pervasive use of this poison is driven by the 

lucrative growing and largely unregulated international trade in live reef food fi sh and the 

marine aquarium industry. The United States is the number-one consumer of MAF, while 

most LRFF are destined for Hong Kong and other Chinese markets. Species targeted by 

cyanide fi shing include nearly all coral reef fi sh species, but its use appears predominant 

among the high-value MAF species such as surgeonfi sh, while LRFF trade frequently targets 

groupers and wrasses. Cyanide causes unacceptable levels of mortality of target species during 

collection and transport. Cyanide also causes mortality to non-target fi shes and invertebrates 

including corals, and is associated with reef degradation as divers break apart coral to extract 

stunned fi shes.

Potential methods for cyanide detection were reviewed during the workshop, focusing on their 

applicability to marine fi sh testing, including colorimetric methods, enzyme-based biosensors, 

cyanide-ion selective electrodes (ISE), and biomarker approaches. The International Marinelife 

Alliance (IMA) created six CDT laboratories and tested over 48,000 specimens under contract 

to the Philippine Bureau of Fisheries and Aquatic Resources (BFAR) from 1993 to 2001. 

BFAR has continued to conduct cyanide testing using the ISE method on a more limited scale 

since 2001. Cyanide testing by the IMA for BFAR, in combination with other initiatives, led to 

a sharp reduction in fi sh testing positive for cyanide over the period 1996 to 1999. 

However, at present only one Cyanide Detection Testing (CDT) laboratory remains operational 

full-time due to funding shortfalls and other issues, and cyanide use has subsequently increased. 

Testing done by the Puerto Princesa, Palawan laboratory found that 49% of the fi sh specimens 

1
tested in 2004 had cyanide present (Rubec, personal comm.). Several alternative methods to 

detect cyanide in marine fi shes have been published, but none of these have been applied to 

date as an enforcement tool. Conservation groups have also attempted to reestablish networks 

of CDT laboratories, with limited success. 

The participants concluded that the most practical way to address cyanide use involves testing 

at points of export, and representatives from the three major exporting countries (Philippines, 

Indonesia, and Vietnam) supported implementation of networks of CDT laboratories in their 

countries. The working group also identifi ed major limitations that need to be overcome for 

testing to be successful. First and foremost, recent studies have questioned the sensitivity of 

various tests, the ease of application by law enforcement in a fi eld situation, and the potential 

diffi culties in detecting cyanide at points of import due to the potential for rapid conversion 

of cyanide to thiocyanate and other metabolites. The participants suggested that the ISE 

method was the preferred test for use at points of export, mainly because it had been regularly 

used for many years. However, they also noted that suffi cient concerns have been raised about 

the application of the ISE method to whole fi sh. For the ISE method to be internationally 

accepted as a standard test for marine fi shes — especially in an enforcement context — the 

methodology used to test fi sh needs independent and careful verifi cation. 

After the conclusion of the plenary speeches, three concurrent working groups were convened 

to tackle questions surrounding cyanide testing and identify pragmatic approaches and 

collaborative initiatives in exporting and importing countries to mitigate the use of cyanide. 

The participants focused on the following topics: 

(1) existing or potential analytical cyanide detection methods that could be applied to marine 

fi shes at collection sites, point of export, and ports of import; 

(2) requirements of an internationally recognized test at points of import that would carry 

weight in a court of law; and

(3) steps, agreements, and/or partnerships that could be established between exporting 

governments, the U.S. government, NGOs, certifi cation agencies, community groups, and 

academic scientists to implement cyanide testing in exporting countries. 

The Field Forensics and Toxicology Working Group concluded serious gaps exist in 

the understanding of the metabolism of cyanide and its major metabolites in marine fi sh. 

A detailed analysis requires that the analyte is stable and at elevated levels in the fi sh long 

enough for it to be delivered to the laboratory and an examination conducted. Without a 

better understanding of these processes in representative fi sh species, the implementation of 

an exposure assessment method that would meet legal requirements would be diffi cult. The 

Working Group identifi ed research as the highest immediate priority to determine the half-life 

of cyanide and major metabolites (especially thiocyanate) in marine fi sh and variations between 

species and life history stages. Other needs included an examination of background levels of 

cyanide (and major metabolites) in marine fi sh collected from different locations and whether 

they are above the limit of quantifi cation, to ensure that the applied test can detect elevated 

levels of cyanide that would indicate exposure by fi sh collectors. To perform this research, 

validated methods for the determination of cyanide, thiocyanate and other metabolites (e.g., 

2
ATCA) in homogenized fi sh tissue must be used. These methods must demonstrate adequate 

sensitivity, specifi city, accuracy, and precision. 

The Export Working Group identifi ed the need for a cohesive program in exporting 

countries to raise awareness among fi shermen and other stakeholders, and improve capacity 

for training, testing, and enforcement. In particular, partnering with local communities, 

government agencies, and NGOs was recommended as the most practical way to address 

an issue of this magnitude and to increase technical capacity and secure fi nancial support. 

Proactive plans must be made “on the ground” to engage the public, retrain and support 

cyanide-free fi sh collectors, and provide solid options for a cyanide-free network of exporters. 

An export testing scheme would require redefi nition of export procedures and licensing to 

ensure testing compliance. Representatives from Indonesia, the Philippines, and Vietnam felt 

it might be useful for the three countries to meet to develop some common protocols for 

testing, enforcement, and education.

The Import Working Group suggested the need for harmonization of standards between 

import and export countries to eliminate policy gaps and tighten enforcement. Exporting 

parties should improve coordination and communication with importing countries, including 

sharing data on collection sites and fi shermen, trade and fi sheries, and exporters. Sharing 

such information could help identify legal and illegal shipments for importing countries, and 

potential collectors, middlemen, or exporters of concern. U.S. enforcement staff would benefi t 

from knowing which companies repeatedly test cyanide-free, as well as those companies that 

tend to use cyanide. The group concluded there is an overall lack of comprehensive knowledge 

of each country’s requirements, resulting in confusion, corruption, and potential loopholes. 

Information on quarantine requirements, documentation needs, and the creation of a unifi ed 

labeling and packaging system were all listed as possible ways to eliminate discrepancies. By 

establishing a tighter import policy and clear standards for compliance, the United States can 

provide stronger incentives for change in the industry.

SUMMARY RECOMMENDATIONS

During the fi nal session, workshop participants discussed and formulated nine key 

recommendations necessary to mitigate the use of cyanide in the capture of coral reef fi shes. 

The recommendations are presented here in priority order. There was general consensus that 

the primary issues to be resolved are gaps in our understanding of the cytokinetics of cyanide 

in marine fi shes. The next critical steps involve validation of the existing ISE method used to 

detect cyanide at points of export, followed by implementation of this test at points of export. 

Further research on possible rapid fi eld tests and tests to detect metabolites in fi sh tissue upon 

import should be undertaken. Upon confi rmation of an internationally recognized and valid 

testing method for cyanide, networks of CDT labs should be established at major points of 

export and profi ciency programs should be established for chemists. 

Recommendation 1: Determine the pharmaco-kinetics of cyanide 

Research is required to determine detectable levels of cyanide and cyanide metabolites in coral 

reef fi shes and how these levels change over time following exposure. Without this knowledge, 

3
it is diffi cult to identify the most appropriate cyanide testing methods that can be applied at 

different stages in the chain of custody, including collection sites, export holding facilities, and 

points of import, possibly 2 to 3 weeks after collection. 

The concentration of cyanide present in the living fi sh will vary depending on the concentration 

of the cyanide solution used during exposure, the length of time of the exposure, time for 

holding and duration of transport, and post-collection treatment. Rates of metabolism may 

also vary depending on species or size. Research should encompass these possible variations 

such that it is possible to quantify measurable levels of cyanide in representative species and 

life history stages of marine fi shes, and how these levels change over time, using at least two 

methods and multiple known concentrations. 

To represent the time from initial collection and transport from the reef to holding facilities 

in exporting countries and then on to import destinations, fi sh samples should be exposed to 

varying concentrations of cyanide, with representative individuals sampled immediately after 

exposure and subsequent defi ned intervals. One aspect should also include a determination 

of detectable background levels, using fi sh from the wild caught with nets as well as captivebred species. In addition, the effect of freezing samples must be determined for both fi eld 

collection and delayed analysis samples to prevent misinterpretation of data obtained from 

frozen samples.

Recommendation 2: Validation of the Ion Selective Electrode (ISE) Cyanide Detection 

Test 

Numerous methods are available to detect cyanide or cyanide metabolites, but most have been 

used only for water samples and blood; only limited testing has been done on whole fi sh and 

fi sh organs. Currently, the ISE method is the only cyanide test that has been applied on a large 

scale for MAF and LRFF at points of export, and testing has not been done substantially 

by enforcement agencies at points of import. The ISE method was successfully used in the 

Philippines from 1993 to 2001, with at least 48,000 fi sh tested; the test produced cyanidepositive results in fi sh up to 5 to 14 days after exposure (Rubec et al., 2003). 

Representatives from Indonesia and the Philippines indicated that the ISE method is the 

preferred testing method, and both countries have secured necessary equipment, supplies, 

and trained staff to establish CDT laboratories. However, several studies have questioned the 

sensitivity of the test and the potential for false readings, and until these issues are resolved 

it is unlikely that importing countries will accept the results of testing using the ISE method. 

Although use of the ISE method for determination of cyanide concentrations has been 

validated through round-robin studies by the American Society of Testing and Materials 

(ASTM) and the American Public Health Association (APHA), these studies have not been 

performed on samples of digested fi sh, using the specifi c digestion method applied in the 

Philippines. IMA and affi liated laboratories have also developed supporting documentation to 

counter concerns expressed in the literature, but this has not been published in peer-reviewed 

journals or verifi ed by an independent group. 

4
Validation of the ISE method applied in the Philippines should be conducted by an independent 

laboratory not currently involved in marine fi sh testing to resolve questions about QA/QC, 

precision, and bias. This should include an examination of the sensitivity of the ISE method 

for fi sh samples, with emphasis on the distillation process and the recovery of cyanide from 

tissue samples. Results should be compared using at least one other recognized method. With 

some discretion exercised to limit the amount of testing, the ISE method can be validated 

in a relatively short time. The same cannot be said for any other method. A large amount of 

research will be necessary to develop methods to detect either thiocyanate or ATCA present 

in imported MAF, and to validate those methods. 

Recommendation 3: Establish networks of CDT laboratories at points of export

A testing strategy based in export countries should focus on airport “choke points” in order 

to sample shipments before export. This approach would require fewer testing stations and 

would create better monitored and controlled testing conditions and procedures. This test 

must be rapid, credible, standard for all countries involved in the marine aquarium fi sh trade, 

and legally defensible, and it must utilize independently collected (e.g., blind) samples from 

shipments. It must also be able to accurately detect cyanide 5 to 14 days after the fi sh are 

exposed, as the time for transport to landing area and then landing area to export point 

frequently falls within this range. These networks should defi ne and oversee their internal 

training and quality assurance programs and work with similar networks in other jurisdictions 

to establish and support an independent profi ciency testing program. 

Existing licensing schemes for exporting aquarium fi sh are often negligible, lax, and insuffi cient 

for impeding the use of poisons. Currently there are no offi cial cyanide testing requirements 

in Indonesia, Vietnam, or the Philippines. It would be desirable to require that an export 

license and/or permit be linked to standards of compliance, incorporating poison-free capture 

and assent to regular cyanide detection testing. If possible, the export license should involve 

a suitable fee that would help underwrite the costs of testing and allow it to become selffi nancing (see Recommendation 6).

Recommendation 4: Obtain funding for establishment and maintenance of CDT 

networks in exporting countries

Funding sources should be explored to support the creation of CDT laboratory networks in 

the exporting countries (Philippines, Indonesia, Vietnam, and Malaysia) where cyanide fi shing 

is most prevalent. Potential funding agencies include the World Bank, Asian Development 

Bank, or international NGOs.

Recommendation 5: Establish Training, Quality Assurance and Profi ciency Programs 

for CDT labs and chemists

Training, quality assurance, and profi ciency programs need to be implemented at cyanide 

testing laboratories. Many of the CDT procedures require extensive training and should be 

maintained to ensure performance consistency and accurate test results. A training program 

must be developed employing both didactic and hands-on training that develops a demonstrated 

acceptable level of performance, as defi ned by the performance characteristics of the specifi c 

test and the needs of the analysis. A quality assurance program should be based on ISO 

5
17025 or similar structures and should at a minimum employ documented Standard Operating 

Procedures (SOPs), bench and blind QC samples, and statistically determined performance 

limits. A profi ciency testing program should be conducted by an independent body and employ 

the use standard samples, unknown to the analyst, analyzed across the network and evaluated 

statistically to identify laboratory results that do not meet network performance standards. 

Frequency of testing should occur no less than “N” times per year. 

Recommendation 6: Explore options for a simple fi eld test at points of collection

Because of the considerable time and cost involved in quantifi cation of concentrations of 

cyanide within fi sh, research is needed to identify and develop a quick, simple fi eld test that 

could be used by enforcement offi cers at points of collection and at holding facilities in local 

communities. This test would only need to identify the presence of cyanide in fi sh immediately 

after capture, and not actual concentration. 

The most feasible options currently include a low-cost colorimetric kit or ion selective 

electrodes (ISE) linked to portable ISE meters. Limited research of these options has been 

undertaken, but several limitations were noted—e.g., an inability to collect suffi cient quantities 

of blood due to the small size of MAF and the need to digest fi sh tissue to liberate the cyanide, 

potential for interfering substances (e.g., sulfur), and other issues. 

A protocol published by the American Society of Testing and Materials (ASTM D 5049), 

modifi ed for use by the U.S. Food and Drug Administration (FDA) for the analyses of various 

foodstuffs including tuna, can successfully detect cyanide liberated from tissue digested in 

sulfuric acid. This simple test detects cyanide using Cyantesmo paper, which changes color 

from light green to blue in the presence of cyanide. This method may provide a rapid screening 

tool for marine fi sh samples at points of collection, allowing rapid analysis of multiple samples 

at low cost. 

While a rapid screening test may be a deterrent, samples that test positive could be sent on 

to a larger testing facility where a more detailed test would be performed to quantify actual 

concentrations and to prosecute violations. 

Recommendation 7: Testing and Accreditation Plan for Cyanide Free Fish in Exporting 

Countries

Exporting countries need a sound, government-instituted export licensing and certifi cation 

scheme tied to cyanide testing at points of export. Similar to an accreditation approach, 

issuance of the license would be tied to confi rmed cyanide-free status from a testing facility. 

The group thought it would be feasible to implement some form of certifi cation program if 

a suitable fee for the license were required. This would create a fund for the testing procedure 

and help make the system fi nancially self-sustaining. In conjunction with export permitting, 

this method provides an incentive to reform any collectors currently using cyanide. Further, a 

database should be designed to track cyanide testing results; over time this information would 

illuminate patterns of use, mitigation success, and repeat offenders. License renewal could 

be linked to the testing program, with repeat offenders being unable to renew their license. 

6
Importing countries should also require testing and verifi cation by the exporting country 

(through certifi cates or other means) that the imported fi sh are cyanide-free. 

Recommendation 8: Implement Complementary Legislation against Cyanide Fishing 

in Importing Countries 

The offi cials of exporting countries at the workshop strongly advocate U.S. legislation 

regulating the ornamentals trade. This legislation would be fundamental for building political 

will and designing regulation and enforcement in exporting countries. The legislation or Code 

of Federal Regulations (CFR) could make U.S. imports of fi sh containing cyanide illegal, as well 

as clearly state standards for permissible imports (e.g., exporting partners must have testing 

system in place, species restrictions, habitat impact assessments and protection, etc.). 

The Lacey Act makes it illegal to import, export, transport, sell, receive, acquire, or purchase 

fi sh, wildlife, or plants taken, possessed, transported, or sold in violation of a federal law, 

treaty, regulation, or Indian tribal law. It also is illegal for a person to import, export, transport, 

sell, receive, acquire, or purchase in interstate or foreign commerce: fi sh or wildlife taken, 

possessed, transported, or sold in violation of a state law, state regulation, or foreign law (16 

U.S.C. §§ 3371-3378). Since most exporting countries have laws banning the use of cyanide 

and other poisons for fi shing, a large portion of the aquarium fi sh imported to the United 

States (an estimated 90%) are illegal under the Lacey Act. However, enforcement of the Lacey 

Act is diffi cult and time-consuming. For example, 

• A successful case must show importer knowledge and intent to import illegal fi sh.

• Origin countries must have good faith enforcement efforts in place.

• A U.S.-based investigation under the Lacey Act must be supported by the nation whose 

law or regulation was violated.

• In the case of cyanide, the fi sh samples would not be analyzed in the United States 

until three weeks after time of import because of backlogs and resource constraints. 

Shipments would have been processed, shipped to retailers, and sold by that point.

• The United States is unlikely to rely on accuracy of a cyanide test performed in 

exporting country.

• The standards for a test to be considered legally enforceable in the United States are 

high: the test would have to be peer reviewed, reliable, and in use for a period of 

time.

• Each case takes hundreds of hours per shipment to prove. An effective enforcement 

effort would immediately overwhelm the current system resources.

Federal legislation may be necessary to support the development and implementation of testing 

methods for thiocyanate and/or ATCA metabolites at the point of import, and to authorize 

funding in support of cyanide testing. A draft bill proposed by Representative Ed Case (Coral 

Reef Conservation and Protection Act of 2004 HR-4928) would have required some type 

of certifi cation demonstrating that MAF imported to the United States were collected by 

sustainable means without the use of poisons such as cyanide. New U.S. legislation could 

require certifi cation of fi shes tied to cyanide testing by countries exporting MAF to the United 

States, but it may also require more comprehensive legislation in exporting countries and a 

7
formal mechanism for evaluation and demonstration by the responsible party that the fi sh are 

in fact cyanide-free. 

Recommendation 9: Develop a cyanide testing program in the United States 

Participants felt that developing a cyanide testing program for points of import was a 

necessary step for deterring cyanide use. Unfortunately, such a system also presents the greatest 

challenge. 

In one year there are over 11,000 shipments of non-CITES listed reef fi shes into the United 

States. Currently, U.S. Fish and Wildlife Service (USFWS) import inspections focus on 

endangered and threatened species listed under ESA and CITES. Inspection of aquarium fi sh 

is a relative low priority, as none of the species of fi sh are considered endangered. In addition 

to a limited capacity to inspect each shipment, verifi cation of cyanide is problematic because 

fi shes may be imported several weeks after being caught with cyanide, which is known to 

rapidly break down into sodium thiocyanate and other metabolites. 

Given testing backlogs for USFWS, samples would likely wait an additional three weeks after 

collection on import before testing. At this time, testing for cyanide metabolites is possible 

but extremely costly and time-consuming. In addition, there are limitations on the amount of 

time a shipment of fi shes can be detained, as these the fi sh will die if not transferred to aquaria 

fairly quickly after arrival in the United States. Therefore, the challenge for the United States is 

multi-fold, and can be confl icting:

• Creation of a comprehensive cyanide detection testing program in the United States 

must address new, supporting legislation as well as increased funding and staffi ng 

needs.

• The U.S. government agencies involved with import law enforcement would need a 

workable, import point CDT test to confi rm that fi shes declared as being cyanide-free 

were in fact not contaminated with cyanide. 

• Without this U.S.-based test, it would not be possible to enforce the Lacey Act or any 

new legislation created to address the use of cyanide. 

• It would be ideal to supplement a U.S. import CDT scheme with cyanide-free 

certifi cation from exporting countries. Although certifi cation alone would not be 

enough for U.S. standards, it could potentially help with enforcing violations. 

8
INTRODUCTION

Cyanide fi shing is a destructive fi shing technique widely used to capture live coral reef fi shes, 

including species destined for the marine aquarium and live reef food fi sh trades. The use of 

cyanide on coral reefs was fi rst documented in the early 1960s in the Philippines to capture 

aquarium fi shes, principally for export to the United States, the United Kingdom, Germany, 

and France. Cyanide fi sheries expanded to the live reef food fi sheries in the 1970s, and over 

the next two decades it spread throughout Southeast Asia and into the Pacifi c islands. Cyanide 

fi shing has been confi rmed in at least 15 countries, including Indonesia, Malaysia, Maldive 

Islands, Papua New Guinea, the Philippines, Sri Lanka, Thailand, and Vietnam (Jones et al., 

1998). 

Most commonly, sodium cyanide is dissolved in seawater in plastic squirt bottles. Divers using 

hookah squirt the milky solution at the target fi sh, which then often retreat into crevices 

in the reef or within coral thickets. These corals may be subsequently broken apart by the 

diver to capture the fi sh. Cyanide tablets may also be secured to sticks and held close to a 

fi sh, or cyanide is mixed with bait and thrown overboard or placed into fi sh traps. There are 

also reports that fi shermen occasionally pump the cyanide into the water from surface boats, 

mainly to target grouper spawning aggregations. The stunned fi sh are then captured with 

hand-nets or attached to lines and hauled to surface support boats, where they may directly 

enter the trade or be held in fl oating cages until export. 

Cyanide is used in two very different live reef fi sheries. The Live Reef Food Fish Trade (LRFF) 

regularly targets large groupers, coral trout, barramundi cod, and humphead wrasse. Although 

it focuses mainly on a small number of species, the actual trade is large in terms of the biomass 

of fi sh collected. At its peak in 1997, the volume of fi sh in trade was estimated at about 

50,000 metric tons. More recently, trade is about 30,000 metric tons per year, with about 60% 

imported into Hong Kong and the remainder destined for mainland China, Taiwan, Japan, 

and other Asian markets. Until the 1970s, LRFF fi sheries were mainly confi ned to areas in the 

South China Sea in close proximity to ports in Hong Kong and mainland China. The trade 

spread from the Philippines to Indonesia in the 1980s, and continued to expand to countries in 

the Pacifi c Ocean and Indian Ocean during the 1990s. Cyanide use has followed the expansion 

of the LRFF trade. One reason for the rapid expansion of this trade is that live fi sh can fetch 

substantially higher prices than dead fi sh of the same species. The total retail value of the 

LRFF was around $350 million per year between 1997 and 2001. By 2002, it increased to about 

$486 million for Hong Kong and $810 million for the entire trade. Individual fi sh can sell for 

up to $180 per kilogram, depending on species, taste, texture, availability, and time of year. 

Unlike the LRFF, the Marine Aquarium Trade (MAT) consists of a high diversity of fi shes, 

most of which are taken from the wild. Over 1,400 species of reef fi sh are traded worldwide 

for home aquaria at an annual volume of about 30 million fi sh, with approximately 16 million 

imported each year into the United States. Between 70 and 80% of these fi sh are from Indonesia 

and the Philippines, where cyanide use is most prevalent. In the MAT, species targeted by 

cyanide fi shing include nearly all coral fi sh species, but its use may be most prevalent among 

9
the high-value fi sh species such as emperor angel-fi sh (Pomacanthus imperator), blue surgeon-fi sh 

(Paracanthurus sp.), and blue ring angelfi sh (Pomacanthus annularis) (Fahrudin, 2003). The marine 

aquarium industry worldwide is worth an estimated $200 to $330 million annually (USCRTF, 

2000. Wabnitz et al., 2003. FAO, 1996-2005). When examined by weight, aquarium fi sh are 

valued at $500 per kilogram or more, which is considerably higher than a similar weight of 

food fi sh (Cato and Brown, 2003. Wabnitz et al., 2003).

Environmental concerns about cyanide fi shing

Although illegal in most countries, the use of cyanide to capture live reef fi sh remains pervasive, 

propelled by the lucrative growing and largely unregulated international trade in live reef food 

fi sh and the marine aquarium industry. The United States is the number-one importer of coral 

reef fi sh for the aquarium trade, and the demand for these species may be one factor driving the 

continued use of cyanide. Cyanide is toxic to fi sh because it interferes with oxygen metabolism 

by blocking the key enzyme system, cytochrome oxidase (Metzler, 2001), and blocks enzymatic 

pathways in the liver (Solomonson, 1981). Once inside the fi sh tissue, cyanide reacts with 

thiosulfate to produce the comparatively nontoxic thiocyanate which is excreted in the urine. 

Rapid detoxifi cation enables animals such as fi sh to ingest high, sub-lethal doses of cyanide 

(Eisler, 1991), although some of the effects are irreversible and may lead to the death of the 

fi sh (Way et al., 1988). 

Several studies have also demonstrated negative impacts of cyanide on non-target coral reef 

species including corals (Cervino et al., 2003. Jones et al., 1998). Exposure of corals to cyanide 

causes rapid signs of stress and bleaching, and at high concentrations, progressive tissue 

sloughing that can lead to colony mortality. Fishermen spray cyanide into crevices and coral 

thickets where fi sh often hide, and then break apart the corals to access the stunned fi shes, 

leading to substantial damage to the habitat. Large percentages of the target fi sh captured 

with cyanide die during collection or in transit due to their weakened state, which requires 

fi shermen to capture signifi cantly higher numbers of fi shes than would otherwise be needed. 

In fact, some studies indicate that as many as 75% of fi sh collected with cyanide die within 

hours of collection, and another 30% die prior to export. In addition, more than half may 

die shortly after arrival in the United States from a combination of the poisons used in the 

capture and stress associated with handling and transport. Cyanide fi shing is also risky for the 

divers, who often go to considerable depths for extended periods without following proper 

dive procedures. 

Conservation approaches to address cyanide fi shing

Although cyanide fi shing is illegal in most countries, poor law enforcement capabilities and 

high levels of corruption have allowed the use of cyanide to continue. In 1989, the Haribon 

Foundation and Ocean Voice initiated a program to train fi shermen in the use of nets as an 

alternative to cyanide. A second, more aggressive program was implemented in the Philippines 

in the early 1990s by the International Marinelife Alliance (IMA), in partnership with the 

Philippine government’s Bureau of Fisheries and Aquatic Resources. Through a combination 

of the right policies and laws, improved enforcement, enhanced public awareness, training of 

cyanide fi shers in cyanide-free fi sh capture techniques, development of livelihood alternatives, 

community-based resource management programs, and cyanide testing of live fi sh exports 

10
through the implementation of a network of cyanide detection laboratories (CDT labs), this 

program successfully reduced cyanide fi shing within the Philippines, at least temporarily. 

Over a period of roughly eight years, IMA tested 48,000 aquarium fi sh and food fi sh for the 

presence of cyanide. Cyanide was detected overall in about 25% of all aquarium fi sh and 44% 

of the food fi sh. The testing appeared to serve as a deterrent, at least in the initial years, as the 

proportion of aquarium fi sh testing positive declined from about 43% in 1996 to 8% in 1999. 

Unfortunately, the numbers of fi sh testing positive for cyanide has increased in recent years, 

and most CDT labs were closed in the mid 1990s. 

In the United States, the issue of unsustainable trade in coral reef species was fi rst highlighted 

in 1998 with Executive Order 13089 on Coral Reef Protection, which called for the creation 

of the U.S. Coral Reef Task Force (USCRTF). Over the next two years, USCRTF members 

developed a road map for coral reef conservation, the National Action Plan to Conserve 

Reefs, which outlines the key threats affecting reefs and identifi es specifi c actions to mitigate 

those impacts. The Plan includes a section on international coral reefs and the role of the 

United States as a major consumer of coral reef species. This section identifi es seven key 

strategies to advance a sustainable marine ornamental fi shery, one of which specifi cally calls 

on the United States to improve enforcement capacity domestically and internationally. As one 

step to implement the recommendations in the Plan, the NOAA Coral Reef Conservation 

Program provided funding in 2003 and 2004 to reestablish a lab in the Philippines and expand 

testing into Vietnam, Indonesia, and Malaysia. These projects were halted in 2006, due to recent 

publications by researchers from the University of Hong Kong (Mak et al., 2006) and reports 

from the Marine Aquarium Council that suggested the Ion Selective Electrode (ISE) method 

used in the Philippines was not sensitive enough to determine cyanide traces in exposed fi sh.

The USCRTF has also adopted three resolutions on trade since 2004. The most recent, in 

May 2006, requested that the U.S. government identify existing or potential cyanide detection 

tests that could determine whether fi sh had been exposed to cyanide and develop a cyanide 

fi shing mitigation strategy. In response to the USCRTF resolution, NOAA convened the 

International Cyanide Detection Testing Workshop (February 6-8, 2008, in Orlando, Florida) 

to advance cyanide detection methods for the live reef fi sh trade. The primary objective of 

this workshop was to review the state of testing methods and identify simple, cost-effective, 

rapid, and internationally accepted tests to detect cyanide or its metabolites at different points 

along the supply chain, from point of collection and export to ports of import in the United 

States. Also identifi ed were key research needs and major steps that need to be implemented in 

importing countries and exporting countries to curtail the use of cyanide. The United States is 

committed to continuing its work with partners to address the recommendations identifi ed in 

the workshop, including science questions pertaining to cyanide kinetics, additional testing and 

validation of cyanide detection methods, and, ultimately, implementation of cyanide detection 

laboratories. 

11
TERMS OF REFERENCE FOR WORKING GROUPS

Working Group 1

The Field Forensics and Toxicology Working Group

Goal 1: Evaluate existing or potential analytical cyanide detection methods or tests and their 

applicability to marine fi sh.

• Develop a table of all cyanide detection methods. See draft format. 

Include tests for:

o cyanide by-products

o biomarkers

o adducts

o morphological changes in tissues

• Provide assessment of existing knowledge of residence time of cyanide in marine 

fi sh

• Summarize methodology, equipment needs, costs, time required for testing, necessary 

scientifi c expertise, reliability and sensitivity. See draft spread format

Goal 2: Provide recommendations on the most practical, reliable, and user-friendly tests that 

could be used in marine fi sh. 

• Provide recommendations for each test with respect to ability to be internationally 

accepted in a court of law

• Provide recommendations for each test with respect to ability to use test at collection 

sites and holding facilities (within hours of collection and with minimal technology)

• Provide recommendations for each test with respect to ability to use test at export sites 

(within days of collection and with minimal technology)

• Provide recommendations for each test with respect to ability to use test at collection 

sites and holding facilities (up to1-2 weeks or more after initial exposure to cyanide)

• Provide recommendations for use of tests that determine cyanide presence versus a 

quantitative determination of cyanide concentration

Goal 3: Identify research needs to refi ne methodologies and/or develop other testing 

approaches.

• List of recommended research to conduct

12
Working Group 2

The Export Working Group

Infrastructure

Goal 1: Outline what to include in a robust scheme for monitoring exports to determine 

whether a fi sh was caught using cyanide. 

• Provide an outline for ideal testing infrastructure including:

o locations of offi ces and labs

o staffi ng 

o budget

o equipment

o training

• List existing cyanide detection facilities

• List locations that do not, but should have cyanide detection facilities

• List names of government bodies involved in each countries’ cyanide detection

Implementation

Goal 2: Identify steps, agreements, and/or partnerships that could be established between 

exporting governments, the U.S. government, NGOs, certifi cation agencies, community 

groups, and academic scientists to implement cyanide testing. 

• List existing partnerships related to cyanide detection

• List the fi ve most important entities (NGOs, governments, scientists) in export 

countries that must be involved in establishment of revised cyanide detection system

• Briefl y describe fi ve most important areas to improve in existing detection system

• List alternate methods and programs that increase non-destructive catch practices

Enforcement

Goal 3: Provide recommendations with respect to the possibility and/or benefi ts of a 

mandatory, government-issued, export certifi cation that states the fi sh were caught with legal 

practices. 

• Provide a brief description of requirements to enforce and or prosecute the use of 

illegal fi shing practices in export countries

• Provide recommendations regarding legal effi cacy of a test that identifi es the presence of 

cyanide versus a test that provides a quantitative estimate of cyanide concentration

13
Working Group 3

The Import Working Group

Infrastructure

Goal 1: Outline what to include in a robust scheme for monitoring imports to determine 

whether a fi sh was caught using cyanide. 

• Provide an outline for ideal testing infrastructure including:

o locations of offi ces and labs

o staffi ng 

o budget

o equipment

o training

• List locations that do not, but should have cyanide detection facilities

• List names of government bodies involved in each countries’ cyanide detection

Implementation

Goal 2: Identify steps, agreements, and/or partnerships that could be established between 

exporting governments, the U.S. government, NGOs, certifi cation agencies, community 

groups, and academic scientists to implement cyanide testing. 

• List existing partnerships related to cyanide detection

• List the fi ve most important entities (NGOs, governments, scientists) in import 

countries that must be involved in establishment of revised cyanide detection system

• Briefl y describe fi ve most important areas to improve in existing detection system

• List of alternate methods and programs that increase non-destructive catch practices

Enforcement

Goal 3: Provide a recommendation with respect to the possibility and/or benefi ts of a 

mandatory, government-issued, export and/or import certifi cation that states the fi sh were 

caught with legal practices. 

• Provide a brief description of requirements to enforce and or prosecute the use of 

illegal fi shing practices in import countries

• Provide recommendations regarding legal effi cacy of a test that identifi es the presence of 

cyanide versus a test that provides a quantitative estimate of cyanide concentration

• Provide recommendations regarding legal effi cacy of a tiered approach to testing 

• Discuss the current procedure for examination of imports of wildlife, and how this 

varies depending on international regulations (e.g., CITES)

• Respond to the following questions:

o If an exporting country requires documentation verifying the absence of 

cyanide, can import countries such as the U.S. prohibit imports of shipments 

without appropriate certifi cates?

14
o Can the U.S. require verifi cation that no illegal fi shing practices were used to 

capture the fi sh included in a shipment under existing legislation? If not, what 

is needed to prevent imports of cyanide caught fi sh? Can the burden of proof 

be on the exporter/importer?

o What would be needed to prosecute violations under the Lacey Act? 

o Are there provisions under Title IV section 403 of the Magnuson Act that can 

help eliminate imports of cyanide caught fi sh? What requirements do we have 

under Magnuson to address illegal, unreported, or unregulated fi shing?

o What would we need to implement prohibitions on imports of aquarium fi sh 

for exporting countries that cannot verify that they are engaging in sustainable 

harvesting practices? How would we make these import restrictions consistent 

with both domestic legal requirements and WTO obligations?

15
Report from Working Group 1

Participants in the Field Forensics and Toxicology Working Group focused their discussions 

on fi ve key topics:

1) Evaluating different cyanide detection methods that are currently available to test for 

cyanide and cyanide metabolites; 

2) The feasibility of applying these methods to detect cyanide exposure in ornamental 

reef fi sh under different export and import scenarios;

3) Type of training and quality control that would be necessary to ensure consistent, 

reproducible results;

4) Modifi cations or additional research that would be necessary to apply existing tests to 

ornamental fi sh at points of collection, export and import; and

5) Research necessary to characterize natural levels of cyanide and patterns of uptake 

and metabolism of cyanide in marine fi shes. 

Goal 1: Evaluate existing or potential analytical cyanide detection methods or tests and 

their applicability to marine fi sh. 

The Working Group identifi ed and reviewed numerous methods that have been used to detect 

cyanide and cyanide metabolites. The most common of these include: electrochemical methods, 

ion-selective electrodes, spectrophotometric and fl uorescence methods, chromatographic 

techniques and biomarkers of cyanide exposure (Table 1). While these are widely used for 

determining cyanide concentrations in water and in certain biological fl uids such as blood, 

very few of these have been applied to marine fi shes, and even fewer have been successfully 

used on fi sh tissue. 

In order to effectively test for cyanide in marine fi sh, particular aspects of testing should be 

addressed. The appropriate method will depend on the point in the chain of custody (e.g., 

point of collection, export or import), as this will dictate the particular analyte being tested 

(cyanide vs. thiocyanate and ACTA) and the type of sample being analyzed (sample matrix 

- blood, tissue or organs). The sample matrix will dictate the amount of sample preparation 

and cleanup required, while the analyte concentration dictates the sensitivity requirement of 

the method. The sensitivity of a method may be further infl uenced by sample preparation and 

interfering substances and the instrumentation used. 

Cyanide detection methods can range in procedure, extent, and methodology. Presented here 

is a brief summary of the approach, benefi ts, and limitations of known testing methods:

Electrochemical methods

Electrochemical analysis of cyanide and thiocyanate concentrations in biological samples, 

including plasma, tissue, and whole blood have been conducted using ion-selective electrodes 

(described below), potentiometry, amperometry, polarography and coulometry. Two clear 

benefi ts of these methods are high and quick analysis time. However, they can be subject to 

multiple interferences from many organic and inorganic ions, including sulfi de, Fe 3+, ClO4-, 

NO2-, N3-, and I-.

16
Polymeric membrane based ion selective electrodes (ISE)

The American Society of Testing and Materials (ASTM) ISE method is the only approach 

that has been widely used to detect cyanide exposure in marine fi shes. The method involves 

an acid digestion of the fi sh to liberate hydrogen cyanide gas, and capture of cyanide ions in 

sodium hydroxide solution after refl ux distillation. Chemicals must be added to help remove 

interfering substances such as chlorine and hydrogen sulfi de. An ISE meter manufactured 

by Thermo-Orion, using a membrane made of Agl or Ag2

S, is then used to analyze cyanide 

concentrations, based on its interaction with silver. ISE methods may also work for the 

detection of thiocyanate.

Spectrophotometric and fl uorescence methods

Both spectrophotometric and fl uorescence methods require extraction techniques to isolate 

cyanide and eliminate interferences from blood. Spectrophotometric methods have been 

used for detection of cyanide, thiocyanate and ACTA. One common approach involves 

the König dye synthesis to form a cyanide halide that is reacted with an aromatic amine to 

produce a glutaconic aldehyde product that is measured in the visible region of the spectrum. 

Spectrophotometric methods have adequate sensitivity, but they may lack specifi city due to 

interferences from other chemical species commonly present during the analysis of cyanide, 

especially thiocyanate and thiosulfate. They also require lengthy preparation times and the 

products may be unstable. A number of fl uorometric assays are available to determine 

cyanide, which have several advantages over spectrophotometric methods, including a lack of 

interference from thiosulfate and greater sensitivity.

Flow injection analysis (FIA)

The FIA method is a simple one with high reproducibility. The approach involves injecting 

a sample solution containing the target molecule into a fl ow tube where it reacts with certain 

chemicals. When the products reach the detector, the target molecules in the sample are 

measured. The user is able to control the measuring conditions precisely and also has the 

capability to continuously measure cyanide.

Biosensors for detecting cyanide ion

These methods primarily evaluate chemical reaction products based on the enzyme inhibition 

of cyanide, cyanide degrading enzymes, and microbial sensors which measure oxygen uptake 

by bacteria, yeast or other microorganisms. Biosensors have a rapid response, high selectivity, 

and a pollution free procedure. Most biosensors have the advantages of being portable, low 

cost, easy to use, and high selectivity. These methods, however, rely on chemical and physical 

procedures that can be slow, complex, and require the use of expensive equipment and 

environmental loading reagents. Other limitations of biosensors include degradation of the 

biological components that make up these sensors, inconsistent electrochemical signals, and 

diffi culty producing suffi cient quantities and activities of enzymes or microbes on which these 

sensors depend. 

Recently, one group applied a biosensor approach to marine fi shes. Organs of the fi sh were 

homogenized with NaOH and a fungal enzyme extract was used to produce formate from 

metal-cyanide complexes; the formate was converted using an enzyme to NADH which was 

17
measured spectrophotometrically. This approach has not yet been applied to thiocyanate or 

ACTA.

Chromatographic methods

Gas Chromatography (GC), high performance liquid chromatography (HPLC), and ion 

chromatography (IC) can all be used for determination of cyanide. These methods employ 

electron capture detectors, electrochemical detectors, UV/VIS detectors, fl uorescence detectors, 

conductivity detectors, or amperometric detectors. Three types of liquid chromatography 

have been used to analyze cyanide: reverse-phase high-performance liquid chromatography 

(RP-HPLC), ion chromatography (IC), and capillary electrophoresis. 

Liquid chromatographic techniques can determine trace amounts of an analyte and can 

effi ciently separate analytes from interfering components in the matrix, offering advantages 

over spectrophotometric, luminescent, and electrochemical methods. Liquid and gas 

chromatographic techniques also have the ability to simultaneously analyze for cyanide and 

thiocyanate. IC methods can determine all species of cyanide by separation. This technique 

obviates the need for distillation to convert cyanide complexes from metal to HCN. A number 

of pretreatment steps have been developed to facilitate the analysis of cyanide, thiocyanate, 

and ATCA using GC. For example, the sampling of cyanide from the sample head space is the 

most common pre-analysis step when using the GC method. 

Simple fi eld test

A number of “dip stick” type tests have been developed, primarily for detecting cyanide in 

water (e.g., the Cyanide ReagentStripTM Test Kit). These include colorimetric kits that are 

dependent on color changes or based on ion selective electrodes (ISE) linked to portable 

ISE meters. While some experimentation has been undertaken to modify these tests for the 

detection of cyanide in fi sh, nothing conclusive has been developed to date. For example, 

cyanide could be detected in fi sh tissue that had been digested in concentrated NaOH using the 

Soudararajan Procedure, but the test appears to give anomalously high cyanide readings possibly 

due to interference by sulfi de. An alternate approach may include digestion of fi sh in sulfuric 

acid in a closed container, with measurement of cyanide liberated as gas using test strips.

18
Goal 2: Provide recommendations on the most practical, reliable, and user-friendly tests 

that could be used in marine fi sh.

The Working Group discussed the practicality of each test, including the cost and equipment 

needs, level of expertise necessary, time required to analyze samples, reliability and reproducibility 

of different tests, and feasibility of implementing testing in either an exporting or importing 

country. Other considerations included factors that may affect the results, such as cellular 

absorption and detoxifi cation kinetics, sampling and analysis time, sample storage time and 

conditions, and the sample matrix. 

Participants also discussed the need for the development of new methodologies or modifi cations 

that could be made to existing tests that would be necessary under different scenarios to 

address 1) testing of small fi sh (fi sh tissues) instead of blood; 2) testing of cyanide versus 

thiocyanate and ACTA; and 3) benefi ts and limitations of rapid tests that could be used to 

detect the presence of cyanide or cyanide metabolites instead of the actual concentration. 

Timing of testing

The working group members could not come to consensus on the amount of time cyanide 

remains detectable in fi shes. Several participants suggested that cyanide is quickly metabolized 

and excreted in a matter of hours, while others maintained that cyanide is retained for longer 

time periods. 

At present, there is considerable information on metabolism of cyanide in blood. When 

analyzing cyanide from blood (the preferred method for determination of cyanide exposure in 

larger species), analysis must occur as soon as possible after exposure since the concentration 

of cyanide in the blood decays within minutes to hours. However, this may not be the case 

for fi sh tissue, as there is evidence that cyanide is concentrated in several organs and tissues, 

where it is slowly converted to SCN. There are also reports of rapid excretion of cyanide and 

SCN in urine. It is important to note that most work done with fi sh has been on freshwater 

species, and the rate of excretion in saltwater fi sh is likely to differ since, being hypo-osmotic 

in relation to seawater, they have a lower rate of urine production. 

The concentration of cyanide present in fi sh over time is dependent on the concentration of 

the cyanide solution used during exposure, the length of time of the exposure, time for holding 

and duration of transport, post-collection treatment, as well as other factors. Considerable 

work has been done on cytokinetics of cyanide in the rainbow trout (a freshwater fi sh) and 

linkages between cyanide exposure and conversion to SCN, but most of this work involved 

chronic exposure to low levels of cyanide instead of a single or pulse-dose of a much higher 

concentration as would be the case with marine fi shes. 

In general, all researchers found a progressive increase in SCN- in plasma over multiple days, 

followed by a decline until a period where it was no longer detectable (from 16 days to 16 

weeks or more). In addition, at least one study reported detectable levels of HCN in plasma 

after 20 days. According to one participant (Rubec), cyanide remained detectable in marine 

fi sh tissue up to 5-14 days after exposure when using the ISE method. 

19
Type of sample

Most cyanide detection tests have been developed for analysis of cyanide in biological fl uids 

in vertebrates such as blood, urine, and saliva, or in other aqueous solutions including water, 

with limited sampling of mammal and fi sh tissues and organs. While it may be feasible to 

obtain suffi cient quantities of blood from larger fi sh collected for the LRFF trade, most 

marine ornamental fi sh are too small to obtain the volume of blood needed for sampling. As 

a result, cyanide testing must be performed on ground fi sh tissue or organs. This, however, 

may present additional complications including 1) interference associated compounds used 

to digest tissue, such as sulfuric acid, which may trigger oxidation of thiocyanate to cyanide; 

and 2) variations in cyanide concentrations between organs/tissue due to differential rates of 

metabolism and variations in the concentration of the enzyme that catalyzes conversion of 

cyanide to thiocyanate.

Substance being tested 

To date, the only large scale testing for cyanide exposure in marine ornamental fi sh has been 

done at points of export using the ISE method to detect cyanide concentrations. However, 

because of concerns of rapid metabolism of cyanide, testing at points of import (and possibly 

points of export) may also require methodologies that can detect thiocyanate, ACTA or 

other metabolites, which is likely to present added challenges. For example, thiocyanate levels 

are normally quite high and can be inconsistent. Large variation in background thiocyanate 

concentrations reported from mammals would present problems when trying to quantify low 

level cyanide exposure, and large and variable concentrations of thiocyanate may indicate that 

thiocyanate is involved in a number of biological processes in addition to cyanide metabolism. 

One advantage of using ATCA is that it is stable in biological samples for months at freezing 

and ambient temperatures, and therefore may be a lasting signature for cyanide exposure. 

Unfortunately, there are few existing techniques to analyze ATCA from biological matrices 

and not many studies have evaluated the relationship between ATCA concentrations and 

cyanide exposure.

Quality control and validation of results

All of the participants recognized the importance of adopting testing methods that generate 

accurate and reproducible results. To be accepted in a court of law the results should vary 

minimally between testing methods and the chemist performing the test. These methods must 

demonstrate adequate sensitivity, specifi city, accuracy, and precision as determined by the “fi t 

for purpose” concept. There was considerable discussion about the possibility of incorrect 

results, as the amount of cyanide detected in a sample may be altered due to interference 

from inorganic and organic ions as well as the possibility of interconversion between cyanide, 

thiocyanate and other metabolites resulting from sample preparation and storage. 

Once a test is adopted, training, quality assurance, and profi ciency programs need to be 

implemented at cyanide testing laboratories and for fi eld testing. Many of the CDT procedures 

require extensive training and chemists conducting these tests need to be certifi ed and undergo 

periodic spot checks to ensure performance consistency and accurate test results. The Chair 

of the working group drafted a white paper which outlines a possible procedure for QA/

QC. He suggested that a training program must be developed employing both didactic and 

20
hands-on training that develops a demonstrated acceptable level of performance, as defi ned 

by the performance characteristics of the specifi c test and the needs of the analysis. A 

quality assurance program should be based on ISO 17025 or similar structures and should 

at a minimum employ documented Standard Operating Procedures (SOPs), bench and blind 

QC samples and statistically determined performance limits. A profi ciency testing program 

should be conducted by an independent body and employ the use standard samples, unknown 

to the analyst, analyzed across the network and evaluated statistically to identify laboratory 

results that do not meet network performance standards. While there is a recognized need 

for QA/QC, one participant was very concerned about the cost and time necessary for the 

detailed validation of the results of the particular test chosen, and how this is likely to delay 

implementation of a network of CDT laboratories. 

The best test

The overall conclusion of the group was that there would be a need for, at minimum, three 

to four different types of tests, depending on the point in the chain of custody where the fi sh 

are tested. There currently only exists a single test that has been demonstrated to work in fi sh, 

shortly after collection and for detection of cyanide only, although there are many available 

methodologies that could be modifi ed to test for presence of cyanide instead of quantity and 

to quantify other cyanide analytes.

It would be extremely valuable to implement a rapid fi eld test used at collection points. This 

test would need to verify the presence of cyanide, but not necessarily the actual concentration. 

Not only could it be used by enforcement offi cers as a deterrent, but it could also help 

identify particular fi sh or particular collectors suspected of using cyanide, with more detailed 

quantitative monitoring done on samples of concern. The preferred methodology is a rapid 

“dip stick” type test; however, additional research is needed on methods to extract cyanide 

from fi sh tissue to allow use of these approaches. At this point, this type of approach has not 

been successfully applied to homogenized fi sh tissue.

The ASTM ISE method was identifi ed by all participants as the optimal choice for a test at 

points of export, mainly because it has been widely applied to marine fi shes and it appeared 

to be effective during early phases of cyanide testing by IMA. However, several concerns 

identifi ed in the peer-reviewed literature must be addressed before it (and subsequent certifi cates 

verifying fi sh are cyanide free) would be accepted by the United States. One participant stated 

that the method has undergone extensive round robin testing which addressed QA/QC, 

precision, and bias. He also pointed out why other researchers obtained confl icting results 

(e.g., they did not follow the methodology or they used a different electrode). Several other 

questions still remain such as 1) the possibility that fi sh exhibit background levels of cyanide 

but these could not be measured using the ISE method; 2) incomplete knowledge on how long 

cyanide is in fact detectable in fi sh; 3) lack of validation outside of the IMA/BIFAR network 

of the methodology used to digest fi sh and whether this results in incorrect readings due to 

interference of other substances. 

A rapid “fi eld” test would be needed for USFWS inspectors at points of import to verify the 

presence of cyanide and identify possible fi sh for more detailed laboratory analysis. This is not 

21
currently available, and cannot realistically be developed until we address questions regarding 

the time after exposure cyanide and cyanide metabolites are detectable.

A quantitative method for laboratory analysis at points of import is needed for forensics 

laboratories. It is likely that the method would need to detect thiocyanate and not cyanide (or 

possibly ACTA or other analytes). Until more research is done, it is unlikely that an appropriate 

test for points of import can be identifi ed.

Detection Method Testing For Approximate Cost

Low <$5K

Mod $5-25K

High>$25K

Technical 

Training 

Required

Equipment 

Required

Sensitivity Reliability Samples/Day

Half-life 1 hour

Location Collection

Headspace Test Strip CN- Low Low Low Poor Unknown Many

Location Export

Ion Chromatography SCN- High Mod-High High Good Unknown 25+

Gas Chromatography ATCA Mod-High Mod-High High Excellent Good 25+

Colorimetric SCN- Low Low Low Good Good 25+

Location Import

Ion Chromatography SCN- High Mod-High High Good Unknown 25+

Gas Chromatography ATCA Mod-High Mod-High High Excellent Good 25+

Colorimetric SCN- Low Low Low Good Good 25+

Half-life 10 hour

Location Collection

Headspace Test Strip CN- Low Low Low Poor Unknown Many

Location Export

Distillation/ISE CN- Low Moderate Moderate Good Good <5

Ion Chromatography CNSCN- High Mod-High High Good Unknown 25+

Gas Chromatography CNATCA Mod-High Mod-High High Excellent Good 25+

Table 1: Summary of cyanide detection testing methods. 

22
Goal 3: Identify research needs to refi ne methodologies and/or develop other testing 

approaches. 

The group summarized some of the pressing biological questions and information gaps needed 

before the most appropriate testing methods could be adopted. The consensus of the Working 

Group was that serious gaps exist in the understanding of the metabolism and elimination 

of the cyanide anion and its major metabolites in fi sh. Without a better understanding of 

these processes, the implementation of an exposure assessment method that would meet legal 

requirements would be diffi cult. Therefore, the highest priority for research involves support 

for a program that will generate the data necessary to determine the availability of cyanide and 

its metabolites for exposure assessment versus the time involved with the local collection of 

fi sh, their transport to a fi xed site laboratory prior to export, and their arrival at the import 

destination prior to the implementation of an analysis method at any point in the delivery.

The concentration of cyanide present in the living fi sh will vary depending on the concentration 

of the cyanide solution used during exposure, the length of time of the exposure, time for 

holding and duration of transport, and post-collection treatment. Rates of metabolism may 

also vary depending on species or size. Research should encompass these possible variations, 

such that it is possible to quantify measurable levels of cyanide in representative species and 

life history stages of marine fi shes, and how this changes over time, using at least two methods 

and multiple known concentrations. Participants also identifi ed a list of additional research 

needs for developing and refi ning cyanide detection testing methods:

• Determine rates of metabolism of cyanide. A detailed analysis requires that the 

analyte is stable and at elevated levels in the fi sh long enough for it to be delivered 

to the laboratory and an examination conducted. Without a better understanding 

of these processes in representative marine fi sh species, the implementation 

of an exposure assessment method that would meet legal requirements would 

be diffi cult. The Working Group identifi ed the highest immediate priority is 

research to determine the half-life of cyanide and major metabolites (especially 

thiocyanate) in marine fi sh, and variations between species, life history stages, and 

the biological matrix examined. To represent the time from initial collection and 

transport from the reef to holding facilities in exporting countries and then on to 

import destinations, fi sh samples should be exposed to varying concentrations of 

cyanide, with representative individuals sampled immediately after exposure and 

subsequent defi ned intervals.

• Identifi cation of the presence of background levels of cyanide (and major 

metabolites) in marine fi sh. This would include an evaluation of different species 

collected from a variety of locations and whether cyanide levels are above the limit 

of quantifi cation. One aspect should also include a determination of detectable 

background levels, using fi sh from the wild caught with nets as well as captive bred 

species. In addition, the effect of freezing samples must be determined for both 

fi eld collection and delayed analysis samples to prevent misinterpretation of data 

obtained from frozen samples. These steps are critical to ensure that the applied 

test can detect elevated levels of cyanide which would be indicative of exposure by 

23
fi sh collectors. To perform this research, validated methods for the determination 

of cyanide, thiocyanate and other metabolites (e.g., ATCA) in homogenized 

fi sh tissue must be used. These methods must demonstrate adequate sensitivity, 

specifi city, accuracy and precision.

• Demonstration of adequate recovery of cyanide in fi sh samples that are digested 

and verifi cation that sulfuric acid or other compounds used to digest the fi sh do 

not interfere with the readings. 

24
Working Group 1 Participants

Bob KOBELSKI, National Center for Environmental Health, Centers for Disease Control 

and Prevention (chair)

Andy BRUCKNER, NOAA National Marine Fisheries Service, Coral Reef Conservation 

Program

Martin FRANT, Thermo-Fisher Inc.

Ken GODDARD, U.S. Fish and Wildlife Service, National Forensics Laboratory

Brian LOGUE, South Dakota State University

Benita MANIPULA, International Marinelife Alliance

Peter RUBEC, Florida Fish and Wildlife Conservation Commission

25
Report from Working Group 2

Participants in the Export Working Group discussed current actions underway to address 

cyanide fi shing, the effectiveness of these activities, and additional efforts that would be needed 

to successfully eliminate cyanide use for the capture of marine fi shes. A comprehensive and 

robust scheme would require six strategic actions:

1) Education for fi shermen and exporters in problems associated with cyanide use and a 

training program in the use of nets; 

2) Improved partnerships between all sectors of the ornamental and live food fi sh trade, 

including industry, government agencies, and conservation groups;

3) Targeted cyanide testing in the fi eld and at points of export; 

4) Improved monitoring and validation of exports as cyanide-free; 

5) Licensing schemes and adequate enforcement measures for offenders; and 

6) Sustainable funding mechanisms to support cyanide detection labs. 

The group also discussed some of the diffi culties in implementing a cyanide testing network, 

pinpointing the largest gaps as insuffi cient funding, lack of trained personnel, and limited 

laboratory capacity. Funding is particularly challenging, as there are discrepancies in monitoring, 

testing, and enforcement under any cyanide use prevention scheme. They also identifi ed 

diffi culties associated with testing due to the large spatial and temporal scale over which the 

trade operates, and limitations of available testing methods. 

The Working Group developed a short list of priorities for cyanide testing in export countries. 

The fi rst priority is to validate the leading cyanide testing methods for accuracy. The chosen 

method must be a credible, proven test that is effective in prosecution. Once there is consensus 

on a valid, defensible test, training in testing procedures and cyanide-free catch techniques 

must take place. In conjunction, a database should be developed to track testing results and 

violations. This database should be linked to the accreditation and licensing scheme, with 

repeat offenders losing their license for collection or export, along with other penalties. 

Goal 1: Outline what to include in a robust scheme for monitoring exports to determine 

whether a fi sh was caught using cyanide.

Considerations for testing

A successful detection test must be sensitive enough to detect cyanide or cyanide metabolites 

for a minimum of 5 to 10 days after collection to accommodate the time period between 

collection and export.

As the demand for live reef fi sh has increased and local fi sh populations have declined, areas 

of collection have expanded. Whereas most collection once occurred on local reefs near 

villages in close proximity to major international airports, collection areas now include remote 

offshore reefs. The average time between collection and transport to a village landing area is 

26
estimated at roughly 3 to 5 days. However, in some locations roving fi shermen may embark 

on collecting trips that last for up to 2 weeks before fi sh are fi nally landed in a local village. At 

holding facilities, aquarium fi sh are typically quarantined for 1 to 4 days before they are sold 

to a middleman or exporter. For live reef food fi sh, the time between collection and export 

can be even longer: collectors may hold fi sh in fl oating cages for several weeks before they 

are purchased by a middleman or exporter. Trade routes can also be quite complex, and may 

include transport via boat, road, local fl ights and international fl ights. Marine aquarium fi sh are 

also typically held by the exporter for an additional period of 2 to 5 days before shipping. 

Optimal targets for cyanide testing 

Identifying the most effective target points for cyanide testing is a priority. Participants thought 

the easiest location to test for the presence of cyanide was points of export at international 

airports, since the majority of fi sh are sent abroad by air. Cyanide detection testing (CDT) 

laboratories should be established close to major regional airports, including:

• Indonesia — Bali, Jakarta, Sulawesi, Ujung Pandang, Manado, Surabaya

• Vietnam — Saigon, Hanoi, Da Nang

• Philippines — Manila, Cebu

Also needed is development of a fi eld test that could be implemented by enforcement offi cers 

at landing areas. As reef fi sh may be transported by land and boat (e.g., live reef food fi sh), 

random testing would ideally be done in every village with a landing area This raises the issue 

of choosing the most appropriate methods for sampling. An effective cyanide testing regime 

must address who would be impartial samplers, how often to sample, what size sample to take, 

and whether to include targeted samples as well as random ones.

Obstacles limiting testing 

Assuming the Ion Selective Electrode (ISE) cyanide test proves valid, additional obstacles 

to using the test centered on funding, geographic barriers and distance, and potential social 

backlash. Testing at fi eld sites and airport points cover both ends of the export chain, and is 

therefore ideal. However, lack of funding might constrain this possibility. Funding for testing 

would initially rely largely on government support, possibly supplemented by international 

donors and importing countries. Longer-term fi nancing may require inspection fees that are 

covered by the exporter as well as taxing particular industry components; this may subsequently 

be passed on to the importer and ultimately the consumer. Funding for testing schemes should 

be self-sustaining, and needs further consideration on how to structure and implement such a 

system on the export side. 

Enforcement considerations

The process of accountability is a challenge. On one side is the issue of test verifi cation, and 

on the other is the issue of prosecution. Creating a system for trustworthy, reliable testing 

must be airtight in order to stand up to exporters who challenge it, especially if it becomes a 

requirement. There must also be incontestable consistency, so that testing at two separate sites 

does not return differing results. Holding someone accountable would have to lie with the 

government, and depends heavily on where along the export chain you wish to target. There 

27
must also be some degree of coordination between export and import nations for a truly 

successful testing system.

Monitoring fi sh at landing areas can also employ indirect evidence of cyanide use. For example, 

if fi shers are catching more fi sh than would be expected under normal circumstances, they 

might be using cyanide. Further, certain species are more often caught using cyanide than 

others; therefore, monitors should be trained to know which species to look for.

Perhaps the most logical and proven method calls for placing the burden on exporters to clean 

up the supply chain, but it is important to note the inherent challenges in this type of approach. 

Testing only at points of export may be complicated, as the exporter may request cyanide-free 

fi sh but be unable to verify this. Because exporters may purchase fi sh from middlemen, and 

fi sh from multiple sources may be mixed within the export facility, it may be challenging to 

demonstrate the connection between the exporter and the collector. Despite these concerns, 

the participants agreed that initiating a cyanide detection testing scheme at export points 

remains the most practical and manageable starting point.

Target points

In addition to some of the limitations described above, the group felt additional measures are 

needed to tackle the actual root causes of the problem. Several participants felt that exporters 

were the cause of the problem, as opposed to the actual fi shers. The exporters may supply 

the cyanide in some cases, and they also provide few incentives to collectors who use nets 

instead of cyanide. However, testing only at airports may be inadequate, as the volume of fi sh 

exported on a daily basis may exceed the limits of testing, thereby allowing many cyanidecaught fi sh to be exported. It is important to recognize that this oversight may occur even in 

the best of schemes. However, if sampling is frequent enough and penalties high enough, it 

should become more economical for exporters to invest in cleaning up the supply chain.

One other alternative could include spot checks at points of landing, solely to verify exposure 

to cyanide, followed by more quantitative testing of fi sh at airports, with repeat offenders 

losing their license (fi sh collectors) or being prosecuted (exporters). This would shift the 

burden to the exporters, who ultimately make most of the profi t and has direct control over 

what they purchase from fi shermen. In the past, the Philippines conducted monitoring at the 

landing areas, and samples were then sent on for further testing at the airport lab. 

In conjunction with testing, educating collectors has been pushed in the Philippines, while 

Vietnam’s approach to the similar issue of dynamite fi shing has been to deputize villagers who 

curtail nighttime use and enforce local fi sheries management regulations. 

28
Goal 2 : Identify steps, agreements, and/or partnerships that could be established between 

exporting governments, the U.S. government, NGOs, certifi cation agencies, community 

groups, and academic scientists to implement cyanide testing. 

Harmonization

Export countries emphasized the need for harmonization of standards between importers 

and exporters to eliminate policy gaps and potentially tighten enforcement. Import countries 

should create clear requirements for shipments, make the policies available to exporting 

countries so they can comply, and make them understandable. Information on quarantine 

requirements, clear documentation requirements, and the creation of a unifi ed labeling and 

packaging system are all ways to reduce discrepancies.

Training and education

Parties emphasized that tying education and enforcement together is critical to a successful 

implementation strategy. This includes educating government offi cials as well. Public awareness 

campaigns featuring celebrities or other popular personalities might help get the general public 

interested and galvanized on these issues. Education programs must be designed carefully and 

with appropriate follow-up, as past education efforts have also made users of cyanide more 

clever about covering their tracks. Re-education and training for collectors will be necessary 

if some are forced out of the trade by new enforcement and regulations. Parties should work 

further on eco-tourism and supplementary livelihood options.

Partnerships

Partnering with multiple stakeholders and interested parties will increase the likelihood of 

a successful cyanide testing regime. Importing parties should improve coordination and 

communication with importing countries. Data on exporters could be shared with government 

agencies in importing countries, including the numbers of legitimate exporters, which ones 

still use cyanide, where the fi sh are caught, and so forth. Sharing such information could help 

identify legal and illegal shipments for importing countries. 

Because the marine aquarium fi sh trade is lucrative for exporting countries, parties should not 

overlook the role of business entities. Garnering support from the business sector could push 

government in the right direction, or achieve faster results than diplomatic avenues. 

Partnering with local communities is extremely important. It is vital that community-based 

initiatives be carried out correctly. Improper training can result in the community becoming 

even more committed to hiding their cyanide use. Licensing is a potential avenue for communitybased cyanide-free fi shing efforts. In the Philippines, parties are re-defi ning things by province 

and talking to community leaders (mayors) for input on how to regulate live fi sh trade in terms 

of licensing.

Partnering with both local and international NGOs can provide transparency and aid in 

enforcement, education, and communication from the provincial level up through the national 

and international levels.

29
Goal 3: Provide a recommendation with respect to the possibility and/or benefi ts of a 

mandatory, government-issued, export certifi cation that states the fi sh were caught with 

legal practices. 

Cyanide detection: current procedures

Efforts to fi nd cyanide in exporter countries are already taking place through boat searches 

using local enforcement or the Navy, and training on where to look for cyanide. However, 

uncovering cyanide use is harder now due to several key behaviors, including users traveling at 

night, cyanide stashes hidden underwater, and a switch from fi shing out an area to collecting 

for two days and then moving on. Inspections already occur in Indonesia and the Philippines, 

but they are insuffi cient. 

Limited cyanide testing is reportedly occuring at airports in the Philippines, but this testing may 

occur several days to two weeks after initial cyanide exposure. Due to the rapid metabolization 

of cyanide, this time frame may be too long for accurate testing. Sampling is either voluntary 

or provided by the exporter; there is no random or obligatory sampling for cyanide testing 

in Indonesia and the Philippines. Even with a sample provided, boxes may not get inspected 

before shipment. Also, if a shipment makes several stops before its fi nal destination, the 

exporter has no control over the shipment at those stops, where shipments might be tampered 

with or altered.

Next steps

The Working Group felt the only viable strategy to accrue evidence of cyanide use hinges 

on a testing scheme; any other strategy would be diffi cult and, while it could be a supporting 

scheme, it could not be the main strategy. An effective test would need to detect prior 

exposure to cyanide at the landing area within a 5- to 7-day frame, and a 7- to 14-day frame at 

international airports. If the ISE test is examined and proven reliable, funding for reinstating 

and signifi cantly enhancing the accompanying infrastructure will then need to be addressed. 

Cyanide-free verifi cation: testing outputs

The Working Group examined numerous options for verifying cyanide-free marine ornamental 

fi sh. Three main approaches were discussed.

1) Accreditation: The possibility of using a cyanide test as an accreditation tool may 

improve industry support as an alternative to a cyanide-free certifi cation scenario. For 

example, if after 10 cyanide-free shipments occur, the company would be accredited 

for a year’s time. Testing would continue even after accreditation, in order to maintain 

enhanced standards and monitoring.

2) Certifi cation: The viability of independent third-party certifi cation schemes is unclear 

at this point. Previously, shipments verifi ed as cyanide-free based on the ISE testing 

method received cyanide-free certifi cates. While this could be a requirement for export 

of fi sh, it is not apparent whether this could be used as a legal toe-hold for prosecution 

between import and export countries. For example, if a shipment comes with a 

30
certifi cate, but tests positive upon arrival in import countries, import enforcement 

offi cers can then testify in export countries.

3) Licensing: Under a licensing scheme, the marine resource oversight organization 

within each export country should determine loss of license for those who fail cyanide 

testing. Due to the contentious nature of prosecution, the scheme would need an 

independent group to do the sampling. However, this puts the pressure again onto the 

quality of the test (given that the law is clear). One suggestion was that the municipal 

government revoke the fi shing license if authorities choose to trace it back to the point 

of collection. It was also suggested that, similar to anti-fencing laws, the government 

should see it as “anyone with stolen goods is guilty.” This would hold the exporter 

accountable if in possession of fi sh testing positive for cyanide. 

31
Working Group 2 Participants

Kristine JOHNSON, Kingfi sher Foundation (chair)

Aquilino ALVAREZ, Philippines Bureau of Fisheries and Aquatic Resources 

Thomas CABAGAY, East Asian Seas and Terrestrial Initiatives

Ferdinand CRUZ, East Asian Seas and Terrestrial Initiatives

Patricia DEBENHAM, SeaWeb

Agus DERMAWAN, Directorate General of Coastal and Small Islands Ministry of Marine 

Affairs and Fisheries, Indonesia

Roy TORRES, NOAA National Marine Fisheries Service

Thu Ho Thi YEN, Center for Marinelife Conservation

32
Report from Working Group 3

The Participants in the Import Working Group discussed the possibility of implementing a 

cyanide testing scheme at points of import, with an emphasis on the United States. Cyanide 

testing for this sector of the live reef trade would primarily address imports of marine aquarium 

fi sh (MAF), because the United States currently imports 50 to 60% of all MAF, whereas live 

reef food fi sh (LRFF) represent a small component of U.S. imports. While the trade in LRFF 

is substantial (up to 30,000 metric tons per year), most imports are destined for Hong Kong 

and other Asian markets. Testing options could differ between these two types of trade as well, 

as LRFF consist of much larger and differing, selective species than MAF. Hence, testing of 

imports of LRFF could focus on detection of cyanide in blood, while MAF testing relies on 

whole digested fi sh. The cytokinetics of cyanide may also differ among LRFF and MAF, in 

terms of rates of breakdown and removal, as cyanide can be removed from the blood much 

faster than from tissues. Larger, more active mobile predatory fi sh may have different rates 

of metabolism. Most MAF are small, site-attached species, whereas larger groupers and other 

species are likely to have a higher metabolism because they are faster swimmers. In addition, 

the background levels may vary considerably among taxa, and could be higher in larger LRFF 

as these are top predators that may be consuming smaller fi shes containing cyanide.

Goal 1: Outline what to include in a robust scheme for monitoring imports to determine 

whether a fi sh was caught using cyanide.

The group suggested that implementation of cyanide testing at points of import would be 

much more diffi cult, costly, and time-consuming than at points of export. They identifi ed 

numerous challenges that need to be addressed before an effective import cyanide testing 

scheme is possible. These include, but are not limited to, the following central issues:

Infrastructure and facilities 

Defi ciencies in staffi ng, funding, equipment, and facility resources remain key issues in 

developing a U.S. import cyanide monitoring system. At present, lead ports for fi sh shipments 

include Los Angeles and Miami, with some support from non-designated ports such as 

Charleston and Tampa. Hundreds of boxes of invertebrates, fi sh, and corals pass through 

U.S. ports daily (Table 1, and Figures 1-2). The U.S. Fish and Wildlife Service (USFWS) Law 

Enforcement staff largely focus on species listed under the Endangered Species Act (ESA) 

and the Convention on International Trade in Endangered Species (CITES), due to specifi c 

legislative mandates for these taxa. In addition, there are no adequate facilities to maintain 

large shipments of fi sh while testing is undertaken, and seizures of live fi sh imports would be 

impractical and likely to result in high levels of mortality. 

33
Identifi cation and testing

The United States currently imports about 16 million MAF per year. Shipments can range from 

20 to 25 or more boxes for small tropical fi sh, while larger shipments can include 50 or more 

boxes. Each box may contain several hundred fi sh, with each fi sh packaged in an individual 

bag or with mixed species in larger bags. Opening the bags to examine a fi sh introduces oxygen 

out of the bag and further threatens survival. This poses a huge challenge for identifying all 

species and possibilities for cyanide testing. The optimal strategy for testing would include a 

rapid, simple diagnostic test for use at the port, such as a “dipstick” method, that would simply 

identify the presence of cyanide (“yes or no” system). The test cannot take more than a few 

minutes, based on shear size of import shipments, lack of manpower, and split attention for 

ESA and CITES identifi ed priority species. If the fi eld test confi rmed the presence of cyanide, 

a shipment could be seized and sent on to a diagnostic laboratory where a more detailed test 

could be performed to quantify levels of exposure. 

No. of 

Shipments

% of Total 

Shipments Quantity

% of Total 

Quantity

Agana, GU 7 0 1,865 0

Atlanta, GA 230 2 435,498 3

Baltimore, MD 1 0 45 0

Blaine, WA 2 0 16 0

Buffalo-Niagara Falls, NY 51 0 293 0

Chicago, IL 257 2 519,547 3

Detroit, MI 74 1 90,430 1

Dallas-Ft.Worth, TX 272 2 295,832 2

Honolulu, HI 1,492 13 584,755 4

Los Angeles, CA 5,548 47 8,994,755 56

Miami, FL 1,898 16 2,442,173 15

Newark, NJ 54 0 70,730 0

New York, NY 946 8 1,570,988 10

Portland, OR 78 1 51,950 0

Seattle, WA 73 1 54,689 0

San Franciso, CA 662 6 954,049 6

Sumas, WA 1 0 2 0

San Diego-San Ysidro 10 0 3,545 0

Tampa, FL 75 1 7,925 0

unknown 20 0 6,896 0

Totals 11,751 100 16,085,983 100

Table 1: Shipments of marine ornamental fi sh in each United States port city between July 2004 and June 2005.

34
0

1,000

2,000

3,000

4,000

5,000

6,000

Agana, GU

Atlanta, GA

Baltimore, MD

Blaine, WA

Buffalo-Niagara Falls, NY

Chicago, IL

Detroit, MI

Dallas-Ft.Worth, TX

Honolulu, HI

Los Angeles, CA

Miami, FL

Newark, NJ

New York, NY

Portland, OR

Seattle, WA

San Franciso, CA

Sumas, WA

San Diego-San Ysidro

Tampa, FL

unknown

Ports

No. of Shipments

0

1

2

3

4

5

6

7

8

9

10

Agana, GU

Atlanta, GA

Baltimore, MD

Blaine, WA

Buffalo-Niagara Falls, NY

Chicago, IL

Detroit, MI

Dallas-Ft.Worth, TX

Honolulu, HI

Los Angeles, CA

Miami, FL

Newark, NJ

New York, NY

Portland, OR

Seattle, WA

San Franciso, CA

Sumas, WA

San Diego-San Ysidro

Tampa, FL

unknown

Ports

 Quantity (in millions)

Figure 1: Number of Shipments by Port from July 2004 to June 2005.

Figure 2: Quantity of Marine Ornamental Fish by Port from July 2004 to June 2005.

35
Goal 2: Identify steps, agreements, and/or partnerships that could be established between 

exporting governments, the U.S. government, NGOs, certifi cation agencies, community 

groups, and academic scientists to implement cyanide testing.

Funding

Suffi cient funding continues to be a serious concern across the board. Addressing existing 

obstacles and implementing new plans must have fi nancial backing to move forward. One 

approach to enhance funding focuses on improved education and awareness about the issue 

and the factors limiting the ability of USFWS to effectively prevent imports of cyanide-caught 

fi sh. One recommended method involves the use of high-profi le celebrities as spokespeople, 

which is likely to raise public concern and ultimately push politicians to alter and enforce 

policies on the aquarium trade. With political power behind them, trade programs could 

receive targeted funding through congressional earmarks and increased support from large 

donors and NGOs. 

Harmonization

The participants emphasized the need for harmonization of standards between importers and 

exporters to eliminate policy gaps and potentially tighten enforcement. Import countries should 

create clear requirements for shipments, make the policies available to exporting countries so 

they can comply, and make them understandable. Information on quarantine requirements, 

clear documentation requirements, and the creation of a unifi ed labeling and packaging system 

are all ways to reduce discrepancies.

Government linkages: exporting countries and importing countries

Exporting parties should improve coordination and communication with importing countries. 

Representatives from Vietnam, Indonesia, and the Philippines indicated that data on exporters 

could be provided (including the numbers of legitimate exporters), which ones still use cyanide, 

where fi sh are caught, and so forth. Sharing such information could help identify legal and 

illegal shipments for importing countries. United States enforcement staff would benefi t from 

knowing which companies repeatedly test cyanide-free, as well as those companies that tend 

to use cyanide. Some degree of coordination must occur between export and import nations 

for a truly successful testing system.

36
Goal 3: Provide a recommendation with respect to the possibility and/or benefi ts of a 

mandatory, government-issued export and/or import certifi cation that states the fi sh were 

caught with legal practices.

Export certifi cation

Although participants from exporting countries suggested the possibility of either governmentissued or third-party certifi cates verifying fi sh as cyanide-free, the United States is unlikely 

to use these for enforcement or prosecution. A certifi cation document would not resolve 

or relieve any of the enforcement issues discussed, and did not seem reliable or establish 

any measure of relief that certifi ed fi sh would be cyanide-free. Third-party certifi cation also 

seemed improbable, as this process is most often used on the consumer side rather than the 

government side. 

Current U.S. procedure for import inspections examination

All wildlife imported into the United States, with a few exceptions, must be declared to the 

U.S. Fish and Wildlife Service (USFWS) and cleared prior to release by U.S. Customs and 

Border Protection. Each shipment imported into the United States requires the importer and/

or broker to fi le a U.S. Fish and Wildlife Declaration (Form 3-177) to the USFWS at the time 

of import. Each shipment includes the fi ling of the USFWS declaration form, which provides 

all relevant information pertaining to the shipment, such as date of import, U.S. importer, 

foreign exporter, the common and scientifi c name for each species of wildlife, quantity and 

origin of species, carrier, number of cartons containing wildlife, etc., and other accompanying 

documentation.

All shipments containing species listed under the Endangered Species Act (ESA), the Convention 

on International Trade in Endangered Species of Wild Fauna and Flora (CITES), or other 

federal wildlife laws must include the appropriate permits. As such, these types of shipments 

usually have a higher priority for physical inspection as opposed to shipments containing nonprotected wildlife. The physical inspection entails verifying the shipment’s contents with the 

information listed on the permits, invoices, and other documentation. Shipments containing 

non-protected species of wildlife are randomly inspected. 

Due to the high volume of wildlife shipments imported into the United States, the mission 

and priorities of the Offi ce of Law Enforcement, and limited staffi ng and resources at the 

present time, it is unlikely that all shipments containing live marine ornamental fi sh can be 

inspected and tested for the presence of cyanide unless new legislation is adopted and staffi ng 

is increased. 

Policy changes and trade restrictions

The United States currently has no legislation banning the import of unsustainably caught 

aquarium fi shes. It was generally agreed during the working group session that existing U.S. 

laws under the Lacey Act could be suffi cient for prosecution, but the execution, funding, and 

support for those regulations needs consideration. Within the United States, the Lacey Act 

drives import inspection and enforcement. If exporting countries impose clear laws against 

exporting cyanide-caught fi sh, the United States could potentially prosecute violations under 

37
the Lacey Act. Such legislation would allow the United States to demand more culpability on 

the importer and, in effect, make the trade of cyanide-caught fi sh an international violation.

Unfortunately, U.S. courts are unlikely to accept any analytical procedure that does not 

conclusively determine both the presence of cyanide and the amount of cyanide in the 

specimen. For example, if only trace amounts of cyanide are detected, the defense could offer 

expert witness testimony that cyanide can be found as a “background” contaminant in many 

ocean waters of the world.

38
Working Group 3 Participants

Eric PUNKAY, SeaWeb/SeaStrategy Network (chair)

Gil ADORA, Philippines Bureau of Fisheries and Aquatic Resources

Eddie MCKISSICK, U.S. Fish and Wildlife Service, Division of Law Enforcement

Glynnis ROBERTS, NOAA Fisheries, Coral Reef Conservation Program

Ambrosius RUWINDRIJARTO, Telapak

Vicky VINA, U.S. Fish and Wildlife Service, Division of Law Enforcement

39

WHITE PAPERS

Introduction

Since the early 1960’s, coral reefs have been 

increasingly exploited by fi shermen-who 

use cyanide to capture reef fi shes alive, in 

order to sell them to the live food fi sh trade 

(sold to restaurants in Hong Kong and 

mainland China) and marine aquarium fi sh 

trade (Rubec 1986, Rubec 1987). Fishermen 

stun the fi sh by squirting hydrogen cyanide 

(HCN) solution onto coral heads and into 

crevices in coral reefs. The sodium cyanide 

(NaCN) tablets used by collectors in the 

Philippines and Indonesia weigh about 20 g 

each (Johannes and Riepen 1995). Fishermen 

who collect ornamental fi sh for aquarium 

tanks generally place one or two 20 gram 

tablets of sodium cyanide (or potassium 

cyanide) into a one-liter plastic squirt bottle 

fi lled with seawater, while food-fi sh collectors 

use three to fi ve tablets (Rubec et al. 2001). 

The tablets sequentially dissolve in the squirt 

bottle as collectors proceed to spray the reefs, 

making it diffi cult to determine the cyanide 

ion (CN-

) concentrations being applied. 

It has been suggested that fi sh collectors 

use concentrations ranging from 1,500 to 

120,000 mg/L (Johannes and Riepen 1995; 

Barber and Pratt 1997, 1998; Pet and Djohani 

1998; Jones et al. 1999). Since not all of the 

cyanide applied from squirt bottles dissolves, 

it is sometimes visible underwater as a whitish 

plume (Rubec et al. 2001). Not all fi shermen 

limit themselves to squirt bottles. Reports 

from the Philippines assert that 55-gallon 

drums of cyanide have been dumped onto 

reefs to kill and capture food fi sh (del Norte 

et al. 1989, Johannes and Riepen 1995).

As far as cyanide testing is concerned, the 

amount of cyanide that might be detected 

is partly a function of the exposure 

concentration of cyanide. If higher doses 

are used in collecting, then higher initial 

concentrations in the fi sh are possible. The 

review conducted by SeaWeb at the CDT 

Workshop quoted a paper by Fahrudin 

(2003) that stated: “One cyanide tablet (2 g) 

is mixed with approximately 3 liters of water 

in a plastic bag or bottle” (SeaWeb 2008). 

Based on this information, Fahrudin (2003) 

calculated the resulting cyanide concentration 

as approximately 6.67 mg/L (ppm). It should 

be noted that the tablets weigh approximately 

20 grams (not 2 grams) and that the tablets 

are usually dissolved in a squirt bottle of 

about one liter volume (Johannes and Riepen 

1995). The minimum exposure concentration 

squirted on a fi sh on the reef exceeds 1500 

mg/L (Cervino et al. 2003).

At the Cyanide Detection Workshop (held 

February 6-8, 2008 in Orlando, Florida) a 

subgroup of experts (Dr. Brian Logue, Dr. 

Robert Kobelski, Dr. Peter Rubec, Dr. Martin 

Frant, Ms. Benita Manipula) met to discuss 

the various methods that have potential 

for measuring cyanide or other metabolic 

byproducts in marine fi sh.

Methods for Detection of Cyanide and 

its Metabolites in Marine Fish

Dr. Peter J. Rubec1

, Dr. Martin Frant2, and Ms. Benita Manipula1

1. International Marinelife Alliance

2. Thermo-Fisher Inc. (formerly Thermo-Orion Inc.)

43
Pharmaco-kinetics of Cyanide In Fish

Dr. Kobelski of the Centers for Disease 

Control and Prevention (CDC) led the 

discussion. He directed the discussion toward 

the need to determine uptake and release rates 

for cyanide ion (CN-

) and/or the thiocyanate 

anion (SCN-

) in marine fi sh. The committee 

was divided between those that believe that 

cyanide is quickly metabolized and excreted 

in a matter of hours (Kobelski and Logue) 

and those that believe that cyanide is retained 

for longer time periods (Rubec, Manipula, 

and Frant). Dr. Logue of South Dakota 

State University has summarized information 

from mammals that indicates that cyanide 

in humans is quickly metabolized (Logue 

and Hinkens 2008). Dr. Kobelski noted that 

he had contacted a marine toxicologist at 

Cornell University that told him there was no 

reason to believe that fi sh would be different 

from mammals, since the pharmaco-kinetic 

processes are similar. The committee agreed 

that CN-

 is concentrated in certain organs 

where it is converted to SCN- by the enzyme 

rhodanese. While there are several pathways 

for cyanide metabolism, most of the SCN- is 

excreted in the urine. There was disagreement 

concerning how quickly these processes occur 

in fi sh. 

Dr. Rubec discussed the available research 

pertaining to fi sh. He also noted that the IMA 

was able to detect cyanide in marine aquarium 

fi sh (MAF) 5-14 days after they were captured 

using cyanide. This suggested that cyanide is 

retained in MAF for longer time periods than 

what has been documented with mammalian 

research (mostly humans).

There has been research conducted concerning 

cytokinetics of cyanide in freshwater fi sh 

(rainbow trout) but almost nothing published 

for marine fi sh. Dr. Rubec mentioned the 

research on rainbow trout by Raymond et al. 

(1986) and Brown et al. (1995). This research 

has been reviewed in the paper concerning the 

cyanide detection testing (CDT) conducted by 

the International Marinelife Alliance (IMA) 

under contract with the Philippines Bureau 

of Fisheries and Aquatic Resources (BFAR) 

from 1993 to 2001 (Rubec et al. 2003).

The concentration of cyanide present in the 

living fi sh over time is dependent on several 

factors e.g., concentration of the cyanide 

solution used during exposure (squirting), 

the length of time of the exposure, time 

for holding and duration of transport, postcollection treatment (changes of water etc.). 

The conversion rate of hydrogen cyanide 

(HCN) to SCN-

 appears to be limited by the 

availability of sulfur (Leduc 1984). Hence, 

both HCN and SCN-

 occur in the blood. 

The link between waterborne CN- exposure 

and plasma SCN- has been established in the 

rainbow trout (Oncorhynchus mykiss) (Raymond 

et al. 1986; Speyer and Raymond 1988; 

Heming et al. 1985; Heming and Blumhagen 

1989; Lanno and Dixon 1993, 1996a, 1996b). 

In situations of chronic cyanide exposure, 

detectable levels of SCN- were present in the 

plasma of trout after one day of exposure to 

0.01, 0.02, or 0.03 mg HCN/L and increased 

over the 20-day exposure period (Raymond et 

al. 1986). Raymond et al. (1986) found that 

there was still enough free HCN present in 

the blood to inhibit the action of cytochrome 

oxidase in the liver of rainbow trout during 

20 days of tests. Lanno and Dixon (1996a) 

also observed the accumulation of SCNin the plasma of trout exposed to 0.006 

or 0.03 mg CN-

/L over a 16-week period. 

Bioconcentration factors (SCN-

/CN-) were 

170 and 88, respectively, for the two exposure 

concentrations. 

Brown et al. (1995) used high-pressure 

liquid chromatography (HPLC) to examine 

the pharmaco-kinetics of plasma SCN- in 

44
rainbow trout exposed to 40 mg SCN-

/L. 

Using depuration rate constants (k2

) ranging 

from 0.29-0.34 day-1, a depuration half-life 

of about 4 days was estimated. Signifi cant 

levels of SCN- were detectable at 8 days and 

declined to below detection limits by 16 days. 

The determination of plasma SCN- levels may 

be a useful biomarker of cyanide exposure.

The studies by Brown, Lanno, and Dixon cited 

in the previous paragraphs generally involved 

exposing rainbow trout to low levels of either 

CN- or SCN- for long time periods (chronic 

exposures). Research is needed in which fi shes 

are pulse-dosed with higher concentrations 

of cyanide over short (acute) time periods 

(less than 90 seconds) to simulate exposures 

similar to those used during cyanide fi shing. 

The conversion rate of CN-

 to SCN- facilitated 

by the enzyme rhodanese is not simply a 

function of enzyme kinetics (Leduc 1984). 

The conversion rate appears to be limited 

by the availability of sulfur present in the 

fi sh. Likewise, the metabolism and excretion 

of SCN- from the fi sh may be related more 

to osmoregulation than to temperaturemediated enzyme kinetics. Freshwater fi sh 

have a higher blood ion concentration (hyperosmotic) in relation to the surrounding water; 

while marine fi sh have a lower blood ion 

concentration (hypo-osmotic) in relation to 

seawater. Hence, freshwater fi sh have a high 

rate of urinary excretion and marine fi sh have 

a low rate of urinary excretion; which helps 

the fi sh to maintain osmotic equilibrium with 

surrounding aquatic environments (Smith 

1982). 

Since the physiology of freshwater and 

marine fi sh is drastically different with regard 

to the maintenance of osmotic balance (Smith 

1982), it stands to reason that the regulation 

of plasma anions may also differ. It is believed 

that marine fi sh retain SCN-

 for a longer 

time period than freshwater fi sh because of 

the lower rate of urinary excretion. Hence, 

the interpretation of results from cyanide 

exposure studies conducted with freshwater 

fi sh to the toxicity and kinetics of cyanide in 

marine fi sh should be conducted with caution. 

Scientifi c studies are needed concerning the 

physiology and pharmaco-kinetics of CNand SCN-

 with marine fi sh.

The committee agreed that there was an urgent 

need for research to determine the pharmacokinetics of cyanide and its metabolites in 

MAF. Without this knowledge, it is diffi cult to 

predict whether or not various cyanide testing 

methods can be applied associated with the 

chain-of-custody from collectors to export 

companies situated the Philippines, Indonesia, 

and Vietnam to importers, wholesalers, and 

retailers situated in importing countries like 

the U.S.A. 

Field Test In Exporting Countries

In response to threats from terrorism, a 

number of fi eld tests for cyanide have 

been developed and evaluated for the USEnvironmental Protection Agency (US-EPA) 

by Battelle. Most of these are either lowcost colorimetric kits (dependent on color 

changes) or based on ion selective electrodes 

(ISE) linked to portable ISE meters. These 

kits mostly detect cyanide in water. One 

colorimetric test kit discussed by Dr. Rubec 

is sold by Industrial Test Systems (ITS) and 

is called the Cyanide ReagentStripTM Test Kit 

(Batelle 2005). Thermo-Fisher markets the 

Thermo Orion 9606 portable ISE equipment. 

There are other companies with similar 

products that also were evaluated by Batelle 

for US-EPA over the past fi ve years.

Mr. Gil Adora (Deputy Director of BFAR 

within the Philippines Department of 

Agriculture) and Mr. Agus Dermawan (Deputy 

Director of the Division of Small Islands 

45
and Marine Parks within the Indonesian 

Ministry of Marine Affairs and Fisheries) 

both expressed, in their presentations at the 

Cyanide Detection Workshop, the need for 

a rapid, easy to use fi eld test to support law 

enforcement efforts. The available kits and/

or meters generally measure CN-

 in solution. 

These kits may be useful to detect cyanide ion 

in solution (such as in seized cyanide squirt 

bottles) or to detect cyanide after suspected 

tablets seized from collectors/fi shers are 

dissolved in water (fresh or saltwater). The 

fi shers/collectors use either sodium cyanide 

(NaCN) or potassium cyanide (KCN) for 

cyanide fi shing. Sodium cyanide is mostly used 

to capture fi sh by fi shers in the Philippines; 

while potassium cyanide is more commonly 

employed for cyanide fi shing in Indonesia. 

The committee agreed that portable test kits 

designed for measuring cyanide in solution are 

unlikely to be effective in detecting cyanide 

bound in the tissues of MAF.

Soudararajan Procedure

Another suggested method for testing fi sh 

samples for the presence of cyanide was 

developed by Dr. Rengarajan Soundararajan 

in 1990 (Rubec and Soundararajan 1991). The 

method involves the extraction of cyanide 

from blended fi sh tissues using concentrated 

sodium hydroxide (NaOH). The method is 

appealing since it is relatively simple and quick 

to conduct. The cyanide ion is released from 

the tissues into sodium hydroxide (NaOH). 

The high pH (pH 12-13) of the solution 

prevents the cyanide from being lost to the 

atmosphere. Cyanide concentrations can then 

be measured with an ion-selective electrode 

(ISE). 

Five fi sh species obtained from Indonesia tested 

in the U.S.A. had cyanide ion concentrations 

ranging from 5.8 to 23 mg/kg (ppm) (Rubec 

and Soundararajan 1991). A Clown Triggerfi sh 

obtained from the Philippines exhibited a 

cyanide ion measurement of 1120 mg/kg. No 

cyanide was detected in two Flame Angelfi sh 

obtained from the Marshall Islands, for two 

French Angelfi sh from the Caribbean, and 

two species of surgeonfi sh obtained from 

Hawaii. Hence, cyanide was detected in fi sh 

from countries known to have collectors 

using cyanide and no cyanide was detected in 

fi sh obtained from countries where cyanide is 

not used for collecting marine aquarium fi sh.

The International Marinelife Alliance (IMA) 

evaluated the technique in 1991, but abandoned 

it after it was found to give anomalously high 

cyanide readings. It was suspected that the 

anomalously high readings found by the IMA 

might have occurred because of false-positive 

readings caused by sulfi de interference with 

the ISE electrode.

Research by Aquarium Systems was conducted 

with a small grant obtained by IMA from the 

Columbus Zoo (Frakes and Studt 1996). The 

basic procedure was to expose the fi sh to 

known concentration of cyanide ion, kill the 

fi sh, then puree the samples in 5 Molar (M) 

NaOH. The NaOH volume was calculated 

to yield a 10% by weight fi sh slurry. This 

slurry was allowed to settle and a clear aliquot 

was diluted with distilled water to produce 

another 10% by weight dilution. Cyanide ion 

(CN-

) concentrations in the fi nal solution, 1% 

fi sh tissue, and approximately 0.5M-NaOH 

were measured in millivolts (mV) recorded 

with an Orion CN-

 ISE linked to an Orion 

ISE meter. 

These readings were compared with a semilog plot (calibration) produced with known 

cyanide concentrations of 0.1, 1.0, and 10 

mg/L levels with mV readings recorded 

as each level (Frakes and Studt 1996). Lead 

carbonate was added to the supernatant 

solution to precipitate sulfi des from solution. 

The fi rst trial compared readings with Atlantic 

46
Blennies, which were net-caught and exposed 

to cyanide. Both the test and the control 

fi sh (not exposed to cyanide) were found to 

exhibit cyanide levels of about 300 mg/L 

(ppm). Hence, the addition of lead carbonate 

to the solution prior to ISE testing did not 

eliminate the anomalously high readings.

 A second experiment evaluated whether 

the high readings might be due to iodide 

interference with the ISE. Again, the 

experiment was inconclusive. In a third 

experiment, test fi sh (4 Caribbean Blue 

Chromis, 1 Atlantic Wrasse, and 3 Goldfi sh) 

were exposed to cyanide concentrations 

ranging from 1.3 to 3.9 ppm CN-

 for times 

ranging from one minute (for the 4 chromis) 

to 14 minutes (for a goldfi sh). Readings for 

control fi sh (1 wrasse and 2 goldfi sh) not 

exposed to cyanide produced higher mV 

readings than fi sh exposed to cyanide. 

Recent analyses by Ms. Benita Manipula 

using the Soundararajan procedure and 

cyanide measurements obtained using an 

Industrial Test Systems (ITS) colorimetric 

Cyanide Reagent StripTM (Cyanide Test Kit 

484003) have also experienced diffi culty in 

obtaining reliable measurements of cyanide 

concentrations in comparison to known 

concentrations of cyanide ion in sodium 

hydroxide solution (lacking slurry). The 

problem does not appear to be related to 

the reliability of the ITS cyanide test strips, 

since they have been demonstrated to be 

sensitive (down to 0.02 mg/L) and reliable 

for measuring cyanide ion concentrations in 

water (Battelle 2005).

The most likely explanation for these results 

is that organics in the digested tissue solutions 

containing sodium hydroxide produced the 

anomalous readings both with the ISE and 

the ITS test strips. Dr. Frant suggested that 

these could be volatile mercaptans from 

sulfur-containing proteins. Further research 

is needed to see whether it is possible to 

measure cyanide ion concentrations in 

solutions obtained from marine fi sh using 

the Soundararajan tissue digestion method. It 

does not appear likely that the Soundararajan 

procedure can be applied as a fi eld test (Rubec 

and Soundararajan 1991, Rubec and Manipula 

2008). 

Cyantesmo Screening Test 

The workshop committee agreed that it 

might be possible to digest fi sh samples in 

sulfuric acid in a closed container to release 

hydrogen cyanide (HCN) from the digested 

sample into the atmosphere of the container. 

Test strips mounted in the container (such as 

near the lid of a jar) might be able to provide 

a colorimetric indication for the presence of 

cyanide ion. This approach would need further 

research and fi eld evaluations. It is likely to 

only work near the point of collection in the 

exporting countries; where cyanide ion is 

most likely to be present in the fi sh at higher 

concentrations.

Actually, there are several testing protocols 

based on this idea. The American Society 

of Testing and Materials (ASTM) published 

a screening method (D-5049) for screening 

cyanides in waste (ASTM 1990). The protocol 

involves adding 5 grams of organic sample 

to a sealed vial also containing concentrated 

sulfuric acid. Hydrogen cyanide released 

from the sample causes a color change 

with Cyantesmo paper suspended over the 

sample. 

A similar screening protocol (Figure 1) is used 

by the U.S. Food and Drug Administration 

(FDA) to analyze various foodstuffs for the 

presence of cyanide-including meat and 

vegetables (Flurer et al. 2005). It has been 

used to detect cyanide with tuna samples 

(both frozen and canned) spiked with cyanide. 

47
The Cyantesmo paper changes color from 

light green to dark blue depending on the 

concentration of hydrogen cyanide liberated.

Several papers have been published using 

Cyantesmo paper. Flinger et al. (1992) used 

it as a screening tool to detect the presence 

of cyanide liberated from blood samples. 

Relia et al. (2004) evaluated the method 

with concentrations of cyanide ion ranging 

from 0.25 to 30 mg/L (ppm). The test strips 

demonstrated incrementally increasing deep 

blue color changes over a progressively longer 

portion of the test strip in less than fi ve minutes 

for each concentration of cyanide including 

1, 3, 10, and 30 mg/L. The concentrations of 

0.25, 0.5, and 0.75 mg/L required more than 

two hours to begin demonstration of a color 

change. 

It is recommended that the FDA protocol be 

evaluated as a screening tool for the detection 

of cyanide in fi sh tissue samples near the 

point of collection in exporting countries. 

The procedure will require the use of regional 

testing laboratories. It has the advantage that 

many samples can be analyzed at low cost. 

BioSensors

Mak et al. (2005a) presented a summary of 

various papers that have developed biosensors 

for detecting cyanide ion. Most of the methods 

are dependent on measuring chemical reaction 

products based on enzymatic reactions (e.g., 

formate production from action of cyanide 

hydratase, cytochrome oxidase inhibition, 

peroxidase inhibition, rhodanese and sulphite 

oxidase reactions). There is also a microbial 

sensor, and the measurement of oxygen 

uptake by bacteria. There are efforts being 

made to link the enzymatic reactions to 

potentiometers to measure the enzymatic 

reaction as changes in electrical potentials. 

The idea is to create a microprobe (containing 

the enzymes) that would be inserted into the 

blood to give a reading based on a change in 

Figure 1. Sealed test tubes used by the U.S. Food and Drug Administration to detect the presence of cyanide 

in foodstuffs using Cyantesmo paper suspended above an acidifi ed sample. Image provided by Dr. Fred Fricke, 

USFDA.

48
electrical potential (similar to an ISE reading). 

Literature searches by Dr. Rubec and Dr. 

Frant indicate that no company is presently 

marketing a microprobe based on biosensors. 

A potentiometric biosensor is reported to be 

under development involving the use of an 

EIS (Electrode-Insulator-Semiconducter) as 

a microprobe (Kuegsen et al. 2004, Turek at 

al. 2007). 

Research presented by Mak et al. (2005c) 

suggests that CN-

 in solution associated 

with blended tissue samples (Figure 2, page 

133) declined to zero in about one hour. 

This may be because the cyanide, present in 

solution was taken up by the tissues in the 

homogenate. It should be noted that the 

above experiment by Mak et al. (2005c) does 

not represent a depuration rate, since they did 

not demonstrate that cyanide was excreted 

from the fi sh in less than one hour.

 A study by Bellwood (1981) using radioactive 

tracers indicated that the cyanide in solution 

was taken up across the gills and the stomach 

of the fi sh. The cyanide in the blood was 

rapidly taken up by the fi sh’s organs; including 

the liver, kidney, heart, spleen, and brain. 

Hence, the CN-

 may only be detectable in the 

blood of marine fi sh for a few hours using 

any of the biosensor methods described 

above. This needs further study. Radioactive 

tracers may provide a rapid method for 

determining depuration rates of cyanide and 

its metabolites in MAF.

Use of ISE Electrode and ISE Meter

Provided that the half life of cyanide ion in 

the fi sh is not too short, it should be possible 

to measure cyanide ion extracted from 

digested fi sh tissue samples in a laboratory 

using the ASTM ISE method (the method 

used by the BFAR/IMA labs) from 5 to 14 or 

more days after the fi sh were captured using 

sodium cyanide or potassium cyanide. The 

fact that the International Marinelife Alliance 

(IMA) was able to do this, with over 48,000 

specimen samples in six BFAR/IMA CDT 

laboratories in the Philippines between 1993 

and 2001 (Rubec et al. 2003), suggests that 

the half-life in marine fi sh is longer than the 

half-life of CN-

 reported for humans. 

First it should be noted that the IMA did not 

develop the ISE method for determination 

of cyanide ion concentrations in solution. 

The IMA adopted the Standard Operating 

Procedure (SOP) published in “Standard 

Methods For The Examination of Water and 

Wastewater”, 18th Edition, published by 

the American Public Health Association 

(APHA), American Water Works Association 

(AWWA), and the Water Pollution Control 

Federation (WPCF) (APHA-AWWA-WPCF 

1992) and in the “1997Annual Book Of ASTM 

Standards” Vol. 11.02, (D2036-91) published 

by the American Society of Testing and 

Materials (ASTM 1997). It is also the method 

used by the US-Environmental Protection 

Agency (US-EPA 1983). It was developed by 

the ASTM in the early 1980s and has been 

repeatedly published by these agencies. 

The method uses refl ux-distillation apparatus 

into which chemicals are added to deal with 

interfering substances (Figure 2). Sulfamic 

acid is added to reduce interference from 

nitrates and/or nitrites. Magnesium chloride 

is added as a catalyst. Concentrated sulfuric 

acid is added and the solution (acid, other 

chemicals, and blended tissue sample) heated 

(using a heating mantle under the fl ask) 

for about an hour to digest the fi sh tissue. 

Hydrogen cyanide (HCN) gas passes through 

the refl ux condenser and CN-

 is captured in 

an absorption tube containing concentrated 

sodium hydroxide solution (Manipula et 

al. 2001b). Lead carbonate added to the 

absorption tube is used to eliminate sulfi de 

interference. The CN- concentration is then 

49
measured using an ISE (Figure 3) linked to a 

pH/ISE meter (Manipula et al. 2001c).

The ISE method for determination of CNconcentrations has been evaluated through 

two separate round-robin comparisons by 

the ASTM and by the APHA (ASTM 1987, 

1997, APHA-AWWA-WPCF 1998). Both the 

ASTM round robin and the APHA round 

robin evaluations were based on six operators 

in fi ve laboratories (ASTM 1987, 1997). 

But, the concentrations tested with water 

samples and the sample sizes evaluated were 

different. 

With the ASTM (1987) round-robin 

evaluation, samples of CN-

 in various water 

matrices were sent as unknowns to fi ve 

laboratories for analyses. The testing done 

on samples with a known concentration 

of 0.03 mg/L were determined to have an 

average concentration of 0.029 ppm (ASTM 

1987, 1997). The limit of detection must 

be well below this level. In fact, Elsholz 

et.al. (1990) found with a fi ve minute time 

response that a CN- concentration of 0.003 

mg/L (ppm) was detectable for two different 

cyanide ISEs. Hence, CN- is detectable below 

0.03 mg/L, but the concentration may be 

diffi cult to determine; since the calibration 

on a semi-log plot deviates from a straightline relationship, determined between known 

CN- concentrations and the electric potential 

readings obtained using the ISE apparatus.

There is no doubt by any of the organizations 

(APHA, ASTM, US-EPA, USGS) that have 

endorsed this method, that the ASTM ISE 

method is a reliable method that can be 

used to determine CN- concentrations in 

solution (Ghosh et al. 2006). There are some 

differences in the methods that have been 

separately published by these organizations, 

but they are minor. 

The methods published by these agencies 

involve an acid digestion and distillation of 

samples, with addition of chemicals to help 

remove interfering substances (Gosh et al. 

2006). Cyanide ion can then be measured in 

solution using a variety of methods (including 

but not limited to colorimetric, ISE, HPLC, 

and mass spectrometric methods). 

The International Marinelife Alliance (IMA) 

conducted cyanide detection testing (CDT) 

Figure 2. Distillation apparatus used by IMA chemists 

to digest tissue samples in order to liberate hydrogen 

cyanide gas, that is then captured as cyanide ion in an 

absorber tube containing sodium hydroxide.

Figure 3. Ion Selective Electrodes (ISE) linked to a 

Thermo-Orion pH/ISE meter used by the IMA to 

measure cyanide ion concentrations in sodium hydroxide after refl ux distillation.

50
in the Philippines under contract with BFAR 

from 1993 to 2001. The ISE method, utilized 

by the IMA has been criticized by several 

groups that attempted to repeat the CDT 

procedures used by IMA (Holthus 1999, Mak 

2003, Mak et al. 2005b). This led some people 

to question whether the ISE method utilized 

by IMA and presently by BFAR chemists is 

reliable.

In 1999, the Marine Aquarium Council 

(MAC) convened a panel of eight scientists to 

review the SOP manual utilized by the IMA 

(Manipula et al. 1995). The SOP is essentially 

the ASTM ISE method discussed above. 

The panel members noted that the SOP did 

not contain quality assurance-quality control 

(QA/QC) procedures (Holthus 1999). The 

IMA submitted a formal response to this 

report (IMA-Philippines 1999). The IMA 

noted that QA/QC procedures were being 

routinely applied by the six BFAR/IMA 

laboratories and agreed to incorporate them 

into a revised SOP. This was done and four 

new SOP manuals were produced (Alban et al. 

2001, Manipula et al. 2001a, 2001b, 2001c).

The main disagreement by IMA (IMAPhilippines 1999) with the MAC report 

(Holthus 1999) pertains to analyses conducted 

by an unspecifi ed chemist contracted by MAC 

to repeat the ISE method. The consultant 

changed the test apparatus (substituted a 50 

ml mini-distillation fl ask) and made other 

changes to the procedure. The main criticism 

by IMA was that the consultant overheated 

the distillation fl ask and infused air into the 

refl ux condenser at too high a rate resulting in 

low percent recoveries of cyanide ion in fi ve 

experiments involving marine aquarium fi sh. 

Tables 2, 3, 4, and 5 indicate percent recoveries 

of cyanide ion ranging from 31-77% (Holthus 

1999). If the ASTM (1997) procedure was 

correctly conducted one should expect 

recoveries of CN-

 near 100%. The published 

procedure states “Equivalent apparatus is 

acceptable provided cyanide recoveries of 

100 “ 4% are documented” (ASTM 1997). 

Hence, the low percent recoveries of CNafter the digestion and distillation of cyanide 

spiked tissue samples reported (Holthus 1999) 

brings into serious question the validity of 

the consultant’s experimental results (IMAPhilippines 1999). The IMA noted that it 

routinely had percent recoveries of cyanide 

greater than 90%. Hence, the low percent 

recoveries experienced by the consultant 

were not a problem for IMA chemists who 

correctly applied the ISE methods described 

in the SOP.

The other main concern (IMA-Philippines 

1999) was that the consultant claimed the 

levels of cyanide measured with the ISE 

were below detectable limits (BDL) (Holthus 

1999). In three experiments (Tables 3, 4 

and 5) cyanide levels recovered from spiked 

samples ranged from 0.0001 to 0.0005 mg/g. 

The consultant claimed that the ISE method 

was invalid because the test results were BDL. 

The IMA pointed out that the consultant was 

wrong because the concentrations should have 

been reported in parts per million wet weight, 

which is expressed as mg/kg. The results 

ranged from 0.1 to 0.5 mg/kg (ppm). Hence, 

the consultant’s results were well within the 

range of detection by the ISE apparatus and 

were not below detectable limits. 

The MAC also contracted Dr. Reinhard 

Renneberg at the Hong Kong University of 

Science and Technology to review cyanide 

detection methods and develop a new cyanide 

detection method. A graduate student in his 

laboratory, Karen Mak attempted to repeat 

the ISE method previously used by the IMA 

(Mak 2003). Unfortunately, the manual she 

followed was not the correct SOP manual 

utilized by the BFAR/IMA laboratories 

(Manipula 1995). She used a handout (IMA51
Philippines 2000) obtained at a Live Food 

Fish Trade meeting held in Hong Kong in 

2000 that only partially described the actual 

SOP used by IMA. No attempt was made by 

either Ms. Mak or Dr. Renneberg to contact 

IMA to obtain the correct SOP manual. 

The assertion by Mak et al (2005b) that 

“The refl ux distillation method together 

with an ISE was not sensitive enough for 

the determination of cyanide traces in postcyanide exposed fi sh” is incorrect (Rubec 

2007). Since, this was associated with a 

discussion of the methods used by the IMA, 

the implication was that the IMA could not 

reliably measure cyanide concentrations 

below 0.26 mg/L. However, Mak (2003) did 

not use the same ISE equipment used by the 

IMA. She used a Pheonix ISE electrode (Cat 

No. CN01503); connected to a Jenway pH/

mV meter (model 3305); which has a range 

of detection on a linear semi-log plot of 

0.26 to 260 ppm (mg/L). The IMA used a 

Thermo-Orion ISE electrode (#9406BN) 

that can reliably detect cyanide concentrations 

down to 0.03 mg/L on a linear semi-log plot 

(ASTM 1997). Likewise, the pH/ISE meter 

used by Mak (2003) differs from that used by 

the BFAR/IMA laboratories (Thermo-Orion 

Model 920A) (Manipula 1995, Manipula et 

al.2001c). 

The Thermo Orion ISE equipment (ISE 

electrodes and meter) is capable of detecting 

cyanide ion in solution with a straight-line 

calibration on a semi-log plot down to 0.02 

mg/L (2 ppb) (Frant et al. 1972; Orion 

Research Inc. 1975, 1997; Sekerka and 

Lechner 1976). This was routinely done with 

daily calibrations conducted by the IMA 

laboratories using the Thermo Orion ISE 

equipment (Manipula et al. 2001c). This is 

similar to round-robin fi ndings by the ASTM 

(1987). 

Mak et al. (2005b) reported that the lower 

range of detection for cyanide ion using a 

colorimetric method was 0.026 ppm. Hence, 

they claimed that the colorimetric method 

had a higher sensitivity than the ISE method 

(actually both have about the same lower limit 

of detection). They claimed that the cyanide 

refl ux distillation method together with the ISE 

was not sensitive enough for the determination 

of traces in post-cyanide exposed fi sh. A series 

of QA/QC experiments were conducted that 

are irrelevant considering that the wrong ISE 

equipment was utilized (Mak 2003). Mak et al. 

(2005b) mistakenly concluded: “The cyanide 

distillation method combined with the ISE 

described in the SOP manual and employed 

by the IMA laboratories requires considerable 

modifi cation and elaboration. A reliable and 

ultra sensitive system for cyanide detection 

in fi sh is urgently needed.” It should also be 

noted that the lower limit of detection for 

the biosensor method that Mak et al. (2005c) 

developed is 0.0286 ppm (mg/L), which is 

about the same as the Thermo Orion ISE 

method on a linear calibration (Sekerka and 

Lechner 1976). Hence, it is not more sensitive 

than the ISE method (ASTM 1997).

The present authors, including Dr. Kobelski 

and Dr. Logue, believe that the ISE method 

associated with the SOP manual used by the 

IMA (Manipula 1995) and by the various 

agencies that also use the method (including 

APHA, ASTM, US-EPA and BFAR) is 

scientifi cally reliable, although this has not been 

demonstrated with fi sh tissue in controlled 

experiments. This is also the opinion of Dr. 

Ellen Gonter who helped develop the ISE 

method for ASTM, Dr. Martin Frant of 

Thermo Orion who developed the cyanide 

ISE electrode, and Dr. George Dixon, an 

expert concerning cyanide physiology in fi sh 

and Vice-Dean of Research at the University 

of Waterloo, Canada (all three provided letters 

52
endorsing the IMA’s use of the ISE method 

after the fl awed MAC review).

There was no need to develop a new method 

(MAC 2004, Mak 2003, Mak et al. 2005b), since 

the SOP using ISE presently being applied 

by BFAR (Manipula 1995, Manipula et al. 

2001b, 2001c) is reliable and has international 

acceptance. Any new method would need to 

go through extensive evaluations and roundrobin comparisons before it could be accepted 

by the scientifi c community and by agencies 

regulating the marine aquarium trade.

Dr. Kobelski near the end of the CDT 

Workshop suggested that round-robin 

comparisons needed to be conducted 

involving the ISE method and that 

comparisons needed to be made with an 

independent cyanide detection method. He 

suggested the use Gas Chromatography 

linked to Mass Spectrometric analyses (GCMS), since this is the method used by the CDC 

(Kage et al.1996, Dumas et al. 2005, Murphy 

et al. 2006) and has recently been accepted 

by the US-EPA for the analysis of cyanide in 

drinking water. A GC-MS apparatus used in 

Texas to measure cyanide is depicted (Figure 

4). This would require that fi sh be dosed with 

cyanide and frozen samples be sent to two or 

three separate laboratories. He suggested that 

a statistically signifi cant number of samples 

should be analyzed by the ISE method. This 

could be time consuming and expensive, if 

done by laboratories in the U.S.A. 

According to Dr. Kobeski there are 46 

laboratories in the CDC network, which 

are qualifi ed for trace analysis of cyanide in 

clinical samples. The Michigan Department of 

Community Health has been conducting fi sh 

exposure studies to detect cyanide using GCMS. Preliminary results from the Michigan 

laboratory found a background level of 

cyanide in freshwater fi sh in the 0.01 mg/L 

(10 ppb) range. The Michigan laboratory 

has cross-validated the headspace GC-MS 

method with the distillation ISE method for 

the US-EPA using cyanide in solution.

Dr. Logue suggested that there may be 

background levels of cyanide in MAF. It is 

possible that the cyanide concentrations 

detected in the fi sh tested by the BFAR/

IMA laboratories were background levels of 

cyanide rather than cyanide resulting from 

cyanide fi shing. Ms. Manipula (Chief Chemist 

for the BFAR/IMA laboratories) pointed out 

that the IMA had tested MAF caught with 

nets and found no detectable level of CN- 

(zero) present in the fi sh. Hence, she believes 

that the cyanide being detected resulted from 

cyanide fi shing. Some testing needs to be done 

to verify that MAF do not have background 

levels of cyanide, or that the background 

concentration is below the detectable limit 

(BDL) determined from the linear calibration 

for the ISE method.

Dr. Kobelski agreed that the ISE method was 

probably reliable (Email: February 21, 2008). 

“I have repeatedly said that I believe that the 

ISE method is probably valid – my failure to 

Figure 4. A gas chromatography-mass spectrometry 

(GC-MS) apparatus used to determine cyanide concentrations. Image provided by Dr. David Klein, Texas 

Department of State Health Services.

53
review all the information is one reason I say 

probably – I fi nd no fl aws with the analysis 

technique. I do have concerns about the use 

of sulfuric acid possibly oxidizing thiocyanate 

to cyanide based on some work I did 20+ 

years ago. This is not likely to be an issue with 

drinking water but with biological samples, it 

is a concern.” 

There is almost nothing in the published 

scientifi c literature concerning the use of 

the ISE method for detection of cyanide 

associated with the digestion and distillation 

of fi sh tissue samples (Dzombak et al. 

2006). Consequently, Dr. Kobelski noted the 

following concerns: 1) the possible oxidation 

of the cyanide by sulfuric acid in the digestion 

fl ask; 2) the lack of demonstration of 

adequate recovery in controlled experiments 

using spiked tissue samples; 3) that unexposed 

MAF might have background levels; 4) that 

the kinetics of cyanide might be too fast for 

cyanide to be detectable at points of export 

or import. Since there is no published data 

available, he proposed that research should be 

conducted to evaluate these questions.

Need for a Field Test For Ports of Entry 

In USA

The U.S. Fish and Wildlife Service (USFWS) 

agents present at the Cyanide Detection 

Workshop expressed the need for a reliable, 

rapid, easy to use fi eld test that could be used 

to detect cyanide or its byproducts at US ports 

of entry such as Los Angeles, San Francisco, 

Miami, and New York (where most marine 

ornamental fi shes are imported). There are a 

number of low-cost test kits that can measure 

CN-

 either using colorimetric methods or 

using ion selective electrodes (ISE) linked to 

portable ISE meters. 

The authors believe that because the CN-

 is 

metabolized to thiocyanate anion (by the 

enzyme rhodanese); it is unlikely that cyanide 

ion would be detectable using the ASTM ISE 

method in importing countries like the USA, 

because of the long time delay before fi sh 

reach U.S. points of entry (by air). However, 

Dr. Kobelski felt that if the kinetics are slow 

enough for cyanide to be retained in the fi sh 

until the time of export, that it might also be 

possible to detect the presence of cyanide 

using the ISE method at points of import to 

the U.S.A. Theoretically is should be possible 

to measure SCN-

 in MAF because it would 

be retained for a longer time period. The 

advantage of measuring thiocyanate is that 

appreciable concentrations of SCN-

 may 

be found following exposure and it has a 

longer half-life than CN-

 (Logue and Hinkens 

2008).

Measuring Thiocyanate in Blood

Two research grants were obtained by Dr. 

Roman Lanno at Ohio State University 

(OSU) to determine the feasibility of 

measuring SCN-

 in the blood of MAF after 

they had been imported into the U.S.A. 

(Lanno et al. 2002a, Lanno et al. 2002b). The 

objective of the proposed research (Lanno 

et al. 2002a, 2002b) was to assess whether 

plasma thiocyanate (SCN-) could be used as 

a biomarker of cyanide exposure in marine 

aquarium fi shes captured using the cyanide 

squirt bottle technique. The objective of 

the study was to be met by conducting three 

experiments: i) Validate and refi ne an existing 

high pressure liquid chromatography (HPLC) 

technique for the determination of SCNin the plasma of marine aquarium fi sh, ii) 

Determine the pharmacokinetics of SCN- in 

a marine aquarium fi sh species exposed to 

sublethal levels of waterborne cyanide, and; 

iii) Determine the toxicokinetics of SCN-

 in 

the plasma of a marine aquarium fi sh species 

exposed to a pulse dose of cyanide that 

simulated exposure during actual collection 

on a coral reef. 

54
The intended products from the research at 

OSU were to include: i) a detailed manual 

documenting methods for monitoring SCNin fi sh tissues that can be used to enforce 

regulations in importing and exporting 

countries, ii) Scientifi c publications describing 

the pharmaco-kinetics (uptake and depuration) 

of SCN- in MAF, outlining the bounds and 

limitations of the technique.

A graduate student (Julie Koontz) commenced 

the research in 2003 under the direction of 

Dr. Lanno. She was able to measure SCNconcentrations in the blood of several MAF 

species (Pseudanthias pleurotaenia and 

Pseudanthias squamipinnis) using an HPLC 

method developed by Chen and Yang (1996). 

This HPLC method is capable of detecting 

thiocyanate in blood plasma at low levels (0.5 

– 1 nmol mL-1). It requires derivatization 

of thiocyanate anion with 3-bromomethyl7-methoxy-1,4-benzoxazin-1-one, then 

separation by a reversed-phase C18 column. 

Quantifi cation is by HPLC with fl uorimetric 

detection. 

Koontz (pers. comm. 2005) experienced 

diffi culty in obtaining enough blood from 

the MAF, because of their small size. The 

original method, as described in Chen and 

Yang (1996), requires 0.5 mL of blood 

plasma. It may be possible to use less plasma 

by evaporating the entire 10 mL plasma eluate 

(rather than a 1.0 mL aliquot) to increase the 

quantity of thiocyanate ion present. The same 

change could also increase the sensitivity of 

the test for the 0.5 mL plasma volume and 

possibly extend the period after exposure that 

thiocyanate is detectable. 

For personal reasons, Ms. Koontz decided to 

drop out of the graduate research program 

at OSU. Hence, the pharmaco-kinetic 

research of SCN-

 dynamics in MAF was not 

completed.

Other Methods for Measuring 

Thiocyanate

There are colorimetric tests for cyanide ion 

and the thiocyanate anion that could be 

applied (APHA-AWWA-WPCF 1992, ASTM 

1997). It might be possible to measure the 

presence of SCN-

 in blood samples taken 

from fi sh using colorimetric methods. A spot 

test for these ions is published by the APHAAWWA-WPCF (1998). Provided there are 

no interfering substances in the sample, 

the presence of CN- can be determined by 

colorimetric methods (e.g., chloramine T, 

pyridine barbituric acid, phenolphthalein 

reagents). The CN-

 can be masked using 

formaldehyde and the sample retested. This 

makes the spot test specifi c for SCN-. Whether 

this approach would work with blood samples 

is unknown. More research is necessary.

It may be possible to measure SCN- levels in 

MAF after their tissues are dissolved in nitric 

acid and sulfuric acid. More acidic solutions 

are required to liberate SCN- from tissues 

than are required to liberate CN-. There 

is an ISE sold by Thermo-Fisher that can 

measure thiocyanate anion concentrations, 

after the tissues are digested. There are many 

colorimetric methods for SCN- and it is likely 

that HPLC or ion chromatography could 

be used. CDC has a liquid chromatographymass spectrometric (LC-MS/MS) method 

for the analysis of SCN-

 in both blood and 

urine for human clinical samples. It is unlikely 

that the thiocyanate ion can be distilled (like 

the ASTM ISE method used for detection 

of cyanide ion). More research is needed to 

determine whether a test for thiocyanate in 

MAF is feasible. 

Dr. Kobelski suggested that measuring 

thiocyanate in the blood of MAF was not 

feasible on a routine basis due to the low 

volume of blood in small ornamental fi sh. 

Whatever test is applied in the USA will 

55
probably start with fi sh tissue or tissue 

homogenates. 

Dr. Logue pointed out, based on mammalian 

research, that thiocyanate can be produced 

from several metabolic pathways (Logue and 

Hinkens 2008). “However, thiocyanate is 

naturally found in biological fl uids, and while 

this is a condition of all cyanide metabolites, 

thiocyanate levels are normally quite high 

and can be inconsistent. Large variation in 

background thiocyanate concentrations (in 

mammals) make it diffi cult to determine low 

level cyanide exposure....Large and variable 

concentrations (of thiocyanate) may indicate 

that thiocyanate is involved in a number of 

biological processes in addition to cyanide 

metabolism.” Hence, even if thiocyanate 

could be detected in MAF being imported, 

it might not be possible to assert in court 

that the presence of thiocyanate detected 

in the fi sh was absolute proof that the fi sh 

were caught using cyanide in the country of 

origin. 

Based on the previous discussion, it is unlikely 

that a rapid fi eld test for thiocyanate can be 

applied. Analyses of frozen samples sent to a 

laboratory (such as the USFWS laboratory in 

Oregon) will probably be necessary. From a 

cost perspective, measuring thiocyanate may 

be much less costly than the ATCA method 

(discussed below).

ATCA Test

The committee agreed that from a theoretical 

standpoint is may be better to use a biomarker 

such as ATCA (Lindquist et al. 2005, Logue et 

al. 2005, Logue et al. 2007, Logue and Hinkens 

2008), especially if the US-based test results 

will be used for prosecution of those in the 

aquarium trade. “A minor metabolic pathway 

is the conversion of cyanide ion to 2-amino2thiazoline-4-carboxylic acid (ATCA). 

Another by product (tautameric form) is 

2-iminothiazolidine-4-carboxylic acid (ITCA). 

The production of ATCA may predominate 

when sulfur donors become depleted or in 

tissues where rhodanese is sparse. ... ATCA 

may be used as an alternative to thiocyanate 

for determination of cyanide exposure. An 

advantage of using ATCA is that it is stable 

in biological samples for months at freezing 

and ambient temperatures and therefore, may 

be a lasting signature for cyanide exposure. 

However, relatively few techniques have been 

described to analyze ATCA from biological 

matrices and relatively few studies have 

been conducted to evaluate the relationship 

between ATCA and cyanide exposure” (Logue 

and Hinkens 2008). Nothing is known about 

the pharmaco-kinetics of ATCA. A lot of 

research is needed before it could be applied 

to support law enforcement. 

Since, Dr. Logue has done research on ATCA 

he may have the equipment. If the USFWS 

laboratory in Oregon was to adopt this test 

method, they would need to purchase the 

equipment. Lundquist et al. (1995) measured 

ATCA in urine using HPLC. Logue et al. 

(2005) measured ATCA in blood and urine 

using GC-MS. It is not clear how ATCA 

would be liberated from fi sh tissues prior to 

analysis. According to Dr. Logue (pers. comm. 

2008) the cost of the GC-MS apparatus to 

measure ATCA with peripheral equipment is 

about $125,000. The cost of the equipment 

and its technical complexity most probably 

precludes its use for measuring ATCA in 

exporting countries. 

Validation Studies

Dr. Rubec questioned the need for additional 

studies to evaluate the ASTM ISE methodology. 

There have already been several round-robin 

studies by the American Society of Testing and 

Materials (ASTM 1987, summarized ASTM 

1997) and by the American Public Health 

Association (summarized APHA-AWWA56
WPCF 1992, 1998). Both of these studies 

have included at least 5 separate laboratories 

and 6 operators but were performed only on 

aqueous samples, not masticated fi sh tissue. 

The concerns about QA/QC, precision, and 

bias were addressed. The IMA also conducted 

round-robin comparisons between the six 

laboratories it ran under contract to BFAR 

(IMA-Philippines 1999, Rubec et al. 2003). If 

round-robin testing needs to be conducted, it 

should be done to validate a U.S.-based test 

for either thiocyanate or the ATCA method. 

It is unlikely that the ASTM ISE method will 

be applied in the USA. It looks like it will be 

used in both the Philippines and in Indonesia. 

An accreditation system tied to CDT analyses 

from export facilities may give the U.S.A. the 

means to prove intent. If fi sh arrive at an 

import facility along with an Accreditation 

Certifi cate, the importers would have to admit 

that they knew the fi sh were accredited as 

being cyanide-free. If a test such as the ATCA 

proves otherwise, there may be grounds 

for prosecution of the importer under the 

Lacey Act. Congressional legislation similar 

to that proposed by Congressman Ed Case 

of Hawaii (House Resolution 4928 for 108th

Congress in 2004) may be necessary to allow 

law enforcement offi cials (from USFWS 

and/or NOAA) to prosecute importers who 

knowingly import fi sh that are proven to 

contain cyanide. The test that would need to 

hold up in court would not be the ASTM ISE 

method. It would be either the thiocyanate 

test or the ATCA test conducted in the U.S.A. 

U.S. offi cials need to investigate the legal 

ramifi cations to see whether this is feasible.

Funding

The IMA conducted CDT in six laboratories 

in the Philippines under contract to BFAR for 

about $100,000 per year. The same testing in 

the U.S.A. would be at least fi ve times more 

expensive based on the disparity of salaries 

between the two countries. 

The Philippines, Indonesia, and Vietnam 

need long-term funding to establish and 

maintain CDT networks in each country. 

Unlike the U.S.A., the CDT laboratories in he 

exporting countries should be used primarily 

as a deterrent to cyanide fi shing. Cyanide 

testing should be conducted regionally using 

the cyanide screening method, so that local 

governments or municipalities can better 

manage their fi sheries. When samples are 

found positive using the screening method, 

frozen specimens would be shipped to CDT 

laboratories established near international 

airports. The CDT laboratories using the ISE 

method need to be tied to a licensing system 

for fi shers and MAF collectors, middlemen, 

and exporters. If fi sh coming from collectors 

or fi shers situated in municipalities, or from 

exporters situated near points of export, are 

found to have cyanide present more than 

3 or 4 times, their licenses can be revoked. 

Prosecution, being expensive, should only 

be used as a last resort. Prosecutions should 

probably only be conducted when either the 

fi shermen/collectors, the middlemen, or the 

exporters are apprehended with cyanide in 

their possession and the fi sh test positive for 

the presence of cyanide.

It should be noted that it becomes more 

diffi cult to prove that fi shes have been 

collected with cyanide by the time the fi sh 

enter the U.S.A. U.S. agencies such as the 

U.S. Agency for International Development 

(USAID) or other agencies, such as the 

World Bank or the Asian Development Bank, 

should fund the creation of cyanide detection 

networks in countries where cyanide use is 

prevalent. It will be cheaper to conduct most 

of the CDT in the exporting countries.

57
The research proposed and the use of an 

ATCA test to support law enforcement in 

the U.S.A. can be justifi ed not only on the 

basis of the aquarium trade (in which cyanide 

contaminated fi sh enter the U.S.A), but also 

from the point of view of counter-terrorism. 

An accurate, reliable test for either the cyanide 

ion or thiocyanate anion, and/or a cyanide 

biomarker like ATCA is needed to protect 

Americans from attacks by terrorists using 

cyanide. The costs for the US-based cyanide 

detection equipment, for the evaluations, 

research, and trainings are higher than testing 

done in the exporting countries. However, 

they can easily be justifi ed based on the 

terrorism threat. 

Acknowledgements

The authors thank Dr. Robert Kobelski and 

Dr. Brian Logue for reviewing the manuscript 

and for providing comments and suggested 

changes.

References Cited

Alban, J., Manipula, B.E. and Rubec, P.J. 

(2001) Standard Operating Procedures For 

Sampling Marine Fish And Invertebrates Used 

By The Philippines Cyanide Detection Test (CDT) 

Laboratory Network. Philippine Department 

of Agriculture-Bureau of Fisheries and 

Aquatic Resources and International 

Marinelife Alliance, 24 pp.

APHA-AWWA-WPCF (1992) Method 4500-

CNE. Standard Methods for the Examination of 

Water and Wastewater, 18th Edition, American 

Public Health Association, American 

Waterworks Association, Water Pollution 

Control Federation, Washington, DC.

APHA-AWWA-WPCF (1998a) Method 4500-

CNE. Standard Methods for the Examination of 

Water and Wastewater, 20th Edition, American 

Public Health Association, American Water 

Works Association, American Pollution 

Control Federation, Washington, D.C.

APHA-AWWA-WPCF (1998b) 4500-CN- K. 

Spot test for sample screening. In Standard 

Methods for the Examination of Water and 

Wastewater, 20th Edition, pp. 4-46 to 4-47, 

American Public Health Association, 

American Water Works Association, 

American Pollution Control Federation, 

Washington, D.C.

ASTM (1987) Research Report D2036, pp 19-

1131, American Society of Testing And 

Materials, Philadelphia, PA.

ASTM (1990) Designation D5049-90. 

Standard test method for the screening of 

cyanide in waste, Test Method C. In 1990 

Annual Book of ASTM Standards, pp 1-4, 

American Society of Testing and Materials, 

Philadelphia, PA.

58
ASTM (1997) Standard test methods for 

cyanides in water. In 1997 Annual Book Of 

Standards, Vol. 11.02, Water (ll), Designation 

D2036-91 (Storer, R.A., et al., Eds.) pp. 

76-92, American Society of Testing and 

Materials, Philadelphia, PA.

Barber, C.V. and Pratt, V.R. (1997) Sullied 

Seas: Strategies For Combating Cyanide 

Fishing in Southeast Asia and Beyond. Report 

prepared by World Resources Institute and 

International Marinelife Alliance, 73 pp, 

Washington, D.C.

Barber, C.V. and Pratt, V.R. (1998) Poison for 

profi ts: cyanide fi shing in the Indo-Pacifi c. 

Environment 40:8, 5-9, 28-34.

Battelle (2005) Industrial Test Systems, Inc. Cyanide 

ReagentStripTM Test Kit, Environmental 

Technology Verifi cation Report by Battelle 

under contract to U.S. Environmental 

Protection Agency, 42 pp.

Brown, D.G., Lanno, R. P., van Den Heuval, 

M.R. and Dixon, D.G. (1995) HPLC 

determination of plasma thiocyanate 

concentrations in fi sh blood: application 

to laboratory pharmakinetic and fi eld 

monitoring studies. Ecotoxicology and 

Environmental Safety 30, 302-308.

Cervino, J. M., Hayes, R. L., Honovitch, 

M., Goreau, T. J., Jones, S. and Rubec, 

P. J. (2003) Changes in zooxanthellae 

density, morphology, and mitotic index in 

hermatypic corals and anemones exposed 

to cyanide. Marine Pollution Bulletin 46, 573-

586.

Chen, S.-H. and Yang, Z.-Y. (1996) 

Determination of thiocyanate anion by 

high performance liquid chromatography 

with fl uorimetric detection. Journal of 

Analytical Toxicology 20 (January/February 

1996), 38-42.

del Norte, A.G.C., Nanola, C.L., McManus, 

J.W., Reyes, R.B., Campos, W.L. and 

Cabansag, J.B.P. (1989) Overfi shing on a 

Philippine coral reef: a glimpse into the 

future. In Coastal Zone _89, In Proceedings 

of the Sixth Symposium on Coastal and Ocean 

Management Vol. 4 (Magoon, O.T., Converse, 

H., Miner, D., Tobin, L.T. and Clark, D., 

Eds.) pp. 3089–3097.

Dixon, D.G. and Leduc, G. (1981) Chronic 

cyanide poisoning of rainbow trout and 

its effects on growth respiration and liver 

histopathology. Archives of Environmental 

Contamination and Toxicology 10, 117-131.

Dzombak, D.A., Bushey, J.T., Drop, S.M. and 

Ghoshi, R.S. (2006) Analysis of cyanide in 

solids and semi-solids. In Cyanide In Water 

And Soil (Dzomback, D.A., Ghosh, R.S. 

and Wong-Chong, W.M., Eds.) pp 155-169, 

CRC Press, Taylor & Francis Group, Boca 

Raton, FL.

Dumas, P., Gringas, G. and LeBlanc, A. 

(2005) Isotope dilution-mass spectrometry 

determination of blood cyanide by 

headspace gas chromatography. Journal of 

Analytical Toxicology 29, 71-75.

Elsholz, O., Frenzel, W., Lin, C.-Y. and Moller, 

J. (1990) Evaluation of experimental 

conditions on the response of fl uoride 

and cyanide selective electrodes in fl ow 

injection potentiometry. Fresenius Journal of 

Analytical Chemistry 338, 159-162.

Fahrudin, A. (2003) Extended Cost Benefi t 

Analysis of Present And Future Use of 

Indonesian Coral Reefs: an Empirical Approach 

to Sustainable Management of Tropical Marine 

Resources. Doctoral Dissertation, Institute 

59
fur Agrarokonomie, 225 pp, Jakarta, 

Indonesia.

Flinger, C.I., Luthi, R., Linkaityte-Weiss, E. 

and Raisys, V.A. (1992) Paper strip screening 

method for detection of cyanide in blood 

using Cyantesmo paper. The American 

Journal of Forsenic Medicine and Pathology 13:1, 

41-44.

Flurer, C.L., Barnes, B.S., Urban, J.R., Roberts, 

J.N. and Toomey, V.M. (2005) Standard 

Operating Procedure-Screen for the Presence of 

Cyanide. U.S. Food and Drug Administration, 

SOP Number: T005(004), 11 pp, Forsenic 

Chemistry Center, Cincinnati, OH.

Frakes, T. A. and Studt, R. (1996) Evaluation 

of Methods for the Cyanide Detection Test for 

Fish Utilizing Alkaline Digestion and Cyanide 

Specifi c Electrode. Report by Aquarium 

Systems associated with IMA grant from 

the Columbus Zoo, 8 pp, Columbus, OH.

Frant, M.S., Ross, J.W. and Riseman, J.H. 

(1972) An electrode indicator technique for 

measuring low levels of cyanide. Analytical 

Chemistry 44, 2227.

Gosh, R.S., Dzombak, D.A., Drop, S.M. 

and Zheng, A. (2006) Analysis of cyanide 

in water. In Cyanide In Water And Soil: 

Chemistry, Risk, and Management (Dzomabak, 

D.A., Ghosh, R.S. and Wong-Chong, G.M. 

Eds.) pp 123-153, Taylor and Francis, Boca 

Raton, FL.

Heming, T.A. and Blumhagen, K.A. (1989) 

Factors infl uencing thiocyanate toxicity 

in rainbow trout Salmo gairdneri. Bulletin of 

Environmental Contamination and Toxicology

43:363-369.

Heming, T.A., Thurston, R.V., Meyn, E.L. 

and Zajdel, R.K. (1985) Acute toxicity of 

thiocyanate. Transactions American Fisheries 

Society 114, 895-905.

Holthus, P. (1999) Peer Review of Cyanide 

Detection Testing Methods Employed in the 

Philippines. 23 pp, Report by the Marine 

Aquarium Council to the World Bank 

Marine Market Transformation Initiative.

IMA-Philippines (1999) Response to the Review 

of the IMA Cyanide Testing Standard Operating 

Procedures Prepared By The Marine Aquarium 

Council. Distributed at the International 

Cyanide Detection Testing Workshop held 

in Orlando, Florida, February 6-8, 2008. 

19 pp, Rubuttal document prepared by the 

International Marinelife Alliance (IMA)-

Philippines. 

IMA-Philippines (2000) Standard Operating 

Procedures for Cyanide Analysis in Fish Tissues. 

Handout provided to participants at a Live 

Food Fish Meeting held in Hong Kong (it 

was not the full SOP manual used by IMA 

chemists), 8 pp, Cyanide Detection Test 

Central Laboratory, Pasig City, Philippines. 

Johannes, R.E. and Riepen, M. (1995) 

Environmental, Economic, and Social Implications 

of the Live Fish Trade in Asia and the Western 

Pacifi c. The Nature Conservancy Report, 87 

pp, Honolulu, HI.

Jones, R.J., Kildea, T and Hoegh Guldberg, 

O. (1999) PAM chlorophyll fl ourometry: a 

new in situ technique for stress assessment 

in scleractinian corals, used to examine the 

effects of cyanide from cyanide fi shing. 

Marine Pollution Bulletin 38, 864-874.

Kage, S., Nagata, T. and Kudo, K. (1996) 

Determination of cyanide and thiocyanate 

in blood by gas chromatography and gas 

chromatography mass spectrometry. Journal 

of Chromatography B 675, 27-32.

60
Kuegsen, M., Kloock, J.P., Knobbe, D.T., 

Junger, M., Krest, I., Goldbach, M., 

Klein, W. and Schoning, M.J. (2004) 

Direct determination of cyanides by 

potentiometric biosensors. Sensors and 

Actuators B 103, 280-385.

Lanno, R.P. and Dixon, D. G. (1993) Chronic 

toxicity of waterborne thiocyanate to 

the fathead minnow (Pimephales promelas). 

Environmental Toxicology and Chemistry 13:9, 

1423-1432.

Lanno, R.P., Fisher, S. and Rubec, P.J. (2002b) 

Development of An Analytical Method for the 

Detection of Thiocyanate in Marine Coral Reef 

Fishes. Proposal funded by the National 

Fish and Wildlife Foundation, 16 pp, 

Washington, D.C.

Lanno, R.P., Fisher, S., Rubec, P.J. and Brittsan, 

M. (2002a) Development of an Analytical 

Method for the Detection of Thiocyanate in 

Marine Coral Reef Fishes. Proposal funded 

by Columbus Zoo-Ohio State University, 

13 pp, Columbus, OH.

Lanno, R.P. and Dixon, D.G. (1996a) The 

comparative chronic toxicity of thiocyanate 

and cyanide to rainbow trout (Oncorhynchus 

mykiss). Aquatic Toxicology 36, 177-187.

Lanno, R.P. and Dixon, D.G. (1996b) Chronic 

toxicity of waterborne thiocyanate to 

rainbow trout (Oncorhynchus mykiss). 

Canadian Journal Fisheries and Aquatic Sciences

53, 2137-2146.

Leduc, G. (1984) Cyanides in water: 

toxicological signifi cance. In Aquatic 

Toxicology, Vol. 2. (Weber, L.J., Ed.) pp 153-

224, Raven Press, New York, NY.

Logue, B.A. and Hinkens, D.M. (2008) The 

Analysis of Cyanide and its Metabolites in 

Biological Samples. Unpublished manuscipt 

distributed at the International Cyanide 

Detection Testing Workshop held in 

Orlando, Florida, February 6-8, 2008.

Logue, B.A., Kirschten, N.P., Petrikovics, 

I., Moser, M..A., Rockwood, G.A. and 

Baskin, S.I. (2005) Determination of the 

cyanide metabolite 2-aminothiozoline-4 

carboxylic acid, in urine and plasma by 

gas chromatography-mass spectrometry. 

Journal of Chromatography, B: Analytical 

Technologies in the Biomedical and Life Sciences

819, 237-244.

Logue, B.A., Masered, W.K., Rockwood, G.A., 

Keebouagh, M.W. and Baskin, S.I. (2007) 

Feasibility of the cyanide metabolite, 

2-amino-2 thiazoline-4-carboxylic acid, as a 

retrospective marker for cyanide exposure. 

Journal of Chromatography B (submitted).

Lundquist, P., Kegedal, B., Nilsson, L. and 

Rosling, H. (1995) Analysis of cyanide 

metabolite 2-aminothiazoline-4-carboxylic 

acid in urine by high performance liquid 

chromatography. Analytical Biochemistry 228, 

27-34.

MAC (2004) Philippines and Indonesia Marine 

Aquarium Market Transformation Initiative 

(MAMTI). Proposal submitted to the 

International Finance Corporation (World 

Bank) Global Environmental Facility 

(GEF) by the Marine Aquarium Council, 

Honolulu, HI.

Mak, K.K.W. (2003) Review of cyanide 

detection methods in the Philippines 

monitoring station. In Novel Bioanalytical 

Tests And Biosensor Systems For Detection of 

Cyanide Traces in Marine Fish, pp 58-79. Ph.D. 

Dissertation, The Hong Kong University 

of Science and Technology, Hong Kong.

61
Mak, K.K.W., Law, A.W.C., Takuda, S., Yanase, 

H. and Renneberg, R. (2005a) Application 

of cyanide hydolase from Klebsiella sp. in 

a biosensor system for the detection of 

low-level cyanide. Applied Microbiology and 

Biotechnology 67, 631-636.

Mak, K.K.W., Yanase, H. and Renneberg, 

R. (2005b) Cyanide fi shing and cyanide 

detection in coral reef fi sh using chemical 

tests and biosensors. Biosensors and 

Bioelectronics 20, 2581-2593.

Mak, K.K.W., Yanase, H. and Renneberg, 

R. (2005c) Novel optical biotest for 

determination of cyanide traces in marine 

fi sh using microbial cyanide hydratase and 

formate dehydrogenase. Microchimica Acta

149, 131-135.

Manipula, B.E., Suplido, E.R., Astillero, 

N.M. (1995) Manual of Standard Operating 

Procedures For Cyanide Analysis. International 

Marinelife Alliance and Philippines 

Department of Agriculture-Bureau of 

Fisheries and Aquatic Resources.

Manipula, B.E., Suplido, E.R., Astillero, N.M. 

(2001a) Standard Operating Procedures For 

Preparation Of Marine Fish And Invertebrate 

Samples For Cyanide Testing, Grouper Research, 

And Assessment Of Blast Fishing. Philippines 

Department of Agriculture-Bureau of 

Fisheries and Aquatic Resources and 

International Marinelife Alliance.

Manipula, B.E., Suplido, E.R., Astillero, N.M. 

(2001b) Standard Operating Procedures For 

Cyanide Testing Used By The Philippines Cyanide 

Detection Test (CDT) Network. Philippines 

Department of Agriculture-Bureau of 

Fisheries and Aquatic Resources and 

International Marinelife Alliance.

Manipula, B.E., Rubec, P.J. and Frant, M. 

(2001c) Standard Operating Procedures For 

Use Of The Thermo Orion Ion-Selective 

Electrode (ISE) And The ISE/pH Meter To 

Assess Cyanide Concentrations. Philippines 

Department of Agriculture-Bureau of 

Fisheries and Aquatic Resources and 

International Marinelife Alliance.

Murphy, K.E., Schantz, M.M., Butler, T.A., 

Benner Jr., B.A., Wood, L.A. and Turk, G.C. 

(2006) Determination of cyanide in blood 

by isotope dilution gas chromatographymass spectrometry. Clinical Chemistry 52 :3, 

458-467.

Orion Research Inc. (1975) Cyanide Ion 

Electrode Instruction Manual, Cambridge, 

Massachusetts, USA. Thermo Orion 

Research Incorporated, Laboratory 

Products Group, Cumming Center, 500 

Cumming Center, Beverly, MA.

Orion Research Inc. (1997) Benchtop pH/

ISE Meter Instruction Manual, Thermo 

Orion Research Incorporated, Laboratory 

Products Group, Cumming Center, 500 

Cumming Center, Beverly, MA.

Pet, J.S. and Djohani, R.H. (1998) Combating 

destructive fi shing practices in Komodo 

National Park: ban the hookah compressor! 

South Pacifi c Commission Live Reef Fish 

Information Bulletin 4 (April), 17-28. http://

www.spc.org.nc/coastfi sh/news/lrf/4/pet.htm

Raymond, P., Leduc, G. and Kornblatt, J.A. 

(1986) Investigation sur la toxicodynamique 

du cyanure et sur la biotransformation chez 

la truit arc-en-ciel (Salmo gaidneri). Canadian 

Journal of Fisheries and Aquatic Sciences 43, 

2017-2024.

Rubec, P.J. (1986) The effects of sodium 

cyanide on coral reefs and marine fi sh in the 

62
Philippines. In Proceedings from the First Asian 

Fisheries Forum (Maclean, Jay L., Dizon, L.B. 

and Hosillos, L.V., Eds.) pp 297-302, Asian 

Fisheries Society, Manila, Philippines.

Rubec, P.J. (1987) The effects of sodium 

cyanide on coral reefs and marine fi sh in 

the Philippines. Marine Fish Monthly 2:2, 

7-8, 17, 20, 27, 34-35, 39, 44, 46-47 and 2:3, 

8-10, 14, 24, 44, 47. 

Rubec, P.J. (2007) Summary of Studies That 

Have Evaluated the ISE Method Used by the 

Bureau of Fisheries and Aquatic Resources 

in the Philippines. Unpublished manuscipt 

distributed at the International Cyanide 

Detection Testing Workshop held in 

Orlando, Florida, February 6-8, 2008.

Rubec, P.J., Cruz, F., Pratt, V., Oellers, R., 

McCullough, B. and Lallo, F. (2001) 

Cyanide-free net-caught fi sh for the marine 

aquarium trade. Aquarium Sciences and 

Conservation 3, 37-51.

Rubec, P.J. and Manipula, B. (2008) Search for 

A Simplifi ed Field Test: The Soundararajan 

Digestion Combined With ISE or Colorimetric 

Methods for Detection of Cyanide Ion 

Concentrations in Marine Fish. Abstract of 

presentation given at the International 

Cyanide Detection Testing Workshop held 

in Orlando, Florida, February 6-8, 2008.

Rubec, P.J. and Soundararajan, R. (1991) 

Chronic toxic effects of cyanide on 

tropical marine fi sh. In Proceedings of the 

Seventeenth Annual Toxicity Workshop: 

November 5-7, 1990, Vancouver, B.C. 

Canadian Technical Report of Fisheries and 

Aquatic Sciences 1774:1 (Chapman, P., et al. 

Eds.) pp 243-251.

Rubec, P.J., Pratt, V.R., McCullough, B., 

Manipula, B., Alban, J., Espero, T. and 

Suplido, E.R. (2003) Trends determined by 

cyanide testing on marine aquarium fi sh in 

the Philippines. In Marine Ornamental Species: 

Collection, Culture & Cultivation (Cato, J.C. 

and Brown, C.L., Eds.) pp 327-340, Iowa 

State Press, Ames, IA.

SeaWeb (2008) Background Paper on Cyanide 

Detection Tests for Live Fish. Unpublished 

manuscipt distributed at the International 

Cyanide Detection Testing Workshop held 

in Orlando, Florida, February 6-8, 2008.

Sekerka, I., and Lechner, J. F. (1976)

Potentiometric determination of low levels 

of simple and total cyanides. Water Research 

10, 479-483.

Smith, L.S. (1982) Osmoregulation. Introduction 

to Fish Physiology, 19-86.

Speyer, M.R. and Raymond, P. (1988) The 

acute toxicity of thiocyanate and cyanate to 

rainbow trout as modifi ed by temperature 

and pH. Environmental Toxicology and 

Chemistry 7, 565-571.

Turek, M., Ketterer, L., Claben, M., Berndt, 

H.K., Elbers, G., Kruger, P., Keugsen, M. 

and Schoning, M.J. (2007) Development 

and electrochemical investigations of an 

EIS (Electrolyte-Insulator-Semiconductor) 

based biosensor for cyanide detection. 

Sensors 7, 1415-1426.

US-EPA (1983) Methods for Chemical Analsis 

of Water and Wastes, Method 335.2. EPA600, 4-79-020, Revised March 1983, U.S. 

Environmental Protection Agency.

63
Validation Methods for the Determination of Cyanide and 

Cyanide Metabolites in Coral Reef Fishes

Robert Kobelski

National Center for Environmental Health 

Centers for Disease Control and Prevention

rmk9@cdc.gov

Background

In a number of locations both ornamental and live food fi sh are captured using techniques 

such as stunning with an aqueous solution containing the cyanide anion (CN-

). This technique 

is destructive to the reef, the individual fi sh and the sustainability of the population and is 

illegal in Indonesia, the Philippines and Vietnam. To determine if fi sh are captured using this 

destructive technique there must be a forensically supportable analysis for cyanide exposure in 

reef fi sh implemented in a timely fashion after capture of the fi sh. 

The length of time between capture and delivery of the fi sh to a capable laboratory can range 

from fi ve to fourteen days. For a laboratory analysis to be viable the analyte must be stable and 

at elevated levels in the sample long enough for the sample to be delivered to the laboratory 

and for the analysis to be conducted. Data from mammals suggests that the cyanide anion is 

readily metabolized to thiocyanante (SCN-

) and 2-aminothiazoline-4-carboxylic acid (ATCA), 

with a half-life of approximately one hour. If metabolism in fi sh is similar there would not 

be an elevated level of cyanide in a fi sh when it reached the laboratory site For there to be an 

elevated level of cyanide after fi ve days the half-life would have to be approximately 10 hours, 

but this toxicokinetics parameter has not been determined.

To determine what analyte can be targeted for forensic analysis the following research needs 

to be conducted:

1) the half-life of cyanide in fi sh has to be determined and the variation in half-life with 

respect to different species must be defi ned,

2) the half-life of thiocyanate, the major metabolite of cyanide, in fi sh must be determined 

as well as the specie-to-specie variation in half-life

The consensus of the Working Group was that there exist serious gaps in the understanding of 

the metabolism and elimination of the cyanide anion and its major metabolites in fi sh. Without a 

better understanding of these processes the implementation of an exposure assessment method 

that would meet legal requirements would be diffi cult. The recommendation of the Working 

Group is that a research program be designed to generate the data necessary to determine the 

determine the availability of cyanide and its metabolites for exposure assessment versus the 

time involved with the local collection of fi sh, their transport to a fi xed site laboratory prior 

to export and their arrival at the import destination prior to the implementation of an analysis 

method at any point in the delivery chain.

64
3) the metabolism of cyanide to ATCA must be demonstrated and if this is a metabolic 

pathway for fi sh the half-life of ATCA must be determined as well as the specie-to specie 

variation in half-life,

4) the background level of cyanide in fi sh must be determined, if it is above the limit of 

quantitation (LOQ) of the analysis technique, to defi ne when an elevated level due to 

cyanide exposure has been encountered.

5) The background level of thiocyanate in fi sh must be determined, to defi ne if an exposure 

to cyanide will result in a statistically meaningful elevation of background thiocycante

6) The background level of ATCA must be determined, if it is above the LOQ of the 

analysis technique, to defi ne when an elevated level due to cyanide exposure has been 

encountered.

To perform this research, validated methods for the determination of cyanide, thiocyanate and 

ATCA in homogenized fi sh tissue must be used. These methods must demonstrate adequate 

sensitivity, specifi city, accuracy and precision as determined by the “fi t for purpose” concept.

3. Outline of Validation Procedure: 

3.1 Method validation is a process by which a laboratory confi rms by examination and the 

provision of objective evidence that the particular requirements for specifi c use are fulfi lled. 

It serves to demonstrate that:

3.1.1 The method can detect, identify and quantitate an analyte(s):

• In all matracies to be analyzed 

• With a demonstrated sensitivity, specifi city, accuracy, reproducibility, ruggedness and 

precision to ensure that results are meaningful and appropriate for decision making 

by the receiving network. 

3.1.2 The method will function reliably for its intended purpose as defi ned by the network.

3.2 The method developer validates a method by conducting experiments to determine or 

verify a number of specifi c performance characteristics that serve to defi ne and quantify 

method performance. 

4. Reference(s):

4.1 Food and Drug Administration, Laboratory Manual, ORA Laboratory Procedure, Volume 

II, ORA-LAB5.4.5, Methods, Method Verifi cation and Validation

4.2 International ANS/ISO/IEC Guide to the Expression of Uncertainty in Measurement, 

1995

4.3 International ANS/ISO/IEC Guide 2 (VIM), 2004

4.4 International ANS/ISO/IEC Guide 8402, Quality Management and Quality Assurance 

65
Vocabulary, 1994

 

4.5 Foundations of Clinical Research, Applications to Practice, Leslie Gross Portney, Mary 

Watkins, Appleton & Lange, 1993

4.6 Validation and Peer Review of U.S. Environmental Protection Agency Chemical Methods 

of Analysis”, Forum on Environmental Measurements, October 14, 2005

4.7 Protocol for the Design, Conduct, and Interpretation of Method-Performance Studies, 

Pure and Applied Chemistry, 67, No. 2, 1995, 331-343. 

4.8 Harmonized Guidelines for Single-laboratory Validation of Methods of Analysis, Pure 

and Applied Chemistry, 2002, 74, 835 - 855.”

5. Specifi c Procedure(s):

5.1 Prior to submitting methods to the network: 

5.1.1 The intended use of the method should be defi ned 

5.1.2 The intended use should be aligned with network capabilities 

5.1.3 A study plan for method validation should be submitted to and approved by the 

involved network for review before starting the validation. 

5.1.4 A written procedure which includes all the following elements must be prepared:

• Intended use and criteria for use 

• Assay principle and safety precautions

• Acceptable sample types, sample collection method, preparation, preservation, 

storage and transportation conditions 

• Description of reagents supplied or directions for preparation and QC

• Description of quality controls to be used. 

• Detailed instructions on how to perform the method. 

• Detailed instructions on how to interpret and report the result. , 

• Any limitations of the method that are known or suspected

• A summary of the performance characteristics of the method

5.1.5 Written directions (specifi cations) for the production and QC of the reagents, if 

provided for the method

5.2 Methods should be validated whenever any of the following occur: 

5.2.1 Submission of a new method to the network for inclusion as an offi cial method.

5.2.2 Expansion of the scope of an existing network method to include additional analytes 

66
or new matrices.

5.2.3 Modifi cation of a network method’s range beyond validated levels.

5.2.4 Modifi cation of a network method that may alter its performance specifi cations. 

This includes: changes to the fundamental science of an existing method, equivalence 

issues such as substitutions of reagents/apparatus, or changes to some instrumental 

parameters. Since it is diffi cult to predict the results of any change, all but the most 

trivial of changes should be evaluated for effects on method performance.

5.3 Performance specifi cations required to validate a method.

5.3.1 Performance specifi cations that should be determined to validate a method will vary 

depending on the intended use, the type of method being validated, and the degree to 

which it has previously been validated. All methods submitted must have all the proper 

controls and the parameters for calibrating and operating the method instrumentation 

included in the written procedure.

5.3.1.1 Typical validation characteristics which should be considered are the following: 

(See Glossary for defi nitions of these characteristics in Appendix 1) 

Characteristics of quantitative methods 

• Method uncertainty 

• Minimum quantifi able concentration

• Detection limit (See MDL and MDC) 

• Applicable analyte concentration range

• Accuracy

• Precision 

• Analytical specifi city

• Linearity

• Ruggedness/robustness

• Clinical sensitivity 

• Clinical specifi city 

5.3.1.2 Validation tools (Ref ORA-LAB SOP# 5.4.5)

The following tools should be used to demonstrate the ability to meet method 

specifi cations of performance:

• Blanks: Use of various types of blanks enables assessment of how much of the 

result is attributable to the analyte and to other causes.

• Reference materials and certifi ed reference materials with typical interferences 

expected. Use of known materials can be incorporated to assess the accuracy of 

the method, as well as for obtaining information on interferences.

• Fortifi ed (spiked) materials and solutions: Recovery determinations can be 

estimated from fortifi cation or spiking with a known amount of analyte. (Note: 

Understanding that spiked recovery may not be truly representative of recovery 

from naturally incurred analytes.)

• Replication: Replicate analyses provide a means of checking for changes in 

precision in an analytical process which could adversely affect the results.

67
• Statistics: Statistical techniques are employed to evaluate accuracy, precision, linear 

range, limits of detection and quantifi cation, and measurement uncertainty.

5.3.1.3 General Validation Protocol Guidance

The following provides guidelines that should be used to determine method 

performance characteristics:

• Quantitative measurements, e.g. determine limit of quantitation (LOQ), linear 

response. Minimally need LOD

• Prepare and analyze spiked blanks, matrix samples of known concentration 

utilizing at least three different concentration levels: low, medium, high based on 

the intended use of the method. These samples are carried through the complete 

sample preparation procedure, extraction and analytical steps of a particular 

method. Matrix effects can also be assessed with these samples. Accuracy and 

precision are calculated from these results; data will also evaluate robustness of 

the method resulting from changes in the sample matrix. (Note: Proper certifi ed 

reference materials and reference standards are used when available.)

• Assure that adequate sample replicates are performed and that results from 

replicate measurements of each analyte are compared.

• Analyze blanks (reagent and matrix) and compare these results to the reported 

limit of detection

• Evaluate interferences: spectral, physical, chemical or memory by analyzing samples 

containing various suspected interferences in the presence of the measurand. 

5.3.1.4 Validation of Methods (Original, New or Modifi ed), should include but not be 

limited to matrix extensions and platform changes. 

• In cases where the sample preparation and/or the extraction procedure/analytical 

method is modifi ed from the existing test procedure and protocol, the new method 

should demonstrate that the modifi cations do not adversely affect the precision 

and accuracy of the data obtained.

• In order to implement the modifi ed method, the standard or existing method is 

fi rst performed. The modifi ed method is then verifi ed against the original method 

validation protocol as defi ned in section 5.3.1.3. 

• For original or new methods the authors should pick a validation level that is 

suitable for their situation as defi ned in section 5.4. 

5.4 Levels of Validation

5.4.1 It is the responsibility of the individual network to determine the level of validation 

that is acceptable for use on the particular method. 

5.4.2 This section references four levels of validations:

Level One: The method is tested in one laboratory for one or more analytes and one or 

more matrices. The laboratory would select a limited number of key characteristic to 

evaluate the method performance. (See tables 1, 2, and 3 for specifi c details for level 

1 validation) 

68
Level Two: The method should be validated in a single laboratory. (See tables 1, 2, and 3 

for specifi c details for level 2 validation) 

 • “Wherever possible and practical a laboratory should use a method of analysis 

that has had its performance characteristics evaluated through a collaborative trial 

conforming to an international protocol. Page 844 Pure and Applied Chemistry, 

2002, 74”

• “Single-laboratory validation requires the laboratory to select appropriate 

characteristics for evaluation from the following: applicability, selectivity, 

calibration, accuracy, precision, range, limit of quantifi cation, limit of detection, 

sensitivity, and ruggedness. Page 845 Pure and Applied Chemistry, 2002, 74.”

This is similar to the MARLAP tiered project method validation approach for radio 

analytical methods. 

Level Three: The method should be validated by testing the applicable performance 

characteristics from a single laboratory validation study using two to seven labs 

with one or more matrices. (See tables 1, 2, and 3 for specifi c details for level 3 

validation) 

Level Four: The method is tested using the criteria for a full collaborative study. The 

study should examine bias, recoveries, applicability, interference, method comparison, 

calibration procedures and all applicable performance characteristics examined in a 

single laboratory validation. (See tables 1, 2, and 3 for specifi c details for level 4 

validation) 

 • For a single type of substance at least 5 materials (test samples) must be used; 

only when a single level of specifi cation is involved for a single matrix may this 

minimum required number of materials be reduced to 3. Page 334, Pure and 

Applied Chemistry, 67, No. 2, 1995. 

 • At least 8 laboratories should report the results for each matrix; only when it is 

impossible to obtain this number may the study be conducted with fewer, but with 

an absolute minimum of 5 laboratories. Page 335, Pure and Applied Chemistry, 

67, No. 2, 1995. 

• The entire protocol is outlined in the article “Protocol for the Design, Conduct, 

and Interpretation of Method-Performance Studies, Pure and Applied Chemistry, 

67, No. 2, 1995, 331-343. 

69
The Analysis of Cyanide and its Metabolites in Biological Samples

Brian A. Logue and Diane M. Hinkens

Department of Chemistry and Biochemistry 

South Dakota State University 

brian.logue@sdstate.edu

Introduction

Cyanide is toxic in humans, animals, and 

fi sh and exposure can occur in various ways. 

Many substances are potential sources of 

cyanide exposure including edible and nonedible plants, industrial operations, fi res, 

and cigarette smoke. Although the primary 

natural source of cyanide poisoning is from 

plants (1-7), other natural sources include 

volcanoes, bacteria, and fungi (1, 8-13). Manmade sources include malfunctioning catalytic 

converters, residential and commercial fi res 

involving the burning of plastics, cigarette 

smoke, as well as illicit uses of cyanide (14). 

Additionally, hundreds of thousands of tons 

of cyanide are manufactured annually in 

the U.S. alone for industrial uses, including 

chemical syntheses, electroplating, plastics 

processing, paint manufacturing, gold and 

silver extraction, tanning, and metallurgy.

Along with many legal industrial uses of 

cyanide, multiple illegal uses of cyanide exist. 

Recently, terrorist acts involving cyanide 

have been the most publicized illicit uses of 

cyanide. In 1982, cyanide was placed in bottles 

of Tylenol in the Chicago area, killing seven 

people (1). In 1995 in Tokyo, an acid and a 

cyanide salt were found in several subway 

restrooms in the weeks following the release 

of nerve agents (2). Another illegal use of 

cyanide is the capture of fi sh for subsequent 

sale in the live fi sh trade (15). Cyanide is 

used at sub-lethal doses to temporarily stun 

fi sh, making them easier to catch (16). The 

practice of using cyanide in this manner has 

been found in a number of countries, and 

has an adverse affect on coral reefs (15). The 

cyanide is toxic to algae that are necessary 

for coral to survive and also produces many 

adverse secondary effects, such as killing 

smaller fi sh species. With this and other 

destructive practices used during capture of 

cyanide stunned fi sh, irreversible damage to 

coral reefs has and continues to occur (17).

As industrial and illicit applications of cyanide 

increase, the need for rapid, sensitive, and 

specifi c analytical methods to assess cyanide 

exposure will be amplifi ed. The goals for this 

review are to briefl y discuss current 

bioanalytical techniques used for verifi cation 

Cyanide is a toxic chemical that may be introduced to living organisms as a result of both 

legal and illicit uses of cyanide. Exposure to cyanide can be verifi ed by analyzing cyanide or 

one of its break-down products from biological samples. This verifi cation is important for 

medical, law-enforcement, forensic, research, and veterinary purposes. This review will identify 

common problems associated with the analysis of cyanide and its metabolites, discuss current 

bioanalytical techniques used for verifi cation of cyanide exposure, and briefl y address the 

metabolism and toxicokinetics of cyanide and its break-down products in biological systems. 

70
of cyanide exposure and to identify common 

problems associated with the analysis of 

cyanide and its metabolites in biological 

samples. This review does not include 

methods to test for cyanide in environmental 

or industrial matrices (e.g., surface waters, 

minerals, process streams). A number of 

related reviews have been published (8, 15, 

18-22).

humans, the following text summarizes 

cyanide metabolism in humans. 

Once absorbed, cyanide is quickly transferred 

to the blood and metabolized through a 

number of processes, as shown in Figure 1. 

The major pathway for cyanide metabolism 

is conversion of cyanide to thiocyanate 

(SCN-

) in the presence of a sulfur donor (e.g., 

thiosulfate) (24). This reaction is catalyzed 

by the enzyme rhodanese (18, 25, 26). About 

80% of the initial cyanide dose is converted 

to thiocyanate, which is subsequently 

excreted in the urine. Other minor metabolic 

the gills or intestine (15). Little is known 

about the metabolism of cyanide in fi sh, and 

more research is necessary to fully understand 

the similarities and differences of mammals 

and fi sh, but considering that cyanide’s toxic 

effects are similar in both mammals and 

fi sh, a number of metabolic pathways may 

also be similar. As the majority of research 

on cyanide metabolism has been done on 

Cyanide metabolism and toxicokinetics

Although there are other chemical forms of 

cyanide, it is hydrogen cyanide (HCN) that 

is the primary toxic agent, regardless of its 

origin. The toxic effects of cyanide can be 

traced to interference of aerobic metabolism 

(18). This occurs when cyanide blocks 

terminal electron transfer by binding to 

cytochrome oxidase for both mammals and 

fi sh (18, 23). For mammals, cyanide ion (CN-

) 

is acquired through ingestion while hydrogen 

cyanide is acquired through inhalation or 

absorption through the mucous membranes 

or skin. For fi sh, cyanide is absorbed through 

S N

NH2

S NH

NH

OH OH

O O

SCN- HCN/CN- Cyanocobalamin

(Vitamin B12)

HCNO

CO2

HCOOH

(Metabolized as other

one carbon compounds)

+ Cystine

+ S donor Hydroxocobalamin

ATCA ITCA

+

Protein Adducts

rhodanese

Figure 1. Human metabolism of cyanide.

71
pathways are the conversion of cyanide 

to 2-amino-2-thiazoline-4-carboxylic acid 

(ATCA; the tautomeric form of ATCA 

is 2-iminothiazolidine-4-carboxylic acid – 

ITCA; Figure 1) in the presence of cystine 

(18, 26-28), and the reversible reaction of 

cyanide with hydroxocobalamin to form 

cyanocobalamin. (Note: Throughout the 

text, the ATCA/ITCA tautomeric pair will 

be referred to as ATCA.) The production 

of ATCA may predominate when sulfur 

donors become depleted or in tissues where 

rhodanese is sparse. Other minor pathways 

for metabolism include the creation of onecarbon metabolites and protein adducts (i.e., 

reaction of a chemical species with a protein 

to form a chemical bond that modifi es the 

parent protein) (18, 29). Each metabolite in 

Figure 1 could potentially be used as a marker 

for cyanide exposure. 

The toxicokinetics and metabolism of an 

analyte must be considered when an analytical 

technique is used to determine exposure to the 

analyte. A main consideration for determining 

a target for confi rmation of exposure is the 

half-life of the biomarker of interest. Table 1 

lists the half-lives of cyanide and thiocyanate 

for acute exposures of a number of mammalian 

species. If the half-life of a toxic agent is 

short, as with cyanide (t1/2 = 0.34-1.28 hours), 

it can be diffi cult to determine exposure by 

direct analysis of the toxic agent if signifi cant 

amounts of time have elapsed. Thiocyanate 

offers a longer half-life (t1/2 = 4.95-5.8 hours). 

The half-lives of ATCA and cyanide-protein 

adducts are unknown, although for proteinadducts, a half-life similar to that of the 

parent protein could be expected (e.g., 20-25 

days for human serum albumin) assuming the 

adduct is stable (30, 31). It should be noted 

that chronic exposure to cyanide increases the 

apparent half-life of cyanide and thiocyanate 

(32). This is most likely due to the depletion 

of sulfur donors over the course of the 

chronic exposure with subsequent reduction 

of cyanide transformation by this metabolic 

pathway. The activity of rhodanese could also 

be reduced over the course of the chronic 

exposure, leading to longer half-lives of 

cyanide.

As seen by the half-lives of cyanide among 

a number of mammals in Table 1, the 

toxicokinetics of cyanide is somewhat species 

dependent. Therefore, although generalities 

concerning the toxicokinetics and metabolism 

of cyanide can be made, half-life values cannot 

be used directly from other species. To more 

fully understand the relationship of cyanide 

metabolism and toxicokinetics of sparsely 

studied species (e.g., most fi sh species), more 

research should be undertaken to determine 

relationships between organisms of interest. 

Cyanide/

Metabolite Species t1/2 (hr) Reference

Cyanide Human 0.34-1.00 (34)

Rat 0.64 (35)

Pig 0.54 (35)

Goat 1.28 (35)

Thiocyanate Rat 5.80 (35)

Pig 4.95 (35)

Goat 13.9 (35)

Table 1. Reported half-lives of cyanide and thiocyanate from acute exposures of cyanide.

72
Special considerations for the analysis of 

cyanide and its metabolites

Considering the typical half-life of cyanide for 

acute exposure (Table 1), it may be diffi cult 

to determine cyanide concentrations in blood 

or tissue for long periods of time following 

exposure. When analyzing cyanide from blood 

(the preferred method for determination of 

cyanide exposure in large species), analysis 

must occur soon after exposure since the 

concentration of cyanide in the blood decays 

within minutes to hours. A number of issues 

limit the direct analysis of cyanide from 

blood, including rapid cyanide detoxifi cation 

processes, the diffi culty of establishing 

steady state cyanide levels with time, and 

the volatility and nucleophilic properties of 

cyanide. Although limited, direct analysis of 

cyanide from blood is still useful in that it may 

be the only biomarker capable of indicating 

exposure to cyanide within the initial minutes 

following exposure (33-35). 

Although traces of cyanide in urine (36, 37), 

saliva (25, 38), and expired air (39-42) have been 

found, direct analysis of cyanide from these 

matrices is even more limited. It is diffi cult 

to assess decay of cyanide concentrations in 

tissues, because levels of rhodanese are highly 

variable between organs (18). Considering the 

highly nucleophilic nature of cyanide ion, free 

cyanide concentrations are likely to be very 

low in tissues, although this does not preclude 

some reservoir of bound cyanide within the 

tissues. Any cyanide analysis technique would 

have to consider conversion of bound cyanide 

to free cyanide prior to analysis. 

As an alternative to direct analysis of cyanide, 

thiocyanate and ATCA may be determined 

in urine, saliva, tissue, and blood. Cyanideprotein adducts have also been found in 

human blood proteins. These markers may 

offer an advantage in half-life (Table 1 for 

thiocyanate), and correlation of thiocyanate 

and ATCA concentrations to cyanide exposure 

have been examined (43-49). Each of these 

markers has advantages and disadvantages 

when considering the detection of cyanide 

exposure. 

The advantages to measuring thiocyanate 

are that appreciable concentrations may be 

found shortly following exposure and it has 

a longer half-life than cyanide (35). However, 

thiocyanate is naturally found in biological 

fl uids, and while this is a condition of all cyanide 

metabolites, thiocyanate levels are normally 

quite high and can be inconsistent (25, 43, 50-

55). Large variation in background thiocyanate 

concentrations makes it diffi cult to determine 

low-level cyanide exposure. Also, Ballantyne 

(56) found that concentrations of thiocyanate 

in blood varied inconsistently during storage 

at a number of different temperatures and 

that analytical recovery of thiocyanate from 

whole blood was diffi cult. Large and variable 

concentration may indicate that thiocyanate is 

involved in a number of biological processes 

in addition to cyanide metabolism. Indeed, 

signifi cant use of thiocyanate by biological 

processes other than cyanide metabolism has 

been established (24, 57, 58).

ATCA may also be used as an alternative 

for determination of cyanide exposure. An 

advantage to using ATCA is that it is stable 

in biological samples for months at freezing 

and ambient temperatures (45, 48). It also has 

been found that ATCA is not metabolized 

further (18, 47, 59) and therefore, may be 

a lasting signature of cyanide exposure. 

However, relatively few techniques have been 

described to analyze ATCA from biological 

matrices (45, 48, 60, 61) and relatively few 

studies have been performed to evaluate 

the relationship between ATCA and cyanide 

exposure (45, 48). With more knowledge of 

ATCAs behavior with relation to cyanide 

exposure, ATCA’s stability and applicability 

73
to sensitive analytical techniques may prove 

benefi cial. 

Recently, cyanide-protein adducts have been 

discovered (29). If these proteins are stable, 

they could serve as long-lived markers of 

cyanide exposure. Disadvantages of this 

analysis include costly instrumentation, 

limited research pertaining to the behavior of 

these adducts, and the diffi culty and length of 

sample preparation. Even considering these 

disadvantages, cyanide adducts are extremely 

promising for providing a long-lived 

biomarker of cyanide exposure, especially 

if a less complex and less costly method of 

analysis can be developed.

Multiple factors must be considered when 

choosing which analyte to target for 

verifi cation of cyanide exposure. Analysis 

of cyanide or any one of its metabolites 

has advantages and disadvantages. Table 2 

compares cyanide and its metabolites in terms 

of some factors important for verifi cation of 

cyanide exposure.

Species considerations for verifi cation of 

cyanide exposure

For humans, determination of cyanide 

exposure has been suggested or attempted 

from blood (20, 25, 37, 43, 45, 62-97), urine 

(36, 37, 45, 48, 89, 95, 98), saliva (25, 38, 51, 

93, 99), expired air (39, 42), and tissue (postmortem) (74, 96, 100) samples. Blood may be 

the most versatile biological sample used to 

determine exposure to cyanide, because the 

analysis of cyanide, thiocyanate, ATCA, and 

cyanide-protein adducts can be performed on 

blood samples. Saliva, urine, or tissue may be 

more appropriate depending on the analytical 

method and other factors, including ease of 

obtaining the sample. If determination of 

cyanide exposure is to be done from nonhuman species, obviously saliva and urine 

samples will be diffi cult to obtain. Therefore, 

blood and tissues are the most likely samples 

to be analyzed to determine cyanide exposure. 

The choice of blood or tissue is determined 

by a number of factors, including the size of 

the species. For example, most fi sh species 

would not have suffi cient quantities of blood 

to analyze, therefore making the analysis of 

Cyanide/

Metabolitea Half-lives Toxicokinetic 

Data

Storage 

Stability

Biological

Sampleb Speciesc

CN Minutes-hours Some in a few 

species Low

Blood, Urine, Saliva, 

Tissue, Expired Air, 

Rumen 

Human, Fish, 

Cow, Mouse, 

Rat, Guinea Pig, 

Goat, Horse

SCN Hours Limited Medium

Blood, Urine, Saliva, 

Tissue, Milk, Gastric 

Fluid, Cerebrospinal 

Fluid

Human, Fish, 

Rat, Mouse, Pig, 

Goat, Horse

ATCA Unknown None High Blood, Urine, Feces, 

Saliva, Tissue

Human, Fish, 

Rat

CN-protein

adducts

Presumably 

days-months None Unknown Blood Human

a CN – cyanide, SCN – thiocyanate, ATCA – 2-amino-2-thiazoline-4-carboxylic acid.

b Cyanide or the metabolite has been analyzed from this matrix with an analytical method reported in at least one research article or in the 

authors’ laboratories.

c Cyanide or the metabolite has been analyzed from this species with an analytical method reported in at least one research article or in 

the authors’ laboratories.

Table 2. Comparison of cyanide and its metabolites for verifi cation of cyanide exposure.

74
tissue samples indispensable. Although tissue 

may be the only choice for certain species, 

most published methods for determination of 

cyanide exposure are for analysis of biological 

fl uids. While some methods developed for 

biological fl uids may fail for tissue analysis, a 

number of these bioanalytical methods can 

be slightly modifi ed for analysis of tissues. 

Another consideration is that rates of cyanide 

metabolism may be inconsistent between 

organs because of variable rhodanese and 

sulfur donor concentrations (18). Therefore, 

careful selection of tissue, depending on the 

analyte to be determined, can be extremely 

important. Also, analytes must be extracted 

from the tissue for most analytical methods. 

Therefore, an extra extraction and solid 

sample processing may be necessary for tissue 

analysis. 

The detection of cyanide and its 

metabolites in biological samples

The determination of cyanide, thiocyanate, 

ATCA, and cyanide-protein adducts in 

biological fl uids and tissues, is useful for 

forensic, clinical, research, law enforcement, 

and veterinary purposes. Methods of analysis 

include spectrophotometric or fl uorescence 

methods (37, 41, 43, 44, 51, 60, 61, 67, 76, 

77, 82, 87, 89, 99-129), electrochemical 

methods (90, 130-139), gas chromatography 

(33, 38, 45, 62, 63, 66, 70, 78, 79, 81, 83, 86, 

94, 124, 140-158), and liquid chromatography 

techniques (37, 46, 48, 69, 80, 84, 88, 89, 93, 

95, 97, 98, 133, 159-171). Choosing from 

the many available types of methods and 

biomarkers of cyanide exposure, complicated 

by numerous discrepancies in the literature 

between these methods makes selection of 

an analytical method for a specifi c purpose 

nontrivial. Factors that will infl uence the initial 

choice of which biomarker and analytical 

technique to use are cellular absorption and 

detoxifi cation kinetics, sampling and analysis 

time, sample storage time and conditions, 

sample matrix, interferences, sensitivity, 

available instrumentation and equipment, 

expertise, and cost. Table 3 describes some 

differences between the groups of methods 

listed above in terms of these considerations.

Sample preparation and storage

Careful sample preparation of biological 

samples containing cyanide or its metabolites 

is a key element to producing accurate results. 

(Note: It is always necessary to consider the 

volatility of HCN when working with samples 

that may have signifi cant concentrations of 

cyanide and the dangers that it may pose to 

laboratory personnel.) A major problem in 

the analysis of cyanide and thiocyanate is their 

interconversion, which occurs during sample 

preparation and storage and leads to inaccurate 

results. The amount of cyanide within the 

sample can be altered during storage by up 

to 66% in 14 days, depending on the storage 

temperature (20, 33, 77, 172-175). A number 

of researchers have attempted to address this 

problem. In preparation of samples for gas 

chromatographic (GC) analysis, Seto et al. 

(79) demonstrated that artifi cial formation 

of HCN from thiocyanate in blood occurred, 

and later showed that ascorbic acid prevents 

artifactual cyanide formation at temperatures 

below 63°C (175, 176). Sano et al. (88) 

found that, under the conditions studied, 

pretreatment of blood samples with water 

and methanol was successful in preventing 

artifactual formation of cyanide from 

thiocyanate. There are a number of methods 

to help prevent artifi cial formation of cyanide 

during storage, and if samples containing 

cyanide are to be stored before analysis, these 

methods should be considered (see Suggested 

procedures for delayed analysis of biological samples

section). The stability of ATCA in biological 

samples under a number of storage conditions 

has been established (45, 48). 

75
Earlier methods of cyanide analysis involved 

extensive sample preparation in which 

the sample was acidifi ed (typically with 

sulfuric or phosphoric acid), and HCN was 

transferred to alkaline solution by distillation 

or microdiffusion. This served to concentrate 

the cyanide and to separate it from potential 

interferences (33, 43, 70, 77, 82, 105-107, 125, 

144, 151). Buffered hydroxocobalamin (67, 

177) or methemoglobin (100) solutions have 

also been used to capture liberated HCN. 

This pre-treatment method can be used 

prior to most analytical techniques for the 

determination of cyanide. For example, this 

procedure has been used prior to ion-selective 

electrode (ISE) analysis (15). For GC analysis 

of HCN, the procedure of liberating HCN 

by acidifi cation (without capture in solution) 

is extensively used prior to headspace analysis 

(33, 70, 94, 144). It should be noted that when 

using methods that liberate HCN, rubber 

septa or stoppers can react with gaseous 

HCN. Therefore, polytetrafl uoroethylene 

septa should be used (64, 151). 

Individual pretreatment steps (i.e., 

derivatization) are generally necessary for 

detection of cyanide by spectrophotometry 

or fl uorescence. For example, Lundquist 

and Sörbo (123) used a modifi cation of the 

König reaction (Figure 2; discussed in more 

depth in the Spectrophotometric, luminescence, 

and atomic absorption methods section) to 

spectrophotometrically determination blood 

cyanide concentrations by high-performance 

liquid chromatography (HPLC) and 2,3-

napthalenedialdehyde (NDA) and taurine 

have been effectively used with HPLCfl uorescence or as a stand-alone fl uorescence 

method to produce highly sensitive methods 

for the determination of cyanide (36, 37, 69, 

84, 88, 159). Other derivatization schemes 

will be discussed with individual analytical 

methods below. 

Thiocyanate sample preparation is normally 

limited to derivatization that is intended 

to increase a specifi c detector’s response to 

the ion. While initial sample preparation 

is not common, ion exchange columns 

could be used to separate thiocyanate from 

biological sample components. Thiocyanate 

is weakly spectrophotometrically active, 

but it is normally derivatized with a strong 

absorber or fl uorophore prior to analysis 

because of its weak absorbance. For example, 

3-bromomethyl-7-methoxy-1,4-benzoxazin2-one has been used effectively for the 

fl uorometric determination of SCN by 

HPLC (163). Others have also used variations 

of the König reaction to produce stronger 

spectrophotometric signals (95, 118, 123). 

Multiple methods have been proposed for 

the simultaneous analysis of cyanide and 

thiocyanate. Methods for simultaneous 

analysis are generally limited to 

chromatographic methods and preparation 

can involve derivatization. For example, Kage 

et al. (83) used pentafl uorobenzyl bromide 

as the derivatizing agent for simultaneous 

GC-mass spectrometric (MS) analysis of 

cyanide and thiocyanate, and Funazo et al. 

(146) quantitatively methylated cyanide and 

thiocyanate for GC analysis with a nitrogenphosphorous detector (NPD). Multiple 

authors have used other derivatization 

techniques for GC analysis of cyanide or 

thiocyanate in biological samples (145, 150, 

153, 154). 

ATCA has been mainly prepared for analysis 

using cation exchange solid-phase extraction 

columns and individual pretreatment steps, 

depending on the analytical technique. 

Lundquist et al. (48) and Logue et al. (45) both 

used cation exchange solid phase extraction 

columns to separate ATCA from a number of 

components in biological samples. Lundquist et 

al. (48) further purifi ed disulfi des from samples 

76
by reduction and subsequent separation with 

another column. Both Bradham et al. (60) and 

Lundquist et al. (48) heated ATCA in strong 

basic solution to open the ring structure of 

ATCA and produce a thiol group. Bradham 

et al. (60) then used hydroxymercuribenzoate, 

and subsequently diphenylthiocarbazone, to 

produce a colored product that was analyzed 

spectrophotometrically. Lundquist et al. (48) 

derivatized ATCA (after opening the ring) 

with a coumarin derivative and analyzed by 

HPLC. After using a cation exchange column 

(discussed above), Logue et al. (45) prepared 

ATCA for GC-MS analysis by derivatizing 

with a silylating agent.

 Sample preparation for cyanide-protein 

adducts involved isolation of the protein of 

interest with subsequent enzymatic digestion 

of the adducted protein. Fasco et al. (29) used 

this technique to analyze protein fragments 

after digestion with trypsin. Although this 

method was time-consuming and required 

highly powerful instrumentation, the authors 

were able to detect protein-cyanide adducts 

from the plasma fraction of human blood.

Spectrophotometric, luminescence, and 

atomic absorption methods 

Early spectrophotometric methods of cyanide 

analysis from biological fl uids were often 

based on the König synthesis (Figure 2) (39, 

101-107, 178). König dye synthesis involves 

oxidation of cyanide using chloramine-T (82, 

109, 118, 125), hypochlorite (87, 110, 123) or 

bromine water (41, 101, 102, 119) to form a 

cyanogen halide (see Figure 2). The cyanogen 

halide is then reacted with an aromatic amine 

(normally pyridine) to produce a glutaconic 

aldehyde product that is measured in the 

visible region. These methods have adequate 

sensitivity, but they lack specifi city due to 

interferences from other chemical species 

commonly present during the analysis of 

cyanide, especially thiocyanate and thiosulfate 

(179). Also, they often require lengthy 

microdiffusion preparations and the products 

are unstable. Modifi cations have been 

developed that yield more stable reagents and 

increased precision for this type of reaction 

(41, 44, 87, 115-117, 119, 125). 

Spectrophotometric analysis for thiocyanate 

is often a variation of the König reaction 

described above. Hypochlorite and 

thiocyanate react to form the cyanogen 

chloride, then either pyridine-malononitrile 

(76) or barbituric acid-pyridine reagent (95, 

118, 123) can be used as coupling agents. 

Cyanogen chloride can also be reacted with 

isonicotinic acid to produce a glutaconic 

aldehyde. Condensation of this aldehyde 

with two molecules of 1,3-dimethylbarbituric 

acid produces a dye which can be analyzed 

spectrophotometrically (89). Other early 

methods (108) involved oxidation of the 

thiocyanate to hydrogen cyanide, with 

aeration into alkali solution, permitting the 

determination of cyanide as described above 

for the König reaction. The modifi ed König 

reaction was also applied to the simultaneous 

analysis of cyanide and thiocyanate (101, 

CN -

H3C S

O

O

N

Cl

CN-l

-

chloramine-T

H3C S

O

O

NH 2

N

CN-H 2 +

O O

Glutaconic aldehyde

+ 2H2O

+ HCl

+ 2H+

Figure 2. König reaction for the spectrophotometric 

analysis of cyanide.

77
102). Nagashima (109) used the differences 

in the rates of the reaction of cyanide and 

thiocyanate with chloramine-T and variations 

in pH dependence for the simultaneous 

spectrophotometric determination of cyanide 

and thiocyanate. 

ATCA has also been analyzed 

spectrophotometrically. Bradham et al. (60) 

analyzed ATCA from urine as described 

above. Limitations for this method include 

interference from a number of species 

(including cyanide ions) and it is time 

intensive.

Fluorescence (37, 110-114) methods have 

also been applied to the determination of 

cyanide in biological fl uids. As with the 

spectrophotometric methods, fl uorescence 

methods also require extraction techniques 

to isolate cyanide and eliminate interferences 

from blood. A number of sensitive 

fl uorometric assays to determine cyanide, free 

of thiosulfate interference (a problem with a 

number of spectrophotometric methods) 

have been developed with greater sensitivity 

than spectrophotometric methods (110, 111, 

114). One specifi c fl uorometric method of 

analysis of cyanide is reaction of cyanide with 

NDA and taurine to create a highly fl uorescent 

molecule that can be used for the fl uorometric 

determination of cyanide (37, 159). Cyanide 

from whole blood has also been determined 

by fl ow injection chemiluminescence (180). 

Acidifi cation and distillation (as mentioned 

above) was used to separate cyanide from 

interfering whole blood components and 

a microchip based reactor was used to mix 

reagents to produce chemiluminescence. 

Although non-chromatographic fl uorescence 

methods for the determination of thiocyanate 

and ATCA have not been published, the 

possibility of derivatization with fl uorescent 

derivatizing agents exists. For instance, 

3-bromomethyl-7-methoxy-1,4-benzoxazin2-one and coumarin dyes have been used 

effectively for the HPLC-fluorometric 

determination of thiocyanate (163) and 

ATCA (48), respectively. Cyanide-protein 

adducts have not been analyzed by fl uorescent 

methods.

Atomic absorption (AA) methods are indirect 

methods of cyanide or thiocyanate analysis. A 

metal is added to a sample and a metal complex 

is formed with the analyte. This complex 

either precipitates or is extracted into an 

organic solvent and subsequently analyzed by 

AA. For example, cyanide has been analyzed 

by complexing an iron(II)-phenanthroline, 

and extracting the complex in chloroform 

for subsequent AA analysis (181). Chattaraj 

and Das (127) used this technique with fl ame 

AA by forming a complex with copper to 

determine thiocyanate from biological fl uids. 

This technique has also not been used to 

determine ATCA or cyanide-protein adducts. 

Electrochemical, ion-selective electrode, 

and biosensor methods 

Many electrochemical methods for the 

detection of cyanide and thiocyanate exist, 

but few of these methods have been applied 

to their analysis in biological samples (15, 

90, 131, 134, 135, 182). Benefi ts of using 

electrochemical methods are high sensitivity 

(for some methods) and quick analysis time. 

However, they can be subject to multiple 

interferences from many organic and inorganic 

ions, including sulfi de, ClO4

-

, NO2

-

, N3

-

, and I-

(130, 183). Electrochemical methods can also 

be hampered by narrow working concentration 

ranges and may require large sample sizes 

(132, 137). Westley and Westley (130) used a 

silver rotating disk electrode, and a dropping 

mercury electrode for the voltammetric 

determination of cyanide and thiocyanate in 

biological samples, including plasma, tissue, 

and whole blood. Electrochemical detection 

78
can also be used for analysis of cyanide and 

thiocyanate with ion chromatography (133).

Polymeric membrane-based ion selective 

electrodes (ISEs) have been developed 

to address some of the issues limiting 

electrochemical analysis of cyanide and 

thiocyanate (21, 132, 136, 183-188). ISEs can 

exhibit rapid response, high sensitivity, wide 

linear range, low cost, and they are usually 

simple to operate. The polymeric membrane 

in ISEs contains an ion carrier that interacts 

selectively with the analyte. To selectively 

analyze cyanide or thiocyanate, the ion carrier 

must strongly interact with the analyte anion 

and weakly interact with other anions. This 

interaction is often enhanced by the use of 

a metal-ligand interaction. Although many 

ISEs exist, they are somewhat more limited 

for anions compared with cations (183). 

ISEs have been developed for cyanide analysis, 

but few have been used for analysis of cyanide 

from biological samples. This is due to their 

interaction with multiple ion interferences. If 

these interferences can be removed, then ISE 

methods can be used for the determination 

of cyanide from biological samples. In fact, 

the standard method for analysis of cyanide 

from fi sh tissues is based on ISEs (184). For 

this method, tissue samples are prepared 

by homogenizing, acidifying, and distilling 

internal organs of fi sh species (similar 

to those mentioned above in the Sample 

preparation and storage section). Cyanide present 

in the homogenized tissue is converted to 

HCN and captured in an alkaline solution 

following distillation. A cyanide ISE is then 

used to analyze for cyanide based on its 

interaction with silver. Another ISE test using 

gold disc electrodes coated with a sulfonated 

tetrafl uoroethylene copolymer was recently 

developed by Lindsay and O’Hare for the 

analysis of cyanide in blood without sample 

pretreatment (185).

Thiocyanate, due to its lipophilicity, is highly 

suited for electrostatic interaction techniques, 

and ISEs have been used successfully for 

the selective determination of thiocyanate 

(21). ISEs have been described that exhibit 

good agreement with values determined 

by ion exchange chromatography (136) or 

spectrophotometric methods (132, 183, 186, 

187), and were used to analyze thiocyanate 

from serum (136), urine (132, 183, 186-

188), and saliva (132, 183, 186, 188). These 

include an ISE based on crystal violet 

thiocyanate or methylene blue thiocyanate 

in nitrobenzene (136), and a highly selective 

thiocyanate polymeric membrane sensor 

that contained a nickel(II)-azamacrocycle 

complex coated on a graphite electrode 

(132). The Ni(II)-electrode showed a great 

enhancement in selectivity coeffi cients and 

detection limits from previously reported 

electrodes and was successfully used for the 

analysis of thiocyanate in urine, saliva and 

milk. Other electrodes which demonstrated 

good selectivity for thiocyanate in biological 

samples were a poly(vinyl chloride) (PVC) 

membrane electrode based on a nickelhexaazacyclotetradecane derivative (186), 

a PVC membrane electrode with an 

unsymmetrical nickel(II) macrocyclic complex 

as an ion carrier (183), and a graphite electrode 

based on iron phthalocyanine membranes 

with sodium tetraphenylborate as a lipophilic 

anionic additive (187). 

Many biosensors (small detection devices 

normally based on biological activity toward an 

analyte) exist for the determination of cyanide, 

including microbial cyanide sensors, sensors 

based on the enzyme inhibition of cyanide, 

and sensors based on cyanide degrading 

enzymes. Most biosensors have advantages 

of being portable, low cost, easy to use, 

and can have high selectivity. Limitations of 

biosensors include degradation of biological 

components that make up these sensors, 

79
inconsistent electrochemical signals, and 

diffi culty producing suffi cient quantities and 

activities of enzymes or microbes on which 

these sensors depend. Most of the biosensors 

developed for cyanide analysis have not been 

applied to the analysis of biological samples, 

although a method for the determination of 

cyanide in fi sh was recently described (15). 

Organs of the fi sh were homogenized with 

NaOH and a fungal enzyme extract was 

used to produce formate from metal-cyanide 

complexes. Then formate dehydrogenase and 

nicotinamide adenine dinucleotide (NAD+) 

were added to convert the formate into CO2

, 

which reduced NAD+ to NADH. NADH 

was monitored spectrophotometrically and 

used to determine the amount of cyanide in 

the sample.

Currently, biosensors have not been applied 

to thiocyanate detection from biological 

samples. Also, electrochemical and biosensor 

methods have not been applied to analyze 

for ATCA and cyanide-protein adducts from 

biological samples.

Liquid Chromatography

The complex nature of biological matrices, 

small concentrations of cyanide and its 

metabolites, and the high number of species 

that interfere with spectrophotometric, 

luminescent, and electrochemical methods 

have necessitated analysis of cyanide and 

its metabolites by more powerful methods. 

Liquid chromatographic techniques can 

determine trace amounts of an analyte and can 

effi ciently separate analytes from interfering 

components in the matrix. Chromatographic 

techniques, both liquid and gas, also have the 

ability to simultaneously analyze for cyanide 

and thiocyanate. For these reasons, they have 

gained popularity in analysis of cyanide and 

cyanide metabolites in biological samples. 

Three types of liquid chromatography have 

been used to analyze cyanide: reverse-phase 

high-performance liquid chromatography 

(RP-HPLC) (36, 46, 48, 80, 84, 88, 93, 159, 

160, 162-164), ion chromatography (IC) (69, 

95, 97, 98, 133, 138, 164-169, 189-191), and 

capillary electrophoresis (139). RP-HPLC 

methods are common, but generally require 

pretreatment steps for each anion or multiple 

post-column reagents. Ion chromatography 

is often used for thiocyanate analysis, and 

although these methods are categorized 

separately, ion chromatography is often a 

modifi ed RP-HPLC method (i.e., a column 

modifi er is added to the mobile phase to create 

an ionic stationary phase). Some common 

detectors used in the liquid chromatographic 

analysis of cyanide or its metabolites include 

spectrophotometric (46, 89, 95, 160, 166), 

fl uorescence (37, 84, 88, 93, 159, 163, 164), 

electrochemical (162, 165, 168, 191), or mass 

spectrometric (29, 80) detection. 

Several groups have adapted the 

spectrophotometric detection of cyanide and 

thiocyanate in blood and urine based on the 

König reaction to RP-HPLC (89, 95, 161, 

166). This reaction has also been used for 

HPLC with fl uorometric detection. Toida et 

al. (84) analyzed cyanide in blood at picomole 

levels with HPLC and fl uorometric detection 

by using a variant of the König reaction, 

replacing pyridine with pyridine-barbituric 

acid. Fluorescence detection was also used 

for the determination of cyanide in human 

erythrocytes and whole blood using RPHPLC with pre-column derivatization with 

NDA and taurine (88). A number of other 

fl uorometric HPLC methods have been 

developed for the analysis of cyanide and 

its metabolites from biological fl uids (69, 

88, 93, 159, 163). Thiocyanate has also been 

analyzed using RP-HPLC with fl uorometric 

detection. Tanabe et al. (93) used HPLC 

with fl uorometric detection to determine 

80
thiocyanate in saliva and serum based on the 

formation of fl uorescent cerium (III) from 

cerium (IV) by a redox reaction. RP-HPLC 

has also been used for the simultaneous 

detection of cyanide and thiocyanate. Using 

pentafl uorobenzylbromide as derivatizing 

agent, Liu and Yun (46) simultaneously 

determined cyanide and thiocyanate in blood 

and milk. Mass spectrometric detection 

was used by Tracqui and Tamura (80) after 

microdiffusion sample preparation followed 

by derivatization with NDA and taurine for 

HPLC-MS analysis of cyanide in blood.

ATCA and cyanide-protein adducts have 

also been analyzed with RP-HPLC (29, 48). 

Lundquist (48) used HPLC with fl uorometric 

detection for the determination of ATCA 

in urine. Although this method was time 

consuming, it was able to detect ATCA in 

the urine of smoking individuals. RP-HPLC 

with mass spectrometric detection has been 

used to determine cyanide and blood-protein 

adducts. Fasco et al. (29) used RP-HPLC 

with tandem mass spectrometric detection 

to analyze cyanide-adducted proteins from 

human plasma. The method involved isolation 

and enzymatic digestion of cyanide-adducted 

human serum albumin.

A number of authors have used ion exchange 

(or ion interaction) chromatography to 

analyze biological fl uids for thiocyanate 

because of the ease of thiocyanate analysis. 

Chinaka et al. (69) used NDA derivatization 

of cyanide with an ion exchange column, 

allowing the simultaneous determination 

of cyanide and thiocyanate. Lundquist 

et al. (95) used an ion exchange column 

with visible spectrophotometric detection 

for the determination of thiocyanate in 

serum and urine. Connolly et al. (98) used 

ion interaction LC with UV detection to 

analyze thiocyanate in urine. Other authors 

have coated normal reverse-phase HPLC 

columns or used ion-pairing agents to analyze 

thiocyanate. Examples of this type of analysis 

include, a RP-HPLC column coated with 

cetyldimethylamine (97, 169), a zwitterionic 

micellar-coated stationary phase (189), and 

bovine serum albumin as the stationary phase 

with tartaric acid as the eluent (190). Brown et 

al. (164) used a cetylpyridinium coated reversephase column to create an ion-exchange 

column for the analysis of thiocyanate from 

rainbow trout plasma in a pharmacokinetic 

study of thiocyanate exposure. Cookeas 

and Efstathiou (191) successfully analyzed 

thiocyanate in saliva with fl ow injection and 

ISE detection using a cobalt-phthalocyanine 

modifi ed carbon paste electrode. ISEs have 

also been used to detect thiocyanate in urine 

following ion chromatography (133).

Capillary electrophoresis (CE) has been used 

successfully for the analysis of thiocyanate 

in biological fl uids (139). Glatz et al. (139) 

analyzed blood, urine, and saliva samples for 

thiocyanate with CE and spectrophotometric 

detection no sample preparation aside from 

dilution. CE methods for cyanide, ATCA, 

and cyanide-protein adducts have not been 

suggested. 

Gas Chromatography

One of the most common methods for analysis 

of cyanide, thiocyanate, and more recently 

ATCA is gas chromatography. Common 

detectors used for analysis of cyanide or its 

metabolites are the electron capture detector 

(ECD) (66, 70, 81, 140, 141, 151, 154, 156, 

192), nitrogen-phosphorus detector (NPD) 

(33, 62, 63, 78, 79, 86, 124, 144-147, 150, 152, 

155), and mass spectrometric (MS) detector 

(45, 83, 94, 148, 153, 158). Although most 

of these methods are used for detection of 

cyanide in blood some groups have applied 

gas chromatography techniques for other 

biological matrices and for the detection of 

thiocyanate and ATCA. 

81
For the detection of cyanide from biological 

matrices, no specifi c derivatization is necessary 

as HCN is volatile. Therefore, the most 

common pre-analysis step in GC analysis 

of cyanide is sampling of cyanide from the 

sample head space. Either equilibrium or 

dynamic headspace methods can be used to 

prepare a sample for GC analysis (63, 66, 

70, 79, 81, 94, 124, 141, 143, 149, 152, 158). 

Another pre-analysis step for cyanide analysis 

is cryogenic oven trapping, which has been 

used to trap liberated HCN into headspace 

with high resolution and sensitivity (86, 147). 

Because thiocyanate and ATCA are nonvolatile, they require chemical modifi cation 

(normally derivatization) to allow analysis by 

GC. Chemical modifi cation is also necessary 

for analysis of cyanide by ECD (because of its 

poor response) or for simultaneous analysis of 

cyanide and thiocyanate. Therefore, a number 

of pretreatment steps have been developed to 

facilitate the analysis of cyanide, thiocyanate, 

and ATCA by GC.

In GC analysis, the NPD permits the 

sensitive and specifi c detection of nitrogencontaining compounds, and has been used 

for the detection of cyanide and thiocyanate 

in plasma, urine and saliva (79, 83, 145, 

146, 150). However, this detector may be 

unstable at times, and is less sensitive than 

some other types of GC detectors. This has 

led to the creation of analytical methods 

that take advantage of more stable and 

sensitive detectors such as the ECD and MS. 

When using ECD detectors in GC analysis 

of HCN, derivatization is required before 

analysis. Derivatization of cyanide has been 

performed by alkylation of cyanide (83) 

or the conversion of HCN into cyanogen 

chloride by choramine-T oxidation (Figure 2) 

(70, 151, 156). Kage and coworkers (83) used 

GC-ECD to simultaneously analyze cyanide 

and thiocyanate using an extractive alkylation 

technique. 

One of the most sensitive methods for 

analysis of cyanide, thiocyanate, and ATCA 

is GC-MS (83, 94). With MS detection, stable 

isotope standards (e.g. 13CN for 12CN) can be 

used to correct for matrix effects common 

to cyanide and cyanide metabolites. This 

can eliminate the need for standard addition 

techniques and matrix matching. Dumas et 

al. (94) used stable isotope standards with 

GC-MS and head space analysis to analyze 

cyanide concentrations. Kage and co-workers 

also used GC-MS to analyze both cyanide and 

thiocyanate simultaneously (83). 

ATCA has also been analyzed by GC-MS. 

Logue et al. (45, 157) analyzed ATCA in plasma 

and urine using GC-MS by fi rst converting 

the non-volatile metabolite into a volatile 

form using trimethylsilyl-trifl uoroacetamide. 

Cyanide-protein adducts have not been 

analyzed by GC-MS.

Suggested procedures for delayed analysis 

of biological samples

For cyanide analysis, a sample should be 

collected quickly after exposure and cyanide 

analysis should be performed as soon as 

possible because of the rapid detoxifi cation 

of cyanide from blood samples (discussed 

above). However, if analysis of cyanide 

cannot be performed quickly and storage of 

biological samples is necessary, the following 

should be considered: 

1) Volatility and nucleophilicity of cyanide. As 

described above, HCN is volatile and 

cyanide ion is nucleophilic. Tightly 

sealed vials, low temperatures, high 

pH, and the addition of preserving 

agents are common procedures that 

have been used to prevent evaporative 

loss of cyanide. Storing samples at low 

temperatures is extremely important 

to reduce evaporative loss, and slow 

biochemical reactions. However, there 

82
are many discrepancies in the literature 

when evaluating the stability of cyanide 

in biological fl uids under various 

conditions (43, 87, 131, 172, 179). 

Generally, nucleophilic losses are reduced 

by adding sequestering agents (e.g., 

hydroxocobalamin) or chemicals that 

produce sequestering agents (e.g. sodium 

nitrite to produce methemoglobin) (87, 

123, 193). One method found to improve 

cyanide stability is the addition of silver 

ions to biological samples (87).

2) Cyanide concentration varies in biological 

components. Cyanide in blood primarily 

resides in erythrocytes (red blood 

cells) (64, 82, 87, 174, 194) by 

binding to methemoglobin, forming 

cyanomethemoglobin. Cyanide may 

also be present in plasma, especially 

if cyanide concentrations exceed 

erythrocyte concentrations (82, 87). To 

ensure accurate cyanide concentrations 

when analyzing blood, collection 

containers that contain anticoagulants 

(e.g., heparin) should be used to prevent 

clotting. Analysis of cyanide from tissues 

requires knowledge of the behavior 

of cyanide in specifi c organs since the 

enzyme that catalyzes conversion of 

cyanide to thiocyanate has highly variable 

concentrations depending on the organ. 

Therefore, in specifi c organs, there will be 

little to no cyanide because of extremely 

fast metabolism to thiocyanate. 

3) Potential for cyanide formation during storage. 

Artifactual formation of cyanide 

may also occur in biological samples 

depending on storage conditions (77, 

172-174). It has been suggested that 

oxyhemoglobin (175), thiocyanate 

oxidase (173, 174), and white blood cells 

(77) may oxidize thiocyanate to cyanide 

and these reactions are dependent 

on the temperature and pH of the 

sample. Microorganisms may also be 

responsible for cyanide production and 

low temperature storage will help to 

eliminate their growth (173). 

These considerations are common to all the 

analytical methods for analysis of cyanide 

from biological samples and certainly 

contribute to discrepancies in similar studies 

in the literature. For post-mortem analysis of 

cyanide, production and transformation of 

cyanide must be considered when interpreting 

results along with other considerations 

discussed above (74, 195-198).

Some of the same issues must also be 

considered for thiocyanate, as a number of 

problems with storage of samples to be 

analyzed for thiocyanate have been found 

(56). This may be due to interconversion of 

thiocyanate and cyanide over time and the 

removal and production of thiocyanate by 

biological processes other than cyanide 

detoxifi cation (24, 58). It has been suggested 

that ATCA is not involved in other biological 

processes and it has been found to be stable 

during storage (18, 45, 47, 48, 59). While 

ATCA may not have the storage issues of 

cyanide and thiocyanate, there is little 

information on its toxicokinetics, which 

currently limits its use as a biomarker for 

cyanide exposure. Also, cyanide-protein 

adducts have recently been discovered, and 

therefore little information about the 

toxicokinetics and stability of these adducts is 

known. 

Conclusions

The analytical determination of cyanide and 

its metabolites is not an easy task due to 

chemical properties, biological activities, and 

limited research. Numerous methods have 

been developed and each has its own 

advantages and disadvantages. However, they 

83
have all provided insight into the verifi cation 

of cyanide exposure from analysis of 

biological samples. Table 3 provides a 

comparison of analytical techniques for 

analysis of cyanide or its metabolites based 

on sensitivity, specifi city, sample size, capacity, 

expertise necessary to perform the method, 

and cost. Some other key pieces of information 

should also be considered prior to choosing a 

method to perform: 1) Was preservation of 

cyanide and its metabolites during storage 

addressed?; 2) Were typical interferences for 

the biological matrix of interest removed?; 3) 

Were analysis procedures that could result in 

the loss of cyanide or its metabolites used 

(i.e., heating or acidifi cation)? 

For the analysis of cyanide, the largest 

inconsistency in the literature is analysis with 

different preservation techniques. Regardless 

of the analytical method, a preserving 

technique needs to be considered so that 

accurate concentrations of cyanide can be 

found (75, 87, 112, 123, 128, 197-200). If 

cyanide is analyzed, biological samples should 

be collected and analyzed as soon as possible 

for confi rmation or refuting cyanide exposure. 

One also needs to consider that all biological 

samples will contain endogenous levels of 

cyanide (and its metabolites). Therefore, 

baseline levels of the analyte measured should 

be known prior to concluding an exposure 

occurred. Problems with direct analysis of 

cyanide, including short half-lives, artifactual 

formation of cyanide, and interconversion 

of cyanide and thiocyanate, contribute to 

diffi culties in the analysis of cyanide for all 

analytical methods designed to determine 

cyanide in biological samples.

While cyanide is most often analyzed to 

determine cyanide exposure, one should 

consider using analytical techniques that 

analyze for cyanide metabolites as well as those 

that analyze for cyanide directly. Although 

cyanide metabolites may offer longer halflives, they also have a number of drawbacks. 

For thiocyanate, the main drawback is large 

and variable background concentrations 

in biological samples. Other disadvantages 

include are interconversion of cyanide and 

thiocyanate, and the use of thiocyanate by 

other biological processes not directly related 

to cyanide metabolism. For ATCA and 

cyanide-protein adducts, the main drawback 

is the limited amount of research available 

on toxicokinetics and relationships of these 

metabolites to cyanide exposure. Care should 

be taken when choosing an analytical method 

to consider not just the parameters of the 

analytical method but also the toxicokinetics 

of cyanide and its metabolites.

84
Techniqueb Sub-category Analytec Matrices Sensitivityd Specifi cityd

Sample 

Sized Capacityd Expertised Costd Refs.

UV-Vis None CN, SCN, ATCA Blood, Urine, Saliva Low Low 0.5-1 mL Medium Low Low

(41, 44, 60, 61, 76, 77, 82, 87, 89, 101-105, 108, 109, 115-119, 123, 178, 179)

Luminescence

Fluorescence CN Blood Medium Medium 0.5-1 mL Medium Medium Low- Medium (77, 110-114, 124)

Chemiluminescence CN Blood Medium- High Medium 3 μL Medium Medium Low (180)

Electrochemistry None CN, SCN Blood, Saliva, Tissue Medium- High Medium- High 1-5 mL Medium- High Low Very Low -Low (15, 21, 90, 130-137, 182, 183, 185-187, 201, 202)

AA Indirect AA SCN Blood, Saliva Medium- High Medium 1 mL Low Medium Medium- High (127)

Biosensor None CN, SCN Blood, Urine, Saliva Medium- High Medium- High 1-5 mL Medium- High Low Very Low -Low (15)

LC

RP-HPLC UV CN, SCN Blood, Urine Medium High 10-100 μL High Medium- High Medium (89, 95, 160, 166)

RP-HPLC 

fl uorescence

CN, SCN, 

ATCA

Blood, Urine, 

Saliva High High 10-100 μL High High Medium (37, 84, 88, 93, 159, 163, 164)

RP-HPLC 

electrochemical SCN Urine Medium- High High 10-100 μL High Medium- High Medium (162, 165, 168)

RP-HPLC mass 

spectrometric CN Blood Very High Extremely High 10-100 μL High Very High High (80)

RP-HPLC MS-MS CN-protein adduct Blood Extremely High Extremely High 10-100 μL Low Extremely High Extremely High (29)

IC CN, SCN Blood, Urine, Saliva Medium High 1-100 μL High High Medium (97, 167, 169)

CE SCN Blood High High 1-10 nL High High Medium- High (138, 167)

GC

GC-NPD CN, SCN Blood, Urine, Saliva High Very High 1-10 μL Medium High High (33, 62, 63, 78, 79, 86, 124, 144-147, 150, 152, 155)

GC-ECD CN, SCN Blood, Urine, Saliva Very High Very High 1-10 μL High High High (66, 70, 81, 140, 141, 151, 154, 156, 192)

GC-MS CN, SCN, ATCA Blood, Urine, Saliva, Tissue Very High Extremely High 1-10 μL High High Very High (45, 83, 94, 148, 153, 158)

a This table is meant to give a general overview of analytical techniques to analyze cyanide and its metabolites along with a general idea about parameters specifi c to each analysis technique. Parameters of specifi c 

methods within a particular analysis technique may be outside of those listed. 

b UV-Vis – ultraviolet visible spectrophotometry, AA – atomic absorption, LC – liquid chromatography, RP – reverse phase, MS-MS – tandem mass spectrometric detection, IC – ion chromatography, CE – 

capillary electrophoresis, GC – gas chromatography, NPD – nitrogen phosphorous detector, ECD – electron capture detector.

c CN – cyanide, SCN – thiocyanate, ATCA – 2-amino-2-thiazoline-4-carboxylic acid. d These parameters are related to the general instrumental technique used and not each individual method of analysis. 

Table 3. Analytical Methods to determine cyanide and its metabolites in biological fl uids.a

85
References

(1) Vetter, J. (2000) Plant cyanogenic glycosides. 

Toxicon 38, 11-36.

(2) Knight, A. P. and Walter, R. G. (2002) 

Plants Causing Sudden Death, In A Guide 

to Plant Poisoning of Animals in North America.

(Knight, A. P. and Walter, R. G., Eds.), Teton 

NewMedia, Jackson, WY.

(3) McGorum, B. C. and Anderson, R. A. (2002) 

Biomarkers of Exposure to Cyanogens in 

Horses with Grass Sickness. Vet. Rec. 151, 

442-445.

(4) Conn, E. E. (1979) Cyanogenic glycosides. 

International Review of Biochemistry 27, 21-43.

(5) Hibbs, C. M. (1979) Cyanide and nitrate 

toxicoses of cattle. Veterinary and human 

toxicology 21, 401-403.

(6) Honig, D. H., Hockridge, M. E., Gould, R. 

M. and Rackis, J. J. (1983) Determination of 

cyanide in soybeans and soybean products. 

Journal of agricultural and food chemistry 31, 

272-275.

(7) Nartey, F. (1980) Toxicological aspects of 

cyanogenesis in tropical foodstuffs, pp 53-

73.

(8) ATDSR. (2004) Toxicological Profi le for 

Cyanide, U.S. Dept. of Health and Human 

Services, Atlanta, GA.

(9) Cicerone, R. J. and Zellner, R. (1983) The 

atmospheric chemistry of hydrogen cyanide 

(HCN). Journal of Geophysical Research, C: 

Oceans and Atmospheres 88, 10689-10696.

(10) Li, Q., Jacob, D. J., Bey, I., Yantosca, R. M., 

Zhao, Y., Kondo, Y. and Notholt, J. (2000) 

Atmospheric hydrogen cyanide (HCN): 

biomass burning source, ocean sink? 

Geophysical Research Letters 27, 357-360.

(11) Way, J. L. (1984) Cyanide intoxication and its 

mechanism of antagonism. Annual Review of 

Pharmacology and Toxicology 24, 451-481.

(12) Towill, L. E., Drury, J. S., Whitfi eld, B. L., 

Lewis, E. B., Galyan, E. L. and Hammons, 

A. S. (1978) Reviews of the environmental 

effects of pollutants. V. Cyanide, 205 pp., 

Oak Ridge Natl. Lab.,Oak Ridge,TN,USA.

(13) Solomonson, L. P. (1981) Cyanide as a 

metabolic inhibitor, pp 11-28.

(14) Way, J. L. (1981) Pharmacologic aspects of 

cyanide and its antagonism, pp 29-49.

(15) Mak, K. K. W., Yanase, H. and Renneberg, R. 

(2005) Cyanide fi shing and cyanide detection 

in coral reef fi sh using chemical tests and 

biosensors. Biosensors and Bioelectronics 20, 

2581-2593.

(16) Rubec, P. J. (1986) The effect of sodium 

cyanide on coral reefs and marine fi sh in 

the Philippines, In The First Asian Fish Forum

(Maclean, J. L., Dizon, L. B. and Hosillos, L. 

V., Eds.) pp 297-302, Asian Fisheries Society, 

Manila, Philippines.

(17) Barber, C. V. and Pratt, V. R. (1998) Poison 

and profi ts: cyanide fi shing in the IndoPacifi c. Environment 40, 4-9, 28-34.

(18) Baskin, S. I., Petrikovics, I., Kurche, J. S., 

Nicholson, J. D., Logue, B. A., Maliner, B. 

J. and Rockwood, G. A. (2004) Insights on 

cyanide toxicity and methods of treatment, 

In Pharmacological perspectives of toxic chemicals 

and their antidotes (Flora, S. J. S., Romano, 

J. A., Jr., Baskin, S. I. and Sekhar, K., Eds.) 

pp 105-146, Narosa Publishing House, New 

Delhi.

(19) Troup, C. M. and Ballantyne, B. (1987) Ch.2 

Analysis of Cyanide in biological fl uids and 

tissues., In Clinical and Experimental toxicology 

of cyanides. (Ballantyne, B. and Marrs, T. C., 

Eds.) pp 22-40, Wright, Bristol.

(20) Lindsay, A. E., Greenbaum, A. R. and 

O’Hare, D. (2004) Analytical techniques for 

cyanide in blood and published blood cyanide 

concentrations from healthy subjects and fi re 

victims. Analytica Chimica Acta 511, 185-195.

(21) Valdes, M. G. and Diaz-Garcia, M. E. 

(2004) Determination of thiocyanate within 

Physiological Fluids and Environmental 

Samples: Current Practice and Future Trends. 

Critical Reviews in Analytical Chemistry 34, 9-23.

(22) Bark, L. S. and Higson, H. G. (1963) A review 

of the methods available for the detection and 

determination of small amounts of cyanide. 

Analyst 88, 751-760.

(23) Metzler, D. E. (2001) Biochemistry: The chemical 

reactions of living cells, Academic Press, San 

Diego.

(24) Isom, G. E. and Baskin, S. I. (1997) 

Enzymes involved in cyanide metabolism., 

In Comprehensive Toxicology (Sipes, I. G., C.A., 

M. and Gandolfi , A. J., Eds.) pp 477-488, 

Elsevier Science, New York.

(25) Tsuge, K., Kataoka, M. and Seto, Y. (2000) 

Cyanide and thiocyanate levels in blood and 

saliva of healthy adult volunteers. Journal of 

Health Science 46, 343-350.

(26) Salkowski, A. and Penney, D. (1994) Cyanide 

poisoning in animals and humans: a review. 

Veterinary and human toxicology 36, 455-466.

(27) Baskin, S. I. and Brewer, T. G. (1997) Cyanide 

86
Poisoning, In Textbook of military medicine, 

Medical aspects of chemical and biological warfare

(Sidell, F. R., Takafuji, E. T. and Franz, D. 

R., Eds.) pp 271-286, Borden Institute, 

Washington.

(28) Borowitz, J. L., Isom, G. E. and Baskin, S. I. 

(2001) Acute and chronic cyanide toxicity, 

In Chemical warfare agents: Toxicity at low levels

(Somani, S. M. and Romano, J. A., Jr., Eds.) 

pp 301-319, CRC Press, Boca Raton.

(29) Fasco, M. J., Hauer III, C. R., Stack, R. F., 

O’Hehir, C., Barr, J. R. and Eadon, G. A. 

(2007) Cyanide adducts with human plasma 

proteins: albumin as a potential exposure 

surrogate. Chem Res Toxicol 20, 677-684.

(30) Noort, D., Benschop, H. P. and Black, R. 

M. (2002) Biomonitoring of exposure to 

chemical warfare agents: A review. Toxicology 

and Applied Pharmacology 184, 116-126.

(31) Turner, P. C., Dingley, K. H., Coxhead, J., 

Russell, S. and Garner, C. R. (1998) Detectable 

levels of serum afl atoxin B1-albumin 

adducts in the United Kingdom population: 

Implications for afl atoxin-B1 exposure in 

the United Kingdom. Cancer Epidemiology, 

Biomarkers and Prevention 7, 441-447.

(32) Dirikolu, L., Hughes, C., Harkins, D., Boyles, 

J., Bosken, J., Lehner, F., Troppmann, A., 

McDowell, K., Tobin, T., Sebastian, M., 

Harrison, L., Crutchfi eld, J., Baskin, S. I. and 

Fitzgerald, T. D. (2003) The toxicokinetics of 

cyanide and mandelonitrile in the horse and 

their relavance to the mare reproductive loss 

syndrome. Toxicology Mechanisms and Methods

13, 199-211.

(33) Calafat, A. M. and Stanfi ll, S. B. (2002) Rapid 

quantitation of cyanide in whole blood by 

automated headspace gas chromatography. 

Journal of Chromatography, B: Analytical 

Technologies in the Biomedical and Life Sciences

772, 131-137.

(34) Hartung, R. (1982) Cyanides and nitriles, In 

Patty’s Industrial Hygiene and Toxicology (Clayton, 

G. D. and Clayton, E., Eds.) pp 4845-4900, 

John Wiley & Sons, New York, NY.

(35) Sousa, A. B., Manzano, H., Soto-Blanco, B. 

and Górniak, S. L. (2004) Toxicokinetics 

of cyanide in rats, pigs and goats after oral 

dosing with potassium cyanide. Archives of 

Toxicology 77, 330-334.

(36) Sano, A., Takezawa, M. and Takitani, S. (1989) 

High performance liquid chromatography 

determination of cyanide in urine by precolumn fl uorescence derivatization. Biomedical 

Chromatography 3, 209-212.

(37) Sano, A., Takezawa, M. and Takitani, S. 

(1989) Spectrofl uorometric determination of 

cyanide in blood and urine with naphthalene2,3-dialdehyde and taurine. Analytica Chimica 

Acta 225, 351-358.

(38) Paul, B. D. and Smith, M. L. (2006) Cyanide 

and Thiocyanate in Human Saliva by Gas 

Chromatography-Mass Spectrometry. Journal 

of Analytical Toxicology 30, 511-515.

(39) Boxer, G. E. and Rickards, J. C. (1952) 

Determination of traces of hydrogen cyanide 

in respiratory air. Archives of Biochemistry and 

Biophysics 39, 287-291.

(40) Raju, J. and Gupta, V. K. (1989) 

Spectrophotometric determination of 

hydrogen cyanide in air and its application 

in biological samples. Asian Environment 11, 

66-72.

(41) Kaur, P., Upadhyay, S. and Gupta, V. K. 

(1987) Spectrophotometric determination 

of hydrogen cyanide in air and biological 

fl uids. Analyst (Cambridge, United Kingdom) 112, 

1681-1683.

(42) Lundquist, P., Rosling, H. and Soerbo, B. 

(1988) The origin of hydrogen cyanide in 

breath. Archives of Toxicology 61, 270-274.

(43) Pettigrew, A. R. and Fell, G. S. (1973) 

Microdiffusion method for estimation of 

cyanide in whole blood and its application 

to the study of conversion of cyanide to 

thiocyanate. Clinical Chemistry (Washington, 

DC, United States) 19, 466-471.

(44) Pettigrew, A. R. and Fell, G. S. (1972) Simplifi ed 

colorimetric determination of thiocyanate 

in biological fl uids, and its application to 

investigation of the toxic amblyopias. Clinical 

Chemistry (Washington, DC, United States) 18, 

996-1000.

(45) Logue, B. A., Kirschten, N. P., Petrikovics, 

I., Moser, M. A., Rockwood, G. A. and 

Baskin, S. I. (2005) Determination of the 

cyanide metabolite 2-aminothiazoline-4-

carboxylic acid in urine and plasma by gas 

chromatography-mass spectrometry. Journal 

of Chromatography, B: Analytical Technologies in 

the Biomedical and Life Sciences 819, 237-244.

(46) Liu, X. and Yun, Z. (1993) High-performance 

liquid chromatographic determination of 

thiocyanate anion by derivatization with 

pentafl uorobenzyl bromide. Journal of 

Chromatography 653, 348-353.

(47) Wood, J. L. and Cooley, S. L. (1956) 

Detoxication of cyanide by cystine. Journal of 

87
Biological Chemistry 218, 449-457.

(48) Lundquist, P., Kagedal, B., Nilsson, L. and 

Rosling, H. (1995) Analysis of the cyanide 

metabolite 2-aminothiazoline-4-carboxylic 

acid in urine by high-performance liquid 

chromatography. Analytical Biochemistry 228, 

27-34.

(49) Cardozo, R. H. and Edelman, I. S. (1952) The 

volume of distribution of sodium thiosulfate 

as a measure of the extracellular fl uid space. 

The Journal of clinical investigation 31, 280-290.

(50) Diem, K., Lentner, C. and Editors (1975) 

Scientifi c Tables-Documenta Geigy. 7th Ed.

(51) Yamanaka, S., Takaku, S., Takaesu, Y. and 

Nishimura, M. (1991) Validity of salivary 

thiocyanate as an indicator of cyanide 

exposure from smoking. Bulletin of Tokyo 

Dental College 32, 157-163.

(52) Jarvis, M. J. (1989) Application of biochemical 

intake markers to passive smoking 

measurement and risk estimation. Mutat Res

222, 101-110.

(53) Maliszewski, T. F. and D.E., B. (1955) True 

and apparent thiocyanate in body fl uids of 

smokers and nonsmokers. Journal of applied 

Physiology 8, 289-291.

(54) Pre, J. and Vassey, R. (1992) Plasma thiocyanate 

and cigarette-smoking status. Medical Science 

Research 20, 2.

(55) Hasuike, Y., Nakanishi, T., Moriguchi, R., 

Otaki, Y., Nanami, M., Hama, Y., Naka, M., 

Miyagawa, K., Izumi, M. and Takamitsu, 

Y. (2004) Accumulation of cyanide and 

thiocyanate in haemodialysis patients. Nephrol 

Dial Transplant 19, 1474-1479.

(56) Ballantyne, B. (1977) Factors in the analysis 

of whole blood thiocyanate. Clinical Toxicology

11, 195-210.

(57) Wood, J. L., Williams, E. F. J. and Kingsland, 

N. (1947) The conversion of thiocyanate 

sulfur to sulfate in the white rat. J Biol Chem

170, 251-259.

(58) Wood, J. L. and Williams, E. F. J. (1949) The 

metabolism of thiocyanate in the rat and its 

inhibition by propylthiouracil. J Biol Chem

177, 59-67.

(59) Weuffen, W., Jess, G., Julich, W. D. and 

Bernhart, D. (1980) Studies on the relationship 

between 2-iminothiazolidine-4-carboxylic 

acid and the thiocyanate metabolism in the 

guinea-pig. Pharmazie 35, 221-223.

(60) Bradham, L. S., Catsimpoolas, N. and 

Wood, J. L. (1965) Determination of 

2-iminothiazolidine-4-carboxylic acid. 

Analytical Biochemistry 11, 230-232, discussion 

232-237.

(61) Baskin, S. I., Petrikovics, I., Platoff, G. E., 

Rockwood, G. A. and Logue, B. A. (2006) 

Spectrophotometric analysis of the cyanide 

metabolite 2-aminothiazoline-4-carboxylic 

acid (ATCA). Toxicology Mechanisms and Methods

16, 339-345.

(62) Levin, B. C., Rechani, P. R., Gurman, J. L., 

Landron, F., Clark, H. M., Yoklavich, M. 

F., Rodriguez, J. R., Droz, L., Mattos de 

Cabrera, F. and Kaye, S. (1990) Analysis of 

carboxyhemoglobin and cyanide in blood 

from victims of the Dupont Plaza Hotel fi re 

in Puerto Rico. Journal of Forensic Sciences 35, 

151-168.

(63) Takekawa, K., Oya, M., Kido, A. and Suzuki, 

O. (1998) Analysis of cyanide in blood 

by headspace solid-phase microextraction 

(SPME) and capillary gas chromatography. 

Chromatographia 47, 209-214.

(64) Rodkey, F. L. and Robertson, R. F. (1978) 

Analytical precautions in measurement of 

blood cyanide. Clinical Chemistry (Washington, 

DC, United States) 24, 2184-2185.

(65) Moriya, F. and Hashimoto, Y. (2001) Potential 

for error when assessing blood cyanide 

concentrations in fi re victims. Journal of 

Forensic Sciences 46, 1421-1425.

(66) Shiono, H., Maseda, C., Akane, A. and 

Matsubara, K. (1991) Rapid and sensitive 

quantitation of cyanide in blood and its 

application to fi re victims. The American 

journal of forensic medicine and pathology : offi cial 

publication of the National Association of Medical 

Examiners 12, 50-53.

(67) Laforge, M., Buneaux, F., Houeto, P., 

Bourgeois, F., Bourdon, R. and Levillain, 

P. (1994) A rapid spectrophotometric 

blood cyanide determination applicable to 

emergency toxicology. Journal of Analytical 

Toxicology 18, 173-175.

(68) Baud, F. J., Borron, S. W., Bavoux, E., Astier, 

A. and Hoffman, J. R. (1996) Relation 

between plasma lactate and blood cyanide 

concentrations in acute cyanide poisoning. 

BMJ (Clinical research ed.) 312, 26-27.

(69) Chinaka, S., Takayama, N., Michigami, Y. and 

Ueda, K. (1998) Simultaneous determination 

of cyanide and thiocyanate in blood by ion 

chromatography with fl uorescence and 

ultraviolet detection. Journal of Chromatography, 

B: Biomedical Sciences and Applications 713, 

353-359.

88
(70) Odoul, M., Fouillet, B., Nouri, B., Chambon, 

R. and Chambon, P. (1994) Specifi c 

determination of cyanide in blood by 

headspace gas chromatography. Journal of 

Analytical Toxicology 18, 205-207.

(71) Clark, C. J., Campbell, D. and Reid, W. H. 

(1981) Blood carboxyhemoglobin and cyanide 

levels in fi re survivors. Lancet 1, 1332-1335.

(72) Vesey, C. J., Saloojee, Y., Cole, P. V. and Russell, 

M. A. H. (1982) Blood carboxyhemoglobin, 

plasma thiocyanate, and cigaret consumption: 

implications for epidemiologial studies 

in smokers. British Medical Journal 284, 

1516-1518.

(73) Shiono, H., Maseda, C., Akane, A. and 

Matsubara, K. (1991) The blood cyanide level 

in fi re victims. Rapid and sensitive quantitation 

of cyanide in blood and its application to 

autopsy cases. Hanzaigaku Zasshi 57, 73-79.

(74) Ballantyne, B. (1975) Blood, brain, and 

cerebrospinal fl uid cyanide concentrations in 

experimental acute cyanide poisoning. Journal 

of the Forensic Science Society 15, 51-56.

(75) Ballantyne, B. (1976) Changes in blood 

cyanide as a function of storage time and 

temperature. Journal of the Forensic Science 

Society 16, 305-310.

(76) Grgurinovich, N. (1982) A Colourimetric 

Procedure for the determination of 

thiocyanate in plasma. Journal of Analytical 

Toxicology 6, 53-55.

(77) Lundquist, P., Rosling, H., Soerbo, B. and 

Tibbling, L. (1987) Cyanide concentrations in 

blood after cigarette smoking, as determined 

by a sensitive fl uorometric method. Clinical 

Chemistry (Washington, DC, United States) 33, 

1228-1230.

(78) Zamecnik, J. and Tam, J. (1987) Cyanide 

in blood by gas chromatography with NP 

detector and acetonitrile as internal standard. 

Application on air accident fi re victims. Journal 

of Analytical Toxicology 11, 47-48.

(79) Seto, Y., Tsunoda, N., Ohta, H. and Shinohara, 

T. (1993) Determination of blood cyanide by 

headspace gas chromatography with nitrogenphosphorus detection and using a megabore 

capillary column. Analytica Chimica Acta 276, 

247-259.

(80) Tracqui, A., Raul, J. S., Geraut, A., Berthelon, 

L. and Ludes, B. (2002) Determination 

of blood cyanide by HPLC-MS. Journal of 

Analytical Toxicology 26, 144-148.

(81) Maseda, C., Matsubara, K., Hasegawa, 

M., Akane, A. and Shiono, H. (1990) 

Determination of blood cyanide using headspace gas chromatography with electron 

capture detection. Nippon Hoigaku Zasshi 44, 

131-136.

(82) Rodkey, F. L. and Collison, H. A. (1977) 

Determination of cyanide and nitroprusside 

in blood and plasma. Clinical Chemistry 

(Washington, DC, United States) 23, 1969-1975.

(83) Kage, S., Nagata, T. and Kudo, K. (1996) 

Determination of cyanide and thiocyanate 

in blood by gas chromatography and gas 

chromatography-mass spectrometry. Journal 

of Chromatography, B: Biomedical Applications

675, 27-32.

(84) Toida, T., Togawa, T., Tanabe, S. and Imanari, 

T. (1984) Determination of cyanide and 

thiocyanate in blood plasma and red cells by 

high-performance liquid chromatography 

with fl uorometric detection. Journal of 

Chromatography 308, 133-141.

(85) Kage, S., Nagata, T. and Kudo, K. (1994) 

Determination of cyanide in blood by GCECD and GC-MS. Japanese Journal of Forensic 

Toxicology 12, 130-131.

(86) Ishii, A., Seno, H., Watanabe-Suzuki, 

K., Suzuki, O. and Kumazawa, T. (1998) 

Determination of cyanide in whole blood by 

capillary gas chromatography with cryogenic 

oven trapping. Analytical Chemistry 70, 

4873-4876.

(87) Lundquist, P., Rosling, H. and Soerbo, B. 

(1985) Determination of cyanide in whole 

blood, erythrocytes, and plasma. Clinical 

Chemistry (Washington, DC, United States) 31, 

591-595.

(88) Sano, A., Takimoto, N. and Takitani, S. (1992) 

High-performance liquid chromatographic 

determination of cyanide in human red 

blood cells by pre-column fl uorescence 

derivatization. Journal of Chromatography 582, 

131-135.

(89) Lundquist, P., Kaagedal, B. and Nilsson, 

L. (1995) An improved method for 

determination of thiocyanate in plasma and 

urine. European Journal of Clinical Chemistry and 

Clinical Biochemistry 33, 343-349.

(90) McAnalley, B. H., Lowry, W. T., Oliver, R. D. 

and Garriott, J. C. (1979) Determination of 

inorganic sulfi de and cyanide in blood using 

specifi c ion electrodes: application to the 

investigation of hydrogen sulfi de and cyanide 

poisoning. Journal of Analytical Toxicology 3, 

111-114.

(91) Baud, F. J., Barriot, P., Toffi s, V., Riou, B., 

89
Vicaut, E., Lecarpentier, Y., Bourdon, R., 

Astier, A. and Bismuth, C. (1991) Elevated 

blood cyanide concentrations in victims of 

smoke inhalation. The New England journal of 

medicine 325, 1761-1766.

(92) Seto, Y., Shinohara, T. and Tsunoda, N. (1990) 

Head-space capillary gas chromatography of 

cyanide in blood. Japanese Journal of Forensic 

Toxicology 8, 56-57.

(93) Tanabe, S., Kitahara, M., Nawata, M. 

and Kawanabe, K. (1988) Determination 

of oxidizable inorganic anions by highperformance liquid chromatography with 

fl uorescence detection and application to 

the determination of salivary nitrite and 

thiocyanate and serum thiocyanate. Journal of 

Chromatography 424, 29-37.

(94) Dumas, P., Gingras, G. and LeBlanc, A. 

(2005) Isotope Dilution-Mass Spectrometry 

Determination of Blood Cyanide by 

Headspace Gas Chromatography. Journal of 

Analytical Toxicology 29, 71-75.

(95) Lundquist, P., Maartensson, J., Soerbo, B. and 

Oehman, S. (1979) Method for determining 

thiocyanate in serum and urine. Clinical 

Chemistry (Washington, DC, United States) 25, 

678-681.

(96) Bogusz, M. (1976) Disappearance of cyanide 

in blood and tissues after fatal poisoning. 

Forensic science 7, 173.

(97) Michigami, Y., Takahashi, T., He, F., 

Yamamoto, Y. and Ueda, K. (1988) 

Determination of thiocyanate in human 

serum by ion chromatography. Analyst 

(Cambridge, United Kingdom) 113, 389-392.

(98) Connolly, D., Barron, L. and Paull, B. (2002) 

Determination of urinary thiocyanate 

and nitrate using fast ion-interaction 

chromatography. Journal of Chromatography, B: 

Analytical Technologies in the Biomedical and Life 

Sciences 767, 175-180.

(99) Lahti, M., Vilpo, J. and Hovinen, J. (1999) 

Spectrophotometric Determination of 

Thiocyanate in Human Saliva. Journal of 

Chemical Education 76, 1281-1282.

(100) Tomoda, A. and Hashimoto, K. (1991) 

The determination of cyanide in water and 

biological tissues by methemoglobin. Journal 

of Hazardous Materials 28, 241-249.

(101) Aldridge, W. N. (1944) New method for the 

estimation of microquantities of cyanide and 

thiocyanate. Analyst 69, 262-265.

(102) Aldridge, W. N. (1945) Estimation of micro 

quantities of cyanide and thiocyanate. Analyst

70, 474-475.

(103) Asmus, E. and Garschagen, H. (1953) Use 

of barbituric acid for the photometric 

determination of cyamide and thiocyanate. 

Fresenius’ Zeitschrift fuer Analytische Chemie 138, 

414-422.

(104) Epstein, J. (1947) Estimation of 

microquantities of cyanide. Anal. Chem. 19, 

272-274.

(105) Tompsett, S. L. (1959) A note on the 

determination and identifi cation of cyanide 

in biologic materials. Clinical chemistry 5, 

587-591.

(106) Feldstein, M. and Klendshoj, N. C. (1957) 

The determination of volatile substances 

by microdiffusion analysis. J. Forensic Sci. 2, 

39-58.

(107) Conway, E. J. (1950) Microdiffusion Analysis and 

Volumetric Error. 3rd ed.

(108) Boxer, G. E. and Rickards, J. C. (1952) 

Determination of thiocyanate in body fl uids. 

Archives of Biochemistry and Biophysics 39, 

292-300.

(109) Nagashima, S. (1984) Simultaneous reaction 

rate spectrophotometric determination 

of Cyanide and Thiocyanate by use of the 

pyridine-barbituric acid method. Analytical 

Chemistry 56, 1944-1947.

(110) Ganjeloo, A., Isom, G. E., Morgan, R. 

L. and Way, J. L. (1980) Fluorometric 

determination of cyanide in biological fl uids 

with p-benzoquinone. Toxicology and Applied 

Pharmacology 55, 103-107.

(111) Morgan, R. L. and Way, J. L. (1980) 

Fluorometric determination of cyanide in 

biological fl uids with pyridoxal. Journal of 

Analytical Toxicology 4, 78-81.

(112) Guilbault, G. G. and Kramer, D. N. (1965) 

Specifi c detection and determination of 

cyanide using various quinone derivatives. 

Anal. Chem. 37, 1395-1399.

(113) Takanashi, S. and Tamura, Z. (1970) 

Fluorometric determination of cyanide 

by the reaction with pyridoxal. Chemical & 

Pharmaceutical Bulletin 18, 1633-1635.

(114) Suzuki, O., Hattori, H., Oya, M. and 

Katsumata, Y. (1982) Direct fl uorometric 

determination of cyanide in human materials. 

Forensic Science International 19, 189-195.

(115) Bark, L. S. and Higson, H. G. (1964) 

Investigation of reagents for the colorimetric 

determination of small amounts of cyanide-I. 

Talanta 11, 471-479.

(116) Lambert, J. L., Ramasamy, J. and Paukstelis, J. 

90
V. (1975) Stable reagents for the colorimetric 

determination of cyanide by modifi ed Koenig 

reactions. Analytical Chemistry 47, 916-918.

(117) Murty, G. V. L. N. and Viswanathan, T. S. 

(1961) Determination of traces of cyanide. 

Analytica Chimica Acta 25, 293-295.

(118) Falkensson, M., Lundquist, P., Rosling, H. 

and Sorbo, B. (1988) A simple method for 

determination of plasma thiocyanate. Annals 

of Clinical Biochemistry 25, 422-423.

(119) Bark, L. S. and Higson, H. G. (1964) 

Investigation of reagents for the colorimetric 

determination of small amounts of cyanideII. A proposed method for trace cyanide in 

waters. Talanta 11, 621-631.

(120) Dunn, W. A. and Siek, T. J. (1990) A rapid, 

sensitive, and specifi c screening technique 

for the determination of cyanide. Journal of 

Analytical Toxicology 14, 256.

(121) Houeto, P., Buneaux, F., Galliot-Guilley, M., 

Baud, F. J. and Levillain, P. (1994) Determination 

of hydroxocobalamin and cyanocobalamin 

by derivative spectrophotometry in cyanide 

poisoning. Journal of Analytical Toxicology 18, 

154-158.

(122) Holzbecher, M. and Ellenberger, H. A. 

(1985) An evaluation and modifi cation of a 

microdiffusion method for the emergency 

determination of blood cyanide. Journal of 

Analytical Toxicology 9, 251-253.

(123) Lundquist, P. and Soerbo, B. (1989) Rapid 

determination of toxic cyanide concentrations 

in blood. Clinical Chemistry (Washington, DC, 

United States) 35, 617-619.

(124) Cardeal, Z. L., Gallet, J. P., Astier, A. and 

Pradeau, D. (1995) Cyanide assay: statistical 

comparison of a new gas chromatographic 

calibration method versus the classical 

spectrophotometric method. Journal of 

Analytical Toxicology 19, 31-34.

(125) Hughes, C., Lehner, F., Dirikolu, L., Harkins, 

D., Boyles, J., McDowell, K., Tobin, T., 

Crutchfi eld, J., Sebastian, M., Harrison, L. 

and Baskin, S. I. (2003) A simple and highly 

sensitive spectrophotometric method for the 

determination of cyanide in equine blood. 

Toxicology Mechanisms and Methods 13, 129-138.

(126) Manahan, S. E. and Kunkel, R. (1973) Atomic 

absorption analysis method for cyanide. 

Analytical Letters 6, 547-553.

(127) Chattaraj, S. and Das, A. K. (1992) Indirect 

determination of thiocyanate in biological 

fl uids using atomic absorption spectrometry. 

Spectrochimica Acta, Part B: Atomic Spectroscopy

47B, 675-680.

(128) Vesey, C. J., McAllister, H. and Langford, 

R. M. (1999) A simple, rapid and sensitive 

semimicro method for the measurement of 

cyanide in blood. Annals of Clinical Biochemistry

36, 755-758.

(129) Guilbault, G. G. and Kramer, D. N. (1965) 

Specifi c fl uorometric method for the 

detection of cyanide. Anal. Chem. 37, 918.

(130) Westley, A. M. and Westley, J. (1989) 

Voltammetric determination of cyanide 

and thiocyanate in small biological samples. 

Analytical Biochemistry 181, 190-194.

(131) Egekeze, J. O. and Oehme, F. W. (1979) Direct 

potentiometric method for the determination 

of cyanide in biological materials. Journal of 

Analytical Toxicology 3, 119-124.

(132) Ganjali, M. R., Yousefi , M., Javanbakht, M., 

Poursaberi, T., Salavati-Niasari, M., HajiaghaBabaei, L., Latifi , E. and Shamsipur, M. (2002) 

Determination of SCN- in urine and saliva of 

smokers and non-smokers by SCN--selective 

polymeric membrane containing a nickel(II)-

azamacrocycle complex coated on a graphite 

electrode. Analytical Sciences 18, 887-892.

(133) Casella, I. G. (1998) Electrooxidation 

of thiocyanate on copper-modifi ed gold 

electrode and its amperometric determination 

by ion chromatography. Analyst (Cambridge, 

United Kingdom) 123, 1359-1363.

(134) Pasciak, J. and Hurek, J. (1984) Catalytic 

effect of thiocyanate ions and possibility of 

their determination by pulse polarography 

with adsorptive concentration on the hanging 

mercury drop electrode. Chemia Analityczna 

(Warsaw, Poland) 29, 571-580.

(135) Cai, X. and Zhao, Z. (1988) Determination 

of trace thiocyanate by linear sweep 

polarography. Analytica Chimica Acta 212, 

43-48.

(136) Below, H., Muller, H., Hundhammer, B. 

and Weuffen, W. (1990) [Determination 

of thiocyanate in serum with ion-selective 

liquid membrane electrodes]. Pharmazie 45, 

416-419.

(137) Shan, D., Mousty, C. and Cosnier, S. 

(2003) Subnanomolar Cyanide Detection 

at Polyphenol Oxidase/Clay Biosensors. 

Analytical Chemistry, A-E.

(138) Bjergegaard, C., Moller, P. and Sorensen, 

H. (1995) Determination of thiocyanate, 

iodide, nitrate and nitrite in biological 

samples by micellar electrokinetic capillary 

chromatography. Journal of Chromatography A

91
717, 409-414.

(139) Glatz, Z., Novakova, S. and Sterbova, H. 

(2001) Analysis of thiocyanate in biological 

fl uids by capillary zone electrophoresis. 

Journal of Chromatography, A 916, 273-277.

(140) de Branander, H. F. and Verbeke, R. (1977) 

Determination of thiocyanate in tissues and 

body fl uids of animals by gas chromatography 

with electron-capture detection. Journal of 

Chromatography 138, 131-142.

(141) Nota, G., Improta, C. and Miraglia, V. R. 

(1982) Determination of cyanides and 

thiocyanates in water as cyanogen bromide 

by headspace gas chromatography. Journal of 

Chromatography 242, 359-363.

(142) Logue, B. A., Masered, W. K., Rockwood, 

G. A., Keebaugh, M. W. and Baskin, S. I. 

(2007) Feasibility of the cyanide metabolite, 

2-amino-2-thiazoline-4-carboxylic acid, as a 

retrospective marker of cyanide exposure. J. 

Chrom. B., submitted.

(143) Cardeal, Z. L., Pradeau, D. and Hamon, M. 

(1993) Determination of HCN by headspace 

gas chromatography using an improved 

method of standardization. Chromatographia

37, 613-617.

(144) Darr, R. W., Capson, T. L. and Hileman, F. D. 

(1980) Determination of hydrogen cyanide in 

blood using gas chromatography with alkali 

thermionic detection. Analytical Chemistry 52, 

1379-1381.

(145) Funazo, K., Kusano, K., Wu, H. L., Tanaka, 

M. and Shono, T. (1982) Trace determination 

of cyanide by derivatization and fl ame 

thermionic gas chromatography. Journal of 

Chromatography 245, 93-100.

(146) Funazo, K., Tanaka, M. and Shono, T. (1981) 

Determination of cyanide or thiocyanate 

at trace levels by derivatization and gas 

chromatography with fl ame thermionic 

detection. Analytical Chemistry 53, 1377-1380.

(147) Watanabe-Suzuki, K., Ishii, A. and Suzuki, 

O. (2002) Cryogenic oven-trapping gas 

chromatography for analysis of volatile 

organic compounds in body fl uids. Analytical 

Bioanalytical Chemistry 373, 75-80.

(148) Thomson, I. and Anderson, R. A. (1980) 

Determination of cyanide and thiocyanate 

in biological fl uids by gas chromatographymass spectrometry. Journal of Chromatography

188, 357-362.

(149) Tsukamoto, S., Kanegae, T., Matsumura, Y., 

Uchigasaki, S., Kitazawa, M., Muto, T., Chiba, 

S., Nagoya, T. and Shimamura, M. (1994) 

Simultaneous measurement of alcohols and 

hydrogen cyanide in biological specimens 

using headspace gas chromatography. Nippon 

Hoigaku Zasshi 48, 336-342.

(150) Jacob, P., III, Savanapridi, C., Yu, L., Wilson, 

M., Shulgin, A. T., Benowitz, N. L., EliasBaker, B. A., Hall, S. M., Herning, R. I. and et 

al. (1984) Ion-pair extraction of thiocyanate 

from plasma and its gas chromatographic 

determination using on-column alkylation. 

Analytical Chemistry 56, 1692-1695.

(151) Maseda, C., Matsubara, K. and Shiono, H. 

(1989) Improved gas chromatography with 

electron-capture detection using a reaction 

pre-column for the determination of blood 

cyanide: a higher content in the left ventricle 

of fi re victims. Journal of Chromatography 490, 

319-327.

(152) McAuley, F. and Reive, D. S. (1983) Rapid 

quantitation of cyanide in blood by gas 

chromatography. Journal of Analytical Toxicology

7, 213-215.

(153) Meiser, H., Hagedorn, H.-W. and Schulz, 

R. (2000) Development of a method for 

determination of cyanide concentrations in 

serum and rumen fl uid of cattle. American 

Journal of Veterinary Research 61, 658-664.

(154) Chen, S. H. and Wu, S. M. (1994) Electroncapture gas chromatographic determination 

of cyanide, iodide, nitrite, sulfi de, and 

thiocyanate anions by phase-transfer-catalyzed 

derivatization with pentafl uorobenzyl 

bromide. Journal of Analytical Toxicology 18, 

81-85.

(155) Yoshida, M., Watabiki, T. and Ishida, N. 

(1989) The quantitative determination of 

cyanide by FTD-GC. Nippon Hoigaku Zasshi

43, 179-185.

(156) Valentour, J. C., Aggarwal, V. and Sunshine, 

I. (1974) Sensitive gas chromatographic 

determination of cyanide. Analytical chemistry

46, 924-925.

(157) Maserek, W. K., Rockwood, G. A., Platoff, 

G. E., Baskin, S. I. and Logue, B. A. (2007) 

Feasibility of using the cyanide metabolite 

2-amino-2-thiazoline-4-carboxylic acid as a 

retrospective marker of cyanide exposure. 

The Toxicologist (submitted).

(158) Frison, G., Zancanaro, F., Favretto, D. 

and Ferrara, S. D. (2006) An improved 

method for cyanide determination in blood 

using solid-phase microextraction and gas 

chromatography/mass spectrometry. Rapid 

Commun. Mass Spectrom. 20, 2932-2938.

92
(159) Felscher, D. and Wulfmeyer, M. (1998) A new 

specifi c method to detect cyanide in body 

fl uids, especially whole blood, by fl uorimetry. 

Journal of Analytical Toxicology 22, 363-366.

(160) Imanari, T., Tanabe, S. and Toida, T. (1982) 

Simultaneous determination of cyanide and 

thiocyanate by high-performance liquid 

chromatography. Chemical & Pharmaceutical 

Bulletin 30, 3800-3802.

(161) Imanari, T., Tanabe, S., Toida, T. and 

Kawanishi, T. (1982) High-performance 

liquid chromatography of inorganic anions 

using iron(3+) as a detection reagent. Journal 

of Chromatography 250, 55-61.

(162) Imanari, T., Ogata, K., Tanabe, S., Toida, 

T., Kawanishi, T. and Ichikawa, M. (1982) 

Determination of thiocyanate, thiosulfate, 

sulfi te and nitrite by high-performance liquid 

chromatography coupled with electrochemical 

detection. Chemical & Pharmaceutical Bulletin

30, 374-375.

(163) Chen, S.-H., Yang, Z.-Y., Wu, H.-L., Kou, 

H.-S. and Lin, S.-J. (1996) Determination of 

thiocyanate anion by high-performance liquid 

chromatography with fl uorometric detection. 

Journal of Analytical Toxicology 20, 38-42.

(164) Brown, D. G., Lanno, R. P., van den Heuvel, 

M. R. and Dixon, D. G. (1995) HPLC 

determination of plasma thiocyanate 

concentrations in fi sh blood: application 

to laboratory pharmacokinetic and fi eldmonitoring studies. Ecotoxicology and 

Environmental Safety 30, 302-308.

(165) Backofen, U., Matysik, F. M. and Werner, 

G. (1996) Determination of cyanide in 

microsamples by means of capillary fl ow 

injection analysis with amperometric 

detection. Anal. Bioanal. Chem. 356, 271-273.

(166) Tominaga, M. Y. and Midio, A. F. (1991) 

Modifi ed method for the determination 

of thiocyanate in urine by ion-exchange 

chromatography and spectrophotometry. 

Revista de Farmacia e Bioquimica da Universidade 

de Sao Paulo 27, 100-105.

(167) Bond, A. M., Heritage, I. D., Wallace, G. G. 

and McCormick, M. J. (1982) Simultaneous 

determination of free sulfi de and cyanide by 

ion chromatography with electrochemical 

detection. Analytical Chemistry 54, 582-585.

(168) Cole, D. E. C., Evrovski, J. and Pirone, 

R. (1995) Urinary thiosulfate determined 

by suppressed ion chromatography with 

conductimetric detection. Journal of 

Chromatography, B: Biomedical Applications 672, 

149-154.

(169) Michigami, Y., Fujii, K., Ueda, K. and 

Yamamoto, Y. (1992) Determination of 

thiocyanate in human saliva and urine by ion 

chromatography. Analyst (Cambridge, United 

Kingdom) 117, 1855-1858.

(170) Olea, F. and Parras, P. (1992) Determination 

of serum levels of dietary thiocyanate. Journal 

of Analytical Toxicology 16, 258-260.

(171) Toida, T., Tanabe, S. and Imanari, T. (1981) 

Fluorometric determination of cyanide and 

thiocyanate by Koenig reaction. Chemical & 

Pharmaceutical Bulletin 29, 3763-3764.

(172) Ballantyne, B. (1977) In vitro production 

of cyanide in normal human blood and 

the infl uence of thiocyanate and storage 

temperature. Clinical Toxicology 11, 173-193.

(173) Seto, Y. (2002) False cyanide detection. 

Analytical Chemistry 74, 134A-141A.

(174) Vesey, C. J. and Wilson, J. (1978) Red cell 

cyanide. Journal of Pharmacy and Pharmacology

30, 20-26.

(175) Seto, Y. (1995) Oxidative conversion of 

thiocyanate to cyanide by oxyhemoglobin 

during acid denaturation. Archives of 

Biochemistry and Biophysics 321, 245-254.

(176) Seto, Y. (1996) Stability and spontaneous 

production of blood cyanide during heating. 

Journal of forensic sciences 41, 465-468.

(177) Cruz-Landeira, A., Lopez-Rivadulla, M., 

Concheiro-Carro, L., Fernandez-Gomez, 

P. and Tabernero-Duque, M. J. (2000) A 

new spectrophotometric method for the 

toxicological diagnosis of cyanide poisoning. 

Journal of Analytical Toxicology 24, 266-270.

(178) Konig, W. (1905) Verfahren zur Darstellung 

neuer stickstoffhaltiger Farbstoffe. Angew. 

Chem. 69, 115-118.

(179) Groff, W. A., Stemler, F. W., Kaminskis, A., 

Froehlich, H. L. and Johnson, R. P. (1985) 

Plasma free cyanide and blood total cyanide: a 

rapid completely automated microdistillation 

assay. Journal of Toxicology, Clinical Toxicology

23, 133-163.

(180) Lv, J., Zhang, Z., Li, J. and Luo, L. (2005) 

A micro-chemiluminescence determination 

of cyanide in whole blood. Forensic Science 

International 148, 15-19.

(181) Danchik, R. S. and Boltz, R. F. (1970) Indirect 

atomic absorption photometric methods for 

the determination of cyanide. Anal. Clin. Acta

49, 567-569.

(182) Du Cailar, J., Mathieu-Daude, J. C., Deschodt, 

J. and Griffe, O. (1976) Thiocyanate ion 

93
plasma levels during perfusion with sodium 

nitroprusside. Annales de l’Anesthesiologie 

Francaise 17, 519-528.

(183) Abbaspour, A., Kamyabi, M. A., Esmaeilbeig, 

A. R. and Kia, R. (2002) Thiocyanate-selective 

electrode based on unsymmetrical benzoN4 

nickel(II) macrocyclic complexes. Talanta 57, 

859-867.

(184) Barber, C. V. and Pratt, V. R. (1997) Sullied 

seas. Strategies for combating cyanide fi shing 

in Southeast Asia and beyond, p 64, World 

Resources Institute, International Marinelife 

Alliance.

(185) Lindsay, A. E. and O’Hare, D. (2006) 

The development of an electrochemical 

sensor for the determination of cyanide in 

physiological solutions. Analytica Chimica Acta

558, 158-163.

(186) Mazloum Ardakani, M., Ensafi , A. A., Niasari, 

M. S. and Chahooki, S. M. (2002) Selective 

thiocyanate poly(vinyl chloride) membrane 

based on a 1,8-dibenzyl-1,3,6,8,10,13-

hexaazacyclotetradecane-Ni(II) perchlorate. 

Analytica Chimica Acta 462, 25-30.

(187) Amini, M. K., Shahrokhian, S. and 

Tangestaninejad, S. (1999) Thiocyanateselective electrodes based on nickel and iron 

phthalocyanines. Analytica Chimica Acta 402, 

137-143.

(188) Hassan, S. S. M., Mohmoud, W. H., Hamad 

Elgazwy, A.-S. S. and Badawy, N. M. (2006) 

A novel potentiometric membrane sensor 

based on aryl palladium complex for selective 

determination of thiocyanate in the saliva 

and urine of cigarette smokers. Electroanalysis

18, 2070-2078.

(189) Hu, W., Tao, H., Tominaga, M., Miyazaki, A. 

and Haraguchi, H. (1994) A new approach for 

the simultaneous determination of inorganic 

cations and anions using ion chromatography. 

Analytica Chimica Acta 299, 249-256.

(190) Zein, R., Munaf, E., Takeuchi, T. and Miwa, T. 

(1996) Microcolumn ion chromatography of 

inorganic UV-absorbing anions using bovine 

serum albumin as stationary phases. Analytica 

Chimica Acta 335, 261-266.

(191) Cookeas, E. G. and Efstathiou, C. E. (1994) 

Flow injection amperometric determination 

of thiocyanate and selenocyanate at a cobalt 

phthalocyanine modifi ed carbon paste 

electrode. Analyst (Cambridge, United Kingdom)

119, 1607-1612.

(192) Fouillet, B., Odoul, M., Chambon, R. and 

Chambon, P. (1992) Specifi c determination 

of cyanide in water by GC/ECD as cyanogen 

chloride. Journal Francais d’Hydrologie 23, 

161-169.

(193) Houeto, P., Hoffman, J. R., Imbert, M., 

Levillain, P. and Baud, F. J. (1995) Relation 

of blood cyanide to plasma cyanocobalamin 

concentration after a fi xed dose of 

hydroxocobalamin in cyanide poisoning. 

Lancet 346, 605-608.

(194) Vesey, C. J., Cole, P. V. and Simpson, P. J. (1976) 

Cyanide and thiocyanate concentrations 

following sodium nitroprusside infusion in 

man. British journal of anaesthesia 48, 651-660.

(195) Ansell, M. and Lewis, F. A. S. (1970) Review 

of cyanide concentrations found in human 

organs. Survey of literature concerning 

cyanide metabolism, “normal”, nonfatal, and 

fatal body cyanide levels. Journal of Forensic 

Medicine 17, 148-155.

(196) Curry, A. S. (1976) American Lecture Series, 

No. 988: American Lectures in Living Chemistry: 

Poison Detection in Human Organs. 3rd Ed.

(197) Bright, J. E., Inns, R. H., Tuckwell, N. J. and 

Marrs, T. C. (1990) The effect of storage 

upon cyanide in blood samples. Human & 

Experimental Toxicology 9, 125-129.

(198) Chikasue, F., Yashiki, M., Kojima, T., Miyazaki, 

T., Okamoto, I., Ohtani, M. and Kodama, 

K. (1988) Cyanide distribution in fi ve fatal 

cyanide poisonings and the effect of storage 

conditions on cyanide concentration in tissue. 

Forensic science international 38, 173-183.

(199) Vesey, C. J. and Langford, R. M. (1998) 

Stabilization of blood cyanide. Journal of 

Analytical Toxicology 22, 176-178.

(200) Goenechea, S. (1982) Hydrogen cyanide: 

losses in samples of stored blood. Zeitschrift 

fuer Rechtsmedizin 88, 97-101.

(201) Gao, D., Gu, J., Yu, R.-Q. and Zheng, 

G.-D. (1995) Substituted metalloporphyrin 

derivatives as anion carrier for PVC 

membrane electrodes. Analytica Chimica Acta

302, 263-268.

(202) Daunert, S., Wallace, S., Florido, A. and Bachas, 

L. G. (1991) Anion-selective electrodes based 

on electropolymerized porphyrin fi lms. 

Analytical Chemistry 63, 1676-1679.

94
Background Paper on 

Cyanide Detection Tests for Live Fish

Patricia Debenham

SeaWeb

Purpose of this Document

The purpose of this document is to provide 

an overview of cyanide detection testing as 

it applies to the capture of live reef fi sh. We 

have provided an introduction to the live 

fi sh aquarium and food trades, as well as the 

techniques that utilize cyanide solutions to 

capture fi sh. We have provided a summary 

of cyanide detection systems currently used 

to determine whether commercial fi sh were 

caught with cyanide. Finally, this document 

includes a summary of cyanide detection 

methods used in medical and environmental 

testing. As part of this last element, we have 

identifi ed scientists and other technical experts 

that are most knowledgeable and experienced 

in cyanide detection methods.

We hope this document provides suffi cient 

background information for those parties 

interested in applying existing methods of 

cyanide detection to the development of a 

detection system that can be used in marine 

fish. To help achieve this goal, we have 

presented a summary of the criteria necessary 

for development and implementation of an 

effective cyanide detection test.

Introduction

The sale of live fi sh from the ocean is a 

valuable international trade. The trade 

includes aquarium fi sh that are sold through 

the hobby market, as well as fi sh sold live to 

restaurants for human consumption (also 

called the live reef food fi sh trade, or LRFFT). 

According to an American Marinelife Dealers 

Association survey, in 1995, about 10 million 

individual marine specimens were sold in pet 

stores throughout the United States for a total 

value of $103.2 million (Marine Aquarium 

Council). The United States imports nearly 

half of the marine aquarium organisms sold 

throughout the world. Other major importers 

are Germany, France, Netherlands, United 

Kingdom, and Japan. Hong Kong is the largest 

consumer of live reef food fi sh and accounts 

for 60% of the world trade (Mak, Yanase et 

al., 2005 citing Johannes and Riepen 1995).

Live marine organisms are harvested from 

the coral reefs of Southeast Asia, the Pacifi c 

Islands, South Asia, Indian Ocean, Australia, 

Hawaii, Mexico, Florida, the Caribbean, 

Brazil, East Africa, and the Red Sea. The 

two dominant countries that supply the 

ornamental fi sh trade are Indonesia and the 

Philippines, accounting for more than half 

of the global marine ornamental fi sh trade 

(Marine Aquarium Council).

In an effort to ensure that this industry has a 

long, healthy future, many governments, nonprofi t organizations, and industry members 

are committed to measures that protect the 

environment that produces the fi sh. One 

organization engaged in such initiatives is 

the Marine Aquarium Council (MAC), whose 

mission is to “conserve coral reefs and other 

marine ecosystems by creating standards 

and certifi cation for those engaged in the 

collection and care of ornamental marine life 

from reef to aquarium.” A variety of other 

organizations focus on issues related to the 

capture of live fi sh sold to restaurants.

95
Cyanide Use in Fish Capture

As in any business, there is a great deal of 

competition and as a result tremendous 

pressure to develop effi cient techniques of fi sh 

capture. One such capture technique involves 

the use of the toxic substance cyanide. Some 

collectors use cyanide to temporarily stun fi sh 

and make them easier to catch. 

Cyanide (CN) occurs naturally in the 

environment and some food substances. 

Industrial activities such as metal processing, 

electroplaters, gold-mining facilities, oil 

refineries, power plants, and solid waste 

combustion can also release cyanide into the 

environment (Eisler, 1991).

Hydrogen cyanide is toxic to the majority 

of living matter and fi sh are no exception 

(Eisler, 1991 citing Marrs and Ballantyne, 

1987). Cyanide is a general respiratory poison, 

although uptake can also occur through 

ingestion or dermal absorption (Eisler, 1991 

citing Towil et al., 1978). In high doses, death 

can occur within minutes (Eisler, 1991). In the 

live fi sh trade, the amount of cyanide used is 

relatively low and recovery is quick. 

Where Is Cyanide Fishing Used?

According to Jones (Jones, Kildea et al., 1999)

cyanide fi shing has been confi rmed in at least 

15 countries or island territories including 

Indonesia, Malaysia, Maldive Islands, Papua 

New Guinea, the Philippines, Sri Lanka, 

Thailand, and Vietnam (Johannes and 

Riepen, 1995 cited in Barber and Pratt, 1997; 

McManus, Reyes et al., 1997). The use of 

cyanide is illegal in most countries; however, 

enforcement resources are low and cyanide 

continues to be a regular practice. The use of 

cyanide is also illegal in Australia, yet the law 

appears to be suffi ciently enforced, making 

the Australian live reef food fi shery cyanidefree (Barber and Pratt, 1997)

Fish Collection Techniques Using Cyanide

While fi shing for aquarium and live food fi sh, 

collectors place sodium or potassium cyanide 

tablets in small plastic bottles fi lled with 

seawater (Jones and Steven, 1997). Detergent, 

milk, or kerosene can also be added to the 

squirt bottle to increase the visibility of the 

solution and to reduce the amount of cyanide 

needed. This has the benefi t of reducing costs 

as well as lowering fi sh death from cyanide 

overdose (Erdman and Pet, 1999). 

The collectors squirt the cyanide solution in 

the direction of a fi sh, which may be hiding 

on or inside the coral reef. Additionally, 

anecdotal reports state that in some cases fi sh 

collectors must break large areas of coral to 

retrieve the stunned fi sh hiding in the coral 

(Mouse, Pet-Soede et al., 2000; Fahrudin, 

2003). Divers then collect the asphyxiated 

fi sh and place it in a bag of fresh seawater for 

transport. Throughout the journey, collectors 

will exchange the seawater frequently 

(Johannes and Riepen, 1995). Other collection 

techniques include placing cyanide inside 

portions of bait or as part of a fi sh paste used 

to attract fi sh (Jones and Steven, 1997). 

How Much Cyanide Is Used?

The Marine Aquarium Council website reports 

that the International Marinelife Alliance 

(IMA) tested 48,000 fi sh in the Philippines to 

fi nd that 25% of aquarium fi sh destined for 

the United States and Europe, and 44% of 

live groupers and humphead wrasse going to 

Hong Kong, were caught using cyanide.

The concentration of cyanide in the squirt 

bottles is not known. Analyses of cyanide 

squirt bottles seized on cyanide fishing 

vessels in Indonesia indicated concentrations 

of 2 g/l (Pet and Djohani, 1998). An analysis 

by Fahrudin (Fahrudin, 2003), reported that 

fi shermen from two islands in Indonesia 

used between 50-60 grams of cyanide per 

96
day. One cyanide tablet (2 g) is mixed with 

approximately 3 liters of water in a plastic bag 

or bottle. Based on this information, Fahrudin 

(Fahrudin, 2003) calculated that the resulting 

cyanide concentration is approximately 6.67 

g/l. 

Other studies suggest that cyanide 

concentrations in squirt bottles vary by almost 

an order of magnitude (Jones, Kildea et al., 

1999): 13 g/l (Pet, 1997), 100 g/l (Barber 

and Pratt, 1997), 30±120 g/l (Johannes and 

Riepen, 1995).

What Species Do Fishers Catch with Cyanide?

In the marine fi sh aquarium trade, species 

targeted by cyanide fi shing include nearly all 

coral fi sh species. However, the collectors 

fi rst target the high price fi sh species, such as 

emperor angelfi sh (Pomacanthus imperator), blue 

surgeonfi sh (Paracanthurus sp.), and blue ring 

angelfi sh (Pomacanthus annularis) (Fahrudin, 

2003).

In the live food fi sh trade, common reef fi sh 

caught are groupers (Serranidae), wrasses 

(Labridae), and snappers (Lutjanidae). The 

humphead (Napoleon) wrasse (Cheilinus 

undulatus), high-finned grouper (Cromileptes 

altivelis), and giant grouper (Epinephelus 

lanceolatus) are traded only in small volumes 

but are particularly valued (Johannes and 

Riepen, 1995; Mak, Yanase et al., 2005)

Impact of Cyanide Fishing

It is hard to fi nd documentation on or to 

estimate the impact of cyanide collection 

on individual fi sh or on the environment 

surrounding the fi sh. In addition, the aquarium 

fi sh trade is highly dynamic and collection 

techniques change quickly, depending on cost 

of materials, ease of use, and individual ability 

(Erdman and Pet, 1999). Below is a summary 

of research results that estimate the impact 

of cyanide fi shing on the fi sh collected as well 

as the surrounding environment.

What Does Cyanide Do to the Fish?

As a general summary, hydrogen cyanide is 

toxic to the majority of living matter (Eisler, 

1991 citing Marrs and Ballantyne, 1987). 

Specifi cally, cyanide appears to be acutely 

toxic to aerobic organisms at concentrations 

greater than 0.1 - 0.3 mg/l causing death 

within 96 hours (Doudoroff, 1980 cited in 

Mak, Yanase et al., 2005). 

Eisler (Eisler, 1991) conducted an extensive 

literature review to evaluate the effect of 

cyanide on a large list of organisms including: 

freshwater and marine aquatic organisms, 

birds, and mammals. The results indicated 

that fi sh were the most sensitive aquatic 

organisms tested under controlled conditions. 

Fish that were exposed to a range of 5.0 - 7.2 

μg/l free cyanide per liter displayed signifi cant 

adverse non-lethal effects, including reduced 

swimming performance and inhibited 

reproduction (Eisler, 1991). Mak et al. (Mak, 

Yanase et al., 2005) report that a 10 μM 

solution of cyanide is non-lethal to fi sh and 

equivalent to what fi sh collectors use on the 

reef.

There is also anecdotal evidence from fi sh 

collectors and the marine aquarium industry 

that fi sh caught without cyanide live longer 

and have lower mortality rates throughout 

shipment. Along with increased mortality 

comes the need to catch more fi sh to respond 

to the demand for aquarium fi sh. Ultimately, 

the increased death caused by cyanide fi shing 

increases the potential of overfi shing.

Metabolism of Cyanide in Fish

Hydrogen cyanide enters a fi sh’s bloodstream 

through the gills and intestine and is rapidly 

distributed to other body tissues. Cyanide is 

toxic to fi sh because it interferes with oxygen 

97
metabolism by blocking the key enzyme 

system, cytochrome oxidase (Metzler, 2001), 

and blocks enzymatic pathways in the liver 

(Solomonson, 1981). Some of the effects, 

such as blocking enzyme functions, are 

irreversible and lead to the death of the fi sh 

(Way, Leung et al., 1988). Once inside the 

fi sh tissue, cyanide reacts with thiosulfate in 

the presence of rhodanese to produce the 

comparatively nontoxic thiocyanate. The 

thiocyanate is excreted in the urine. Rapid 

detoxifi cation enables animals, such as fi sh, 

to ingest high, sub-lethal doses of cyanide 

(Eisler, 1991).

Chu (Chu, Liu et al., 2001) used attenuated 

total reflectance and transform infrared 

microspectroscopy to evaluate the molecular 

mechanisms of cyanide toxicity. The results 

showed changes in structural conformation 

of biomolecular/protein components of gill 

tissue and ultimate loss of function. 

It is not possible to defi nitively identify the 

half-life of cyanide in marine fi sh. However, 

because of the volatile nature of cyanide, 

it is known that cyanide breaks down very 

rapidly (Logue, Kirschten et al., 2005). This is 

particularly true in fi sh and even more so in a 

salt-water environment. Logue et al. (Logue, 

Kirschten et al., 2005) state that depending 

on the route and duration of exposure, in 

humans cyanide is typically eliminated from 

blood within 20 minutes post-exposure. 

Exposure of marine fish to a 10 μM 

concentration of potassium cyanide for 15 

minutes is considered to be a sub-lethal dose 

(Mak, Yanase et al., 2005). Preliminary tests 

to examine cyanide concentrations present 

in marine fi sh after exposure to cyanide 

identifi ed a range of 0.04 ppm to 1.02 ppm 

(Hodgson, pers. com.).

Eisler (Eisler, 1991) reported baseline levels 

of cyanide in freshwater fi sh at less than 1 

μg/kg or 1 ppb fresh weight in gills. However, 

values up to 50 μg/kg occurred occasionally 

in fi sh found in natural streams (Eisler, 1991). 

Cyanide concentrations in fi sh from streams 

that were deliberately poisoned with cyanide 

ranged between 10 and 100 μg/kg (10-100 

ppb) total cyanide whole body fresh weight 

(Wiley, 1984). Total cyanide concentrations in 

gill tissues of salmonids under widely varying 

conditions of temperature, nominal water 

concentrations, and duration of exposure 

ranged from about 30 μg/kg fresh weight 

to greater than 7,000 μg/kg (Holden and 

Marsden, 1964; Eisler, 1991).

What Does Cyanide Do to the Coral Reef?

It is even more diffi cult to assess the impact 

of cyanide use on the reef environment. 

Ultimately, the dose of cyanide experienced 

by corals and surrounding organisms is 

a function of cyanide concentration, the 

proximity to target fi sh to the cyanide source, 

and the local hydrological conditions. Under 

conditions of cyanide fi shing, corals are likely 

to experience initially high (10–1 to 10–2 M), 

rapidly fl uctuating concentrations of cyanide 

that ultimately fall to very low levels (10–5 to 

10–6 M) (Jones and Steven, 1997). In some 

cases, water currents can dissipate cyanide and 

carry the toxin away from the reef. However, 

research using dyed water showed that water 

can be trapped in stagnant zones behind coral 

heads (Wolanski and Jones, 1980). Under 

such conditions, cyanide fi shing could result 

in coral mortality (Jones and Steven, 1997).

Early research showed that photosynthesis 

and calcification of staghorn corals were 

inhibited at concentrations greater than 1 

x10–5 M cyanide (Chalker and Taylor, 1975). 

More recently, Jones and Steven (Jones and 

Steven, 1997) showed that respiratory rates 

of Pocillopora damicornis were inhibited by 

98
10–90% following exposure to cyanide but 

recovered to pre-exposure levels within 1–2 

hours after transfer to clean seawater. These 

researchers also observed that corals died 

after exposure of cyanide at the highest doses 

used by fi sh collectors. After medium doses, 

the corals lost their symbiotic algae resulting 

in discoloration or bleaching. After exposure 

to the lowest doses they lost zooxanthellae but 

not in suffi cient numbers to cause noticeable 

discoloration. 

Cervino (Cervino, Hayes et al., 2003) also 

found that cyanide exposure can be lethal to 

corals. Cervino (Cervino, Hayes et al., 2003) 

exposed a variety of coral and anemone species 

in the lab and in the fi eld to the following 

concentrations of cyanide solution for 1-2 

minutes: 50, 100, 300, and 600 mg/l. Upon 

exposure, corals and anemones immediately 

retracted their tentacles and mesenterial 

fi laments, and discharged mucus containing 

zooxanthellae. Gel electrophoreses techniques 

found changes in protein expression in both 

zooxanthellae and host tissue. Both corals 

and anemones showed an immediate increase 

in mitotic cell division of their zooxenthellae, 

and a decrease in zooxanthellae density. In 

contrast, zooxanthellae cell division and 

density remained constant in controls. Other 

changes included gastrodermal disruption, 

mesogleal degradation, and increased mucus 

in coral tissues. Zooxanthellae showed 

pigment loss, swelling, and deformation. 

Cervino (Cervino, Hayes et al., 2003) reported 

mortality occurred at all exposure levels, and 

concluded that exposure to cyanide causes 

mortality to corals and anemones, even when 

applied at lower levels than those used by 

fi sh collectors. Cervino (Cervino, Hayes et 

al., 2003) also stated that even brief exposure 

to cyanide caused slow-acting and long-term 

damage to corals and their zooxanthellae.

Jones et al. (Jones, Kildea et al., 1999) measured 

the effects of cyanide on coral photosynthesis 

through a series of experiments conducted in 

the fi eld. These measurements were made in 

situ and in real time using a submersible pulse 

amplitude modulation (PAM) fl uorometer. 

In Stylophora pistillata, exposure to cyanide 

resulted in an almost complete cessation in 

photosynthetic electron transport rate (Jones, 

Kildea et al., 1999).

Jones et al. (Jones, Kildea et al., 1999)concluded 

that, based on these studies, cyanide-induced 

bleaching has been documented in fi ve species 

of corals. The species studied represent a 

variety of coral types, including encrusting, 

massive, and branching growth forms. 

This experimental evidence of bleaching is 

supported by observations (Erdman and PetSoede, 1996) who report bleached and dead 

corals surrounding holes or recesses on reefs 

where cyanide fi shing had occurred (Jones, 

Kildea et al., 1999).

Cyanide Fishing in Context

This document outlines that exposure to 

cyanide presents a temporary stress on fi sh, 

corals, and surrounding organisms. Overall, 

the literature suggests that these impacts 

have cumulative, potentially long-lasting 

effects; however they are diffi cult to quantify. 

Conservation and Community Investment 

Forum (CCIF) presented one estimate of 

overall reef-degrading capacity of the cyanide 

fi shery for food fi sh. In Indonesia the loss of 

live coral cover is approximately 0.052 m2

 per 

100 m2

 of reef per year (Conservation and 

Community Investment Forum, 2001).

It is important to include the work of some 

researchers that state that the aggregate 

impact of cyanide fi shing is minimal. Mouse 

et al. (Mouse, Pet-Soede et al., 2000) state 

that the toxicity of cyanide to corals under 

experimental conditions is, in itself, no proof 

99
of degradation on the scale of a reef. For 

example, Mouse et al. (Mouse, Pet-Soede 

et al., 2000) state that blast fi shing accounts 

for a loss in live coral cover of 3.75 m2

 per 

100 m2 of reef each year (Pet and Pet-Soede, 

1999), which is about 75 times more than one 

estimate of cyanide impact, and still about 5–6 

times more than the ‘worst-case’ estimate for 

the loss of coral cover due to cyanide fi shing 

for food fi sh.

Other Toxins Used to Catch Fish

Cyanide is the most widely used chemical in 

marine fi sh capture. However, other poisons 

are utilized as well. Clove oil (or eugenol in 

its purifi ed form) is a moderately well-known 

anesthetic for small fi shes and crustaceans 

(eg. Soto and Burhanuddin, 1995; Munday 

and Wilson, 1997; Erdman and Pet, 1999). 

Quinaldine is another narcotic associated 

with the ornamental industry. It is reported 

to be less dangerous than cyanide, but 

capable of killing fi sh during collection when 

concentrations are high or exposure time is 

long (Randall, 1987). Bleach, formalin, and 

gasoline were reported to be used occasionally 

to catch aquarium fi sh in Puerto Rico in the 

early 1990s (Sadovy, 1992), but the extent to 

which they are used elsewhere is not known. 

There are no data available that quantify the 

extent of use of these chemicals.

Non-Toxic Fish Collection

An alternative to fi shing with toxic substances 

such as cyanide is to catch fi sh with small nets 

or to use hook and line. Numerous regional 

organizations train collectors in how to use 

these non-destructive methods. Despite the 

training, collectors have been slow to switch 

to using nets because they can earn more 

using cyanide. In some cases it is possible 

to pay the collector a premium price for fi sh 

caught in a non-destructive manner (Barber 

and Pratt, 1997).

Existing Cyanide Detection Tests

Because of the fact that cyanide can damage 

the marine environment and potentially 

decrease the mortality of fi sh sold in the 

commercial trade, the use of cyanide to 

capture fi sh is illegal in nearly all countries 

where it is commonly used. In order to enforce 

the law and monitor the presence of cyanide 

in fi sh, the International Marinelife AlliancePhilippines (IMA) and the Philippine Bureau 

of Fisheries and Aquatic Resources (BFAR) 

have developed a cyanide detection test (CDT) 

to determine the presence of cyanide in livecaught fi sh. The American Analytical Testing 

Laboratory supported the development of 

the test (Mak, Yanase et al., 2005).

The fi rst CDT laboratory was established 

at BFAR headquarters in Manila in 1992 

and now includes six laboratories and three 

monitoring inspection stations. The IMA 

CDT uses ion selective electrodes (ISEs) to 

detect the concentration of cyanide in the 

distillate (Mak, Yanase et al., 2005, see Table 

1). In brief, internal organs of a fi sh, where 

cyanide is rapidly absorbed, are homogenized 

with water in a blender. Then the homogenate 

is strongly acidified in a 1-hour reflux 

distillation. If cyanide is present in the sample, 

it is liberated as hydrogen cyanide (HCN) and 

absorbed into a sodium hydroxide solution. 

Finally, the presence of cyanide in absorbing 

solution is detected using ISE. Each CDT lab 

is capable of running at least 20 tests per day. 

Each test takes a total of 1.5 hours including 

the procedures described as above. After the 

test, the result is given back to the owner 

of the source of the sample in the form of 

certification (Barber and Pratt, 1997; Mak, 

Yanase et al., 2005).

Possibilities to Improve Cyanide Detection 

Tests

Recently, researchers from the University 

of Hong Kong simulated and reviewed the 

100
existing cyanide detection methods used 

in fi sh samples. The aim was to answer the 

following questions: (1) is the current cyanide 

detection method appropriate and effective 

and (2) what kinds of improvements can 

be done to achieve a higher sensitivity and 

accuracy? (Mak, Yanase et al., 2005).

The results indicate that the current method 

could not measure cyanide concentration 

below 1 × 10-5 m. However, much lower 

cyanide concentration in seawater was 

enough to suffocate a 500 g marine food 

fi sh (Mak, Yanase et al., 2005). Therefore, 

the researchers concluded that the cyanide 

detection method currently used was not 

sensitive enough for the determination of 

cyanide traces in post cyanide-exposed fi sh. In 

other words, these results indicate the amount 

of cyanide necessary to kill fi sh is well below 

the amounts that can reliably be determined 

using the existing procedures (Mak, Yanase et 

al., 2005).

Ultimately, an effective cyanide detection test 

will include the following criteria:

• Sufficient sensitivity to detect low 

quantities of cyanide in marine fish 

hours and perhaps up to days after 

exposure

• Ability to distinguish between 

background levels of cyanide exposure 

versus concentrations resulting from 

illegal fi shing practices

• Use of technology that is available in 

countries where marine fi sh collection 

occurs

• Ability to detect cyanide exposure 

prior to metabolic processing by fi sh

• Technology that is possible for use by 

non-specialized fi eld staff

• Ability to replicate the procedure in 

many locations

• Ability for rapid reporting of 

results to industry and government 

representatives

• Affordable costs

• The ability to test fi sh at the location 

of import country — which could 

take place several weeks after original 

exposure to cyanide

It may be necessary to develop a hybrid 

approach that utilizes a less sensitive screening 

method in the fi eld. Field-based test samples 

that indicate a positive result for cyanide 

use can be followed up with more sensitive 

and verifi able lab-based tests done on the 

same fi sh sample. The techniques that are 

appropriate in the fi eld include colorimetric 

and some bioassay procedures. If the test 

includes chromatography-related methods 

such as gas chromatography and high 

performance liquid chromatography/mass 

spectrometry, it will be necessary to conduct 

this work in a laboratory.

Preliminary Work to Improve Cyanide 

Detection in Fish

Mak and Law (Mak, Law et al., 2005) 

conducted preliminary experiments to 

develop a biosensor system and satisfy the 

necessary detection criteria. Their methods 

include the pretreatment of cyanide with 

cyanide hydrolase and then detection by 

spectrophotometric formate sensor. The 

researchers report that the system has a 

detection limit at 7.3 μmol/l although 

theoretically it is possible to increase the 

sensitivity of the biosensor by injecting a 

larger volume of the pre-incubated sample 

(i.e. more formate) into the measuring cell.

The researchers identify the following 

advantages of this system over other potential 

cyanide detection tests:

101
- cyanide hydratase converts the toxic 

cyanide into non-toxic products for 

measurement

- the combination of enzymes makes 

the system highly specifi c

- there is no need for vigorous sample 

pre-treatment

- no use of hazardous chemicals 

To take this work a step further, Mak et al. 

(Mak, Yanase et al., 2005) used the microbial 

sensor system to measure cyanide levels in 

marine fi sh. They exposed fi sh to sub-lethal 

doses of 10 μM potassium cyanide in seawater 

for 15 minutes and then transferred the fi sh 

to clean seawater for recovery. After different 

recovery times (0-180 minutes), fi sh were 

tested for residual cyanide using the biosensor 

system. Results are expected in a forthcoming 

publication (Mak et al., in prep).

Cyanide Testing Protocols Used in NonFish Settings

Because of the fact that cyanide is a toxic poison 

and the fact that it can be an environmental 

pollutant, there is a large scientifi c literature 

on cyanide detection. 

Table 1 summarizes the most common 

cyanide detection tests as well as advantages 

and disadvantages of each test. Table 2 

presents a sampling of cyanide detection tests 

in human, fi sh, and mouse blood, urine, and 

tissue samples. In addition, Table 2 presents 

some publications reporting on a variety of 

cyanide detection tests.

It is interesting to note that there is a large 

amount of activity in the U.S. federal 

government to develop rapid cyanide 

detection in humans in order to prepare for 

a potential bioterrorist attack using cyanide. 

It is possible to explore these methods and 

experts to fi nd avenues through which to 

improve the current cyanide detection system 

for marine fi sh.

The current method used to test cyanide 

concentrations in fish employs the Ion 

Selective Electrode (ISE) methodology. This 

method is not highly sensitive and it is subject 

to a tremendous amount of interference 

with samples found in seawater. Tables 1 and 

3 present a wide range of methodologies, 

each with varying degrees of cost, sensitivity, 

and potential effectiveness for marine fi sh 

samples. 

Because cyanide breaks down so quickly in 

fi sh, it may be necessary to develop a test 

that measures a by-product of cyanide. Some 

of the possibilities to explore in this arena 

include:

- Thiocyanate. Thiocyanate is a 

non-toxic by-product of cyanide 

degradation.

- Other biological by-products such as:

o formate (Mak, Law et al., 

2005)

o a cyanide/hemoglobin adduct 

(Kobeleski, pers. com.)

o 2-aminothiazoline-4-

carboxylic acid (ATCA) 

(Logue, Kirschten et al., 

2005)

o a gill protein that is changed 

upon cyanide exposure (Chu, 

Liu et al., 2001)

- Cytochrome oxidase — dose-related 

reductions in cytochrome c oxidase 

activity were detected in various 

organs of rats exposed to oral doses 

of potassium cyanide (Ikegaya, 

Iwase et al., 2001). This marker was 

suggested as a method of diagnosis 

for samples taken within two days 

post-mortem.

102
- Plasma lactate — elevated plasma 

lactate concentrations, resulting from 

the shift to anaerobic metabolism, 

have been used to assess the severity 

of cyanide poisoning in humans 

(Baud, Borron et al., 2002)

Thiocyanate is the major metabolite of 

cyanide, and a number of sensitive methods 

are available for its measurement in biological 

fl uids (see Table 2 and Logue, Kirschten et al., 

2005). Thiocyanate can be a good marker for 

cyanide exposure because it is non-volatile 

and can be analyzed in blood, urine, and saliva. 

However, analytical recovery of thiocyanate 

from whole blood is not quantitative and 

thiocyanate concentrations in blood varied 

inconsistently during storage at various 

temperatures over a period of two weeks 

(Logue, Kirschten et al., 2005). In addition, 

thiocyanate may be formed by other modes 

of metabolism besides cyanide intoxication or 

metabolism (Logue, Kirschten et al., 2005). 

These same limitations to measuring 

thiocyanate as a marker of cyanide 

concentration could equally be true 

for other metabolites such as formate and 

ATCA. All systems that are developed to 

detect cyanide will have to demonstrate that 

they can differentiate between background, 

endogenous sources of cyanide, and that 

their concentrations directly correlate with 

external cyanide exposure.

One lab (Logue, Kirschten et al., 2005) 

is developing a promising possibility for 

cyanide detection by looking at the cyanide 

metabolite 2-aminothiazoline-4-carboxylic 

acid (ATCA). ATCA is stable for months in 

biological samples at freezing and ambient 

temperatures (Logue, Kirschten et al., 2005). 

The method analyzes synthetic urine and 

swine plasma through cyanide derivatization 

and subsequent gas chromatography–mass 

spectrometry (GC–MS) analysis. The study 

identifi ed a detection limit of 25 ng/ml. This 

lab has also conducted preliminary tests of 

this method of fi sh samples (Logue, pers. 

com.). However, further research is needed 

to verify the background levels of ATCA in 

fi sh samples, the metabolic clearance rate of 

ATCA in fi sh, and the correlation between 

cyanide exposure and subsequent ATCA 

concentrations.

Summary

The publication of the Mak et al. (Mak, 

Yanase et al., 2005) paper has initiated 

a discussion regarding the possibility of 

developing an improved cyanide detection 

test for marine fi sh. However, it is important 

to acknowledge that the detection of cyanide, 

cyanide compounds, or cyanide metabolites 

in fi sh tissues is quite diffi cult. Once exposed 

to cyanide, the fi sh rapidly incorporate and 

convert cyanide into thiocyanate (SCN-

). 

Shortly thereafter, the thiocyanate is excreted. 

The half-life of the cyanide is extremely 

short. As a result, detection in the fi sh tissues 

will be a factor of the amount of time that 

passes before a test can be performed. Mak et 

al. (Mak, Yanase et al., 2005) point out that it 

may be that detection of total cyanide in fi sh 

would occur only under “optimal conditions” 

such as extreme cyanide exposure followed by 

rapid implementation of a cyanide detection 

test.

103
Method Advantages

Microbial Biosensors

Use of microbial cells or enzymes to convert cyanide 

into by-products. The electrochemical signal produced 

by the cyanide by-products correlates with original 

cyanide concentration.

- inexpensive 

- simple to operate

- products are nontoxic

- fast processing time

- no interference

- samples easily affected 

by nutrients

- limited life time of 

electrode

- sample preparation may 

be required (2 hrs.)

Ion Selective Electrodes

Ion Selective Electrodes (ISE) are membrane 

electrodes that respond selectively to ions in the 

presence of others. The voltage is theoretically 

dependent on the logarithm of the ionic activity, 

according to the Nernst equation.

- does not require 

extensive sample 

preparation

- inexpensive

- equipment is 

durable in fi eld 

settings

- electrode loses 

sensitivity with use

- requires frequent 

recalibration

- results vary with 

temperature

- iodide levels in seawater 

will cause interference

- prone to interference 

from other ions

Fluorometric

Fluorescence spectroscopy or fl uorometry is a type of 

spectroscopy used for analyzing compounds that have 

the ability to fl uoresce. Generally, this fl uorescence 

is directly proportional to the concentration of the 

material in question.

- reduced 

interference

- high sensitivity 

- sample preparation by 

microdiffusion required

- impurities may diminish 

fl uorescence

- interference potential 

still exists without a preseparation mechanism 

.e.g. chromatography

Colorimetric

Colorimetry is a method to measure the concentration 

of a known constituent of a solution by comparison 

with colors of standard solutions of that constituent.

- rapid detection

- cost effective

- interference from 

thiosulfate ions 

- sample preparation 

often required

- interference 

Mass Spectrometry

Mass spectrometry measures the masses and relative 

concentrations of atoms and molecules by using the 

basic magnetic force on a moving charged particle.

- very sensitive and 

selective

- provides 

confi rmatory 

structural 

information

- machines may not be 

available in source 

countries

- relatively expensive

- needs expertise to run 

and maintain instrument

Electron Capture Detection 

ECD detects electron-absorbing components in 

the output stream of a gas chromatograph. The 

concentration is proportional to electron capture.

- selective detection 

combined with 

chromatographic 

detection

- see comments on GC

Nitrogen/ Phosphorous Detection

Detects electron-absorbing components in the output 

stream of a gas chromatograph.

- selective detection 

combined with 

chromatographic 

separation

- See comments on GC

Table 1. Summary of Cyanide Detection Test Methods

104
Method Advantages Disadvantages

High Performance Liquid Chromatography/Mass 

Spectrometry (HPLC-MS)

The combination of a mass spectrometer and a 

liquid chromatograph makes a powerful tool for 

the detection of trace quantities of non-volatile 

compounds.

- “gold standard” 

analytical technique 

for quantitative 

analysis of complex 

biological and 

environmental 

samples

- high sensitivity

- may be most 

suitable to 

investigating 

metabolites of 

cyanide exposure

- potential of 

detecting evidence 

of earlier cyanide 

exposure

- may require 

derivatization (sample 

pre-treatment) of 

analyte of interest (i.e. 

cyanide metabolites)

Gas Chromatography (GC) - very sensitive and 

selective

- possibly expensive

- machines may not be 

available in source 

countries

- needs expertise to 

run and maintain 

instrument

Direct GC 

Gas chromatography is a type of chromatography 

in which the mobile phase is a carrier gas, usually an 

inert gas, and the stationary phase is a microscopic 

layer of liquid on an inert solid support, inside a 

column.

- minimal sample 

preparation but, 

depending on 

analyte of interest, 

extraction of 

sample may be 

required

- see above comments 

on GC

Headspace GC 

Headspace analysis is most suited for the analysis 

of the very light volatiles in samples that can be 

effi ciently portioned into the headspace gas volume 

from the liquid or solid matrix sample. Complex 

sample matrices, which would otherwise require 

sample extraction or preparation, or be diffi cult to 

analyze directly, are ideal candidates for headspace 

since they can be placed directly in a vial with little or 

no preparation. 

- minimal/no sample 

preparation

- relatively easy to 

use once validated 

protocol is 

established

- see above comments 

on GC

105
Paper Method Matrix Detection Limits

(Mak, Yanase et al., 

2005) Microbial Biosensor Fish Tissue

1 x 10-5 – 1 x 10-4

; = 1.1 

μM = 0.286 ppm

(Mak, Yanase et al., 

2005) Microbial Biosensor Fish Tissue 7.3 μmol/l

(Mak, Law et al., 2005) Microbial Biosensor Fish Tissue 7.3 μmol/l

(Ikebukuro, 

Shimomoura et al., 

1996)

Microbial Biosensor Aqueous 

Solutions 0-1.5 x 10-5

(Groom and Luong, 

1991) Microbial Biosensor Aqueous 

Solutions 5 x 10-6 – 1 x 10-3

(Lee and Karube, 

1995) Microbial Biosensor Aqueous 

Solutions

0.1 – 1 ppm in 50 mM 

phosphate buffer (pH 8)

(Smit and Cass, 1990) Biosensor (Peroxidase-based) Aqueous 

Solutions 1.0 x 10-7 (limit of detection)

BFAR Test for Live 

Fish Trade (Romero, 

Rodolfo et al., 2003)

Ion Selective Electrodes (ISE) Fish Tissue Greater than 1 ×10- 5 M 

(Mak et al., 2005)

(Egekeze and Oehme, 

1979)* Ion Selective Electrodes (ISE) Blood and Liver 5 g/l

(Murphy, Schantz et 

al., 2006)

Gas Chromatography (Headspace)/ 

Mass Spectrometry Blood

60 ppb with uncertainty 

of 8%; = 0.7 ng/g = 

0.007 ug/g

(Ishii, Seno et al., 

1998) Gas Chromatography (Direct) Blood 2.0 ng/ml 

(Levin et al, 1990)* Gas Chromatography (Headspace) Not Available Not Available

(Odoul et al., 1994)* Gas Chromatography (Headspace) Not Available Not Avaiilable

(Tsuge, Kataoka et al., 

2000)

Gas Chromatography 

Spectrophotometry Blood and Saliva 0.05 uM

(Hughes, Lehner et al., 

2003)

Gas Chromatography (Headspace) 

and Spectrophotometry Equine Blood 2 ng/ml

(La Forge et al, 1994)* Mass Spectrometry Blood 0.07 g/ml

Table 2. Summary Published Cyanide Detection Tests

106
Paper Method Matrix Detection Limits

(Tomoda and 

Hashimoto, 1991)* Mass Spectrometry Blood 0.4 g/ml

(Cruz-Landeira et al., 

2000)* Mass Spectrometry Urine 28 ng/ml

(Morgan and Way, 

1980)* Spectrophotometry Blood 0.1 ppm

(Tracqui, Raul et al., 

2002)

High Performance Liquid 

Chromatography - Mass 

Spectrometry

Whole Human 

Blood 5.0 ng/ml (abstract only)

(Sano, Takezawa et al., 

1989)

High Performance Liquid 

Chromatography – Fluorometric Urine 30 pmol/ml

(Deschamps, 2004) Colorimetric Aqueous 

Solutions

0.2 ppm and higher, not 

effective for detection of 

0.1 ppm or lower

(Rella, Marcus et al., 

2004) Colorimetric Aqueous 

Solutions Greater than 1.0 ug/ml

(Fligner, Luthi et al., 

1992)

Colorimetric Human Whole 

Blood 0.2 mg/l

(Ganjeloo, Isom et al., 

1980) Colorimetric, Fluorometric Mouse Blood 0.025 ppm (0.001 umol/

ml)

(Pettigrew and Fell, 

1973) Colorimetric (with microdiffusion) Blood

(Sano, Takezawa et al., 

1989) Spectrofl uorimetric Blood and Urine 0.03 nmol/ml

(Sano, Takezawa et al., 

1989) Fluorometric Whole Human 

Blood and Urine

0.03 nmol/ml (abstract 

only)

(Chinaka, Takayama et 

al., 1998) Ion Chromatography/Fluorometry

Cyanide and 

Thiocyanate in 

Blood

107
The following are some methods to detect thiocyanate, a biological by-product of cyanide

(Liu and Yun, 1993) High Performance Liquid 

Chromatography -Mass Spectrometry 

Thiocyanate in 

Saliva 2 ng

(Pettigrew and Fell, 

1973) Colorimetric Thiocyanate in 

Blood and Urine

Approximately 0.07 

ppm1

(Chanttaraj and Das, 

1992)* Spectrometry (fl ame absorption)

Thiocyanate in 

Blood, Urine, 

Saliva

4 ng/ml

(Li et al, 1993)* Spectrophotometry Thiocyanate in

Blood 0.3 ug/ml

(Chen et al., 1994)* Gas Chromatography (Headspace) Thiocyanate in

Urine, Saliva 0.0115 nmo (in 0.2 ml)

(Michigami et al., 

1992)* Ion Chromatography Thiocyanate in 

Urine, Saliva 20 ng/ml

(Tominaga and Midio, 

1991)* Spectrophotometry Thiocyanate in 

Urine 2.5 umol/l

*

 Data obtained from published literature and not original paper

Half-life of cyanide in fi sh Unknown, but possibly 1-3 days

Concentration of cyanide 

in squirt bottles before exposure Wide range: 6.6-120 g/l (see text)

Concentration of cyanide 

in fi sh after exposure 0.04 ppm to 1.02 ppm (Hodgson, pers. com.)

Detection limit of Ion Selective Electrodes currently 

used Greater than 1 × 10- 5 M (Mak, Yanase et al., 2005)

Background level of cyanide in fi sh (freshwater) less than 1 μg/kg or less than 1 ppb (Eisler, 1991)

Background level of cyanide in humans 50 ppb

Toxic level of cyanide in humans

2.5 ppm (Kobeleski, pers. com.) 

0.1 – 0.3 mg/l (Doudoroff, 1980 cited in Mak, Yanase 

et al., 2005). 

Cyanide concentration that produces non-lethal, 

negative impacts in fi sh 5.0 - 7.2 μg/l (Eisler, 1991)

Table 3. Key Cyanide Detection Facts

108
Table 4 lists experts identifi ed to date related 

to cyanide detection. We predict there are two 

possible ways that some of these experts can 

support efforts to develop a cyanide detection 

system in marine fi sh:

- Conduct research to develop the 

laboratory protocols necessary for 

cyanide detection

- Serve as peer reviewers and advisors 

to evaluate and advise the protocol 

development

The following individuals expressed interest 

in reviewing a request for proposals and 

potentially conducting analytical research to 

develop the protocols:

- Dr. Reinhard Renneberg Laboratory, 

Hong Kong University of Science 

and Technology

- Dr. Brian Logue Laboratory, South 

Dakota State University

- A.C.S Laboratory, Cleveland, Dr. 

Mike Makelov

- Haereticus Laboratory, Clifford, VA, 

Dr. Craig Downs

The following individuals have expressed 

interest in providing advisory services in the 

form of review of a request for proposal or 

review of analytical techniques. Several of 

these individuals have a personal connection 

with the issue and are, or have been, aquarium 

hobbyists.

- Dr. Stephen Lowes, Advion and 

owner of coral growing business

- Dr. Bob Kobeleski, Center for Disease 

Control and lead to develop cyanide 

response network

- Dr. Steve Baskin, Aberdeen Proving 

Ground

- Dr. Terry Mudder, Editor, Cyanide 

Monograph

The far left column of the table provides an 

informal indication of the current status of 

communication with the expert. “C” indicates 

an intention to call the individual; “F” means 

we are attempting to fi nd the experts; “Em” 

means we have sent an email; “X” means 

we have completed contact; “M” means we 

have left a message; “H” means we have had 

communication and the contact is on hold for 

the moment; and “P” means this expert is a 

priority for future activities.

109
Name Affi liation Contact Information Notes

P Dr. Stephen 

Lowes Advion 

607-266-0665, ext. 

106/306

607-844-5587 (home)

lowess@advion.com

Rec. by Jack Henion. Owns coral 

growing business and is chemist for 

Advion. Happy to help on this project.

P Bob 

Kobeleski

CDC

Research Chemist

770-488-4686

rmk9@cdc.gov

Leading team of public health labs to 

develop emergency cyanide terrorism 

response network. Very knowledgeable. 

Karen Mak Hong Kong University

Renneberg Laboratory Karen@ust.hk Developed biosensor system to detect 

cyanide in fi sh.

P Steve Baskin Aberdeen Proving 

Ground

410-436-2378

steven.baskin@amedd.

army.mil

Medical/chemical aspects of chemical 

warfare. Rec. by Karen Murphy. Very 

knowledgeable. Writing a review of 

cyanide detection methods.

P Brian Logue

South Dakota State 

University, Department 

of Chemistry and 

Biochemistry

954-629-2134

brian.logue@sdstate.edu

Developed test to detect ATCA- a CN 

metabolite. Uses GC-MS method and 

trying to develop an HPLC method. Set 

of 10 samples takes 4 hrs (15 minutes 

for each GC-MS sample).

G Terry Mudder Editor, Cyanide 

Monograph.

Cyunara@aol.com

www. Cyantists.com

Mining and cyanide expert. Very 

helpful, as is the website. Also Lead of 

Cyantists, Coordinator of Cyanide in 

the Environment Conference.

C

Tim Van 

Weingarden

Ask for other 

experts.

ACZ labs

ACZ Laboratories, Inc.

Phone: 970-879-6590

Toll Free: 800-334-5493

Fax: 970-879-2216

Terry Mudder says best lab in country 

that works on cyanide. Ask for lab 

manager. Lab says it does not carry out 

R&D, so can’t develop a protocol.

G Ben Blount

Lead Research Chemist

VOC and Perchlorate 

Laboratory,

CDC

770-488-7894

bkb3@cdc.gov

Bob Kobeleski says he is looking at 

thiocyanate in urine. Has a proposal 

with a pharma house to look at CN 

exposure in fi re victims.

G Mike Makelov A.C. S. Labs

Cleveland, Ohio

216-642-8515

headspace@earthlink.net

Physical chemist.

Absolute expert on headspace GC.

X Barry Logan State Toxicologist,

Washington State

206-262-6000

barry.logan@wsp.wa.gov

Worked to developed a quick test of 

human blood with Cyantesmo paper 

strip.

C Steve Burden

Retired from BP

ran CN testing lab, was 

a marine biologist

419-657-6304

419-657-6853 Maybe would collaborate with Makelov.

Table 4. Cyanide Experts

110
X Jerry Thomas CDC 770-488-7279

ciq1@cdc.gov

Board-certifi ed toxicologist. Working on 

a fi re exposure antidote. Interfaces with 

Merck Germany. Rec. by Bob K.

Research Triangle 

Institute

Charlie Sparacino Spar@

rti.org

919-541-6581

Em 8/22/06

Edgar Cook, retired (M 

8/21/06

540-885-2945

919-541-6000

Working with EPA

Em Steve Borron UT? Sborron@intoxicon.com

Boron@uthscsa.edu

Setting up surveillance of CN in hour 

fi re victims. Rec. by Jerry Thomas.

X Jack Henion Adivon 607-266-9162 x308

henionj@advion.com

Veterinary toxicologist. Former 

professor at Cornell Vet school. Has his 

own company. Might know someone in 

fi eld. 

X Todd 

Anderson

Texas Tech

Institute of 

Environment and 

Human Health

806-885-0231

todd.anderson@tiehh.ttu.

edu

Ecotoxicologist with big perchlorate lab 

in fi sh tissue. Like CN, it is an ion that 

you measure with same techniques. He 

uses ion chromatography method. 

C Alex 

Krynitsky

FDA

Research Chemist 301-436-2098 Measures perchlorate in food.

Em Yasuo Seto

National Research 

Institute of Police 

Science, 4th Chemistry 

Section

Seto@nrips.go.jp

81-471-35-8001

F James Way Previously at Texas 

A&M Jlway@medicine.tamu.edu Was on HHS panel (ask him about others).

F Bryan 

Ballantyne

Retired.

Previously, Dow 

or Union Carbide, 

Director of Applied 

Toxicology

Charleston, West Virginia

True expert on Cyanide. Wrote 

“Toxicology of Cyanide.”

Determining if still active.

C Tim Miller 

Morgan

Oregon State 

University,

Asst. Professor, 

Aquatic Pets

Hatfi eld Marine Science 

Center

541-867-0265

541-270-4218

tim.miller-morgan@

oregonstate.edu

Collaborating with MAC. They have 

done some cyanide testing in cattle.

F University of Michigan According to Paul Holthus they have a 

specialty of cyanide testing in animals.

111
F Gary Isom Purdue University Recommended by Steve Baskin.

F Joe Borowitz Purdue University Recommended by Steve Baskin.

F Gary 

Rockwood

Aberdeen Proving 

Ground Recommended by Steve Baskin.

F Randal Baselt

Biomedical 

Publications 

Corporation. Forensic 

toxicologist.

650-344-2256

X Prof. Paul 

Bowser

Cornell College of 

Veterinary Medicine, 

fi sh pathologist

607-253-4029

prb4@cornell.edu

Rec. by Jack Henion. He is happy to 

help, it is a bit off-fi eld for him.

Em Michael Peat

Editor, Journal of 

Forensic Sciences, 

toxicologist

Michael.peat@att.net

Jfs.editor@att.net

Previous Director of Center for Human 

Toxicology

Em

Bruce 

Goldberger Editor, Journal of 

Analytical Toxicology

Rocky Point Labs

Offi ce: 352-265-0680 

Bruce-goldberger@ufl .edu

H Ed 

Pfannkock Gerstel Laboratories Support@gerstelus.com410-204-7251

Providing technical support to 

cyanide terrorism network. Assisted 

in equipment development. Makes the 

automated sampler (cost is $30K) used 

by CDC.

F Ronald Eisler

National Fish and 

Wildlife Service, 

perhaps now at NOAA

Reisler@fi sheries.org

301-897-8616

Evaluated toxic environmental impact 

of NFWS cyanide application program.

X JR 

Deschamps

U.S. Navy, Laboratory 

for Structure and 

Matter

Deschamps@nrl.navy.mil Field test for water.

Em Craig Downs Haereticus Laboratory haereticus1@hughes.net 

Phone/Fax: 434-263-5740

Has conducted cyanide test on fi sh in 

the Middle East.

X Bill McShane Toxicologist, CDC 770-488-4311

wmm9@cdc.gov

Participant on CDC cyanide terrorism 

project. Not able to talk to us.

H Cyanide Reagent Strip Used by EPA

Not good for fi eld

H ES Deocadiz

Environmental 

Management Bureau 

(EMB), DENR, 

Philippines

Wrote ASEAN marine water quality 

criteria for cyanide.

H Peter Scott Previously with MAC Managed process with MAC and Merck 

to evaluate current BFAR test.

112
References

Barber, C. and Pratt, V. (1997) Sullied Seas: 

Strategies for Combating Cyanide Fishing in 

Southeast Asia and Beyond. World Resources 

Institute and International Marinelife 

Alliance, Washington, D.C.

Baud, F. J., Borron, S. W., et al. (2002) Value 

of Lactic Acidosis in the Assessment of 

the Severity of Acute Cyanide Poisoning. 

Clinical Investigations 30:9, 2044-2050.

Cervino, J. M., Hayes, R. L., et al. (2003) Changes 

in Zooxanthellae Density, Morphology, and 

Mitotic Index in Hermatypic Corals and 

Anemones Exposed to Cyanide. Marine 

Pollution Bulletin 46:5, 573.

Chalker, B. E. and Taylor, D. L. (1975) LightEnhanced Calcifi cation, and the Role of 

Oxidative Phosphorylation in Calcifi cation 

of the Coral Acropora cervicornis. Proceedings of 

the Royal Society of London, Series B: Biological 

Sciences 190:1100, 232-331.

Chinaka, S., Takayama, N., et al. (1998) 

Simultaneous Determination of Cyanide 

and Thiocyanate in Blood by Ion 

Chromatography with Fluorescence 

and Ultraviolet Detection. Journal of 

Chromatography, B Biomedical Sciences and 

Applications 713:2, 353-359.

Chu, H. L., Liu, T. Y., et al. (2001) Effect of 

Cyanide Concentrations on the Secondary 

Structures of Protein in the Crude 

Homogenates of the Fish Gill Tissue. 

Aquatic Toxicology 55:3-4, 171.

Conservation and Community Investment 

Forum (2001) Analysis of Destructive Reef 

Fishing Practices in the Indo-Pacifi c. San 

Francisco, CA.

Deschamps, J. R. (2004) Navy Cyanide Test 

Kit. 2004 NRL Review. J. D. Bultman, 

NAVSEA, 115-116.

Eisler, R. (1991) Cyanide Hazards to Fish, 

Wildlife, and Invertebrates: A Synoptic Review.

U.S. Fish and Wildlife Service.

Erdman, M. V. and Pet, J. S. (1999) Krismon 

& DFP: Some Observations on the Effects 

of the Asia Financial Crisis on Destructive 

Fishing Practices in Indonesia. SPC Live 

Reef FIsh Information Bulletin 5, 22-26.

Erdman, M. V. and Pet-Soede, L. (1996) How 

Fresh Is Too Fresh? The Live Reef Food 

Fish Trade in Eastern Indonesia. NAGA, 

the International Centre for Living Aquatic 

Resource Management Quarterly 18, 4-8.

Fahrudin, A. (2003) Extended Cost Benefi t 

Analysis of Present and Future Use of 

Indonesia Coral Reefs: an Empirical Approach 

to Sustainable Management of Tropical Marine 

Resources. Institut fü r Agrarokonomie,

Christian-Albrechts-Universitat zu 

Kiel, Doktorgrades der Agrar-und 

Ernahrungswissenschaftlichen Fakultat, 

Jakarta, Indoneisa.

Fligner, C. L., Luthi, R., et al. (1992) Paper 

Strip Screening Method for Detection 

of Cyanide in Blood Using Cyantesmo 

Test Paper. The American Journal of Forensic 

Medicine and Pathology 13:1, 81-84.

Ganjeloo, A., Isom, G. E., et al. (1980) 

Fluorometric Determination of Cyanide 

in Biological Fluids with p-Benzoquinonel. 

Toxicology and Applied Pharmacology 55, 103-

107.

Groom, C. A. and Luong, J. H. T. (1991) A 

Flow Through Analysis Biosensor System 

for Cyanide. Biotechnology 21, 161-172.

113
Holden, A. V. and Marsden, K. T. (1964) Cyanide 

in Salmon and Brown Trout. Department of 

Agriculture and Fisheries for Scotland, 

Edinburgh, Scotland.

Hughes, C., Lehner, F., et al. (2003) A Simple 

and Highly Sensitive Spectrophotometric 

Method for the Determination of Cyanide 

in Equine Blood. Toxicology Mechanisms and 

Methods 13:2, 129-138.

Ikebukuro, K., Shimomoura, M., et al. (1996) 

A Novel Biosensor System for Cyanide 

Based on a Chemiluminescence Reaction. 

Analytical Chimica Acta 329, 111-116.

Ikegaya, H., Iwase, H., et al. (2001) Diagnosis 

of Cyanide Intoxication by Measurement 

of Cytochrome C Oxidase Activity. 

Toxicology Letters 119:2, 117-123.

Ishii, A., Seno, H. , et al. (1998) Determination 

of Cyanide in Whole Blood by Capillary 

Gas Chromatography with Cryogenic 

Oven Trapping. Analytical Chemistry 70, 

4873-4876.

Johannes, R. and Riepen, M. (1995) 

Environmental, Economic and Social Implication 

of the Live Reef Fish Trade in Asia and 

the Western Pacifi c. 87pp, The Nature 

Conservancy, Honolulu, HI.

Jones, R. J., Kildea, T., et al. (1999) PAM 

Chlorophyll Fluorometry: a New In 

Situ Technique for Stress Assessment in 

Scleractinian Corals, Used To Examine the 

Effects of Cyanide from Cyanide Fishing. 

Marine Pollution Bulletin 38:10, 864.

Jones, R. J. and Steven, A. L. (1997) Effects of 

Cyanide on Corals in Relation to Cyanide 

Fishing on Reefs. Marine and Freshwater 

Research 48, 517-522.

Lee, J. and Karube, I. (1995) A Novel Microbial 

Sensor for the Determination of Cyanide. 

Analytica Chimica Acta 313, 69-74.

Liu, X. and Yun, Z. (1993) High-performance 

Liquid Chromatographic Determination 

of Thiocyanate Anion by Derivatization 

with Pentafl uorobenzyl Bromide. Journal of 

Chromatography 653:2, 348-353.

Logue, B. A., Kirschten, N. P., et al. (2005) 

Determination of the Cyanide Metabolite 

2-Aminothiazoline-4-Carboxylic Acid in 

Urine and Plasma by Gas ChromatographyMass Spectometry. Journal of Chromatography, 

B, Analytical technologies in the biomedical and 

life sciences 819:2, 237-244.

Mak, K. K. W., Yanase, H., et al. (2005) 

Novel Optical Biotest for Determination 

of Cyanide Traces in Marine Fish Using 

Microbial Cyanide Hydratase and Formate 

Dehydrogenase. Michrochimica Acta 149, 

131-135.

Mak, W., Law, C., et al. (2005) Application 

of Cyanide Hydrolase from Klebsiella sp. in 

a Biosensor System for the Detection of 

Low-Level Cyanide. Applied Microbiology and 

Biotechnology 67, 631-636.

Mak, W., Yanase, H., et al. (2005) Cyanide 

Fishing and Cyanide Detection in Coral 

Reef Fish Using Chemical Tests and 

Biosensors. Biosensors & Bioelectronics 20:12,

2581.

Marine Aquarium Council (2006) 

Marine Aquarium Council. http://www.

aquariumcouncil.org/.

McManus, J. W., Reyes, R. B. J., et al. (1997) 

Effects of Some Destructive Fishing 

Methods on Coral Cover and Potential 

114
Rates of Recovery. Environmental Management 

21, 69-78.

Metzler, D. E. (2001) Biochemistry: The Chemical 

Reactions of Living Cells. Academic Press, 

San Diego, CA.

Mouse, P., Pet-Soede, L., et al. (2000) Cyanide 

Fishing on Indonesian Coral Reefs for 

the Live Food Fish Market- What is the 

Problem? SPC Live Reef Fish Information 

Bulletin 7, 1-8.

Munday, P. L. and Wilson, S. K. (1997) 

Comparative Effi cacy of Clove Oil and 

Other Chemicals in Anaesthetization of 

Pomacentrus amboinensis, a Coral Reef Fish. 

Journal of Fish Biology 51, 931-938.

Murphy, K., Schantz, M., et al. (2006) 

Determination of Cyanide in Blood by 

Isotope-Dilution Gas ChromatographyMass Spectometry. Clinical Chemistry 52, 

458-467.

Pet, J. (1997) Destructive Fishing Methods in 

and around Komodo National Park. SPC 

Live Reef Fish Information Bulletin 2, 1-5.

Pet, J. and Djohani, R. (1998) Combating 

Destructive Fishing Practices in 

Komodo National Park: Ban the Hookah 

Compressor! SPC Live Reef Fish Bulletin 4, 

1-12.

Pet, J. and Pet-Soede, L. (1999) A Note on 

Cyanide Fishing in Indonesia. SPC Live 

Reef Fish Bulletin 5.

Pettigrew, A. R. and Fell, G. S. (1973) 

Microdiffusion Method for Estimation of 

Cyanide in Whole Blood and its Application 

to the Study of Conservation of Cyanide 

to Thiocyanate. Clinical Chemistry 19:5, 466-

471.

Randall, J. E. (1987) Collecting Reef Fishes 

for Aquaria. In Human Impacts on Coral Reefs: 

Facts and Recommendations (B. Salvat, Ed.) 30-

39, French Polynesia, Antenne MuseumEPHE.

Rella, J., Marcus, S., et al. (2004) Cyanide 

Detection Using the Cyantesmo Kit. 

Journal of Toxicology: Clinical Toxicology 42:6,

897-900.

Romero, M., Rodolfo, M., et al. (2003) Cyanide 

Detection Testing Validation Report Program 

Phases 1 and 1a. Philippines Department 

of Agriculture Bureau of Fisheries and 

Aquatic Resources (BFAR).

Sadovy, Y. (1992) A Preliminary Assessment 

of the Marine Aquarium Export Trade 

in Puerto Rico. Proceedings of the Seventh 

International Coral Reef Symposium 2, 1014-

1022.

Sano, A., Takezawa, M., et al. (1989) 

Spectrofl uorometric determination 

of cyanide in blood and urine with 

naphthalene-2,3-dialdehyde and taurine. 

Anal Chim Acta 225, 351-358.

Smit, M. and Cass, E. (1990) Cyanide Detection 

Using a Substrate Regenerating, Peroxidebased Biosensor. Analytical Chemistry 62, 

2429-2436.

Solomonson, L. P. (1981) Cyanide as a 

Metabolic Inhibitor. In Cyanide in Biology 

(Vennesland, E. E. C. B., Knowles, C.J., 

Westley, J. and Wissing, F., Eds.) 11-

28, Academic Press, London, United 

Kingdom.

Soto, C. G. and Burhanuddin (1995) Clove 

Oil as a Fish Anaesthetic for Measuring 

Length and Weight of Rabbitfi sh (Siganus 

lineatus). Aquaculture 135, 149-152.

115
Tracqui, A., Raul, J. S., et al. (2002)

Determination of Blood Cyanide by 

HPLC-MS. Journal of Analytical Toxicology 

26:3, 144-148.

Tsuge, K., Kataoka, M., et al. (2000) Cyanide 

and Thiocyanata Levels in Blood and Saliva 

of Healthy Adult Volunteers. Journal of 

Health Science 46:5, 343-350.

Way, J. L., Leung, P., et al. (1988) The 

Mechanism of Cyanide Intoxication and 

its Antagonism. CIBA Foundation Symposium 

140, 232-243.

Wiley, R. W. (1984) A Review of Sodium 

Cyanide for Use in Sampling Stream 

Fishes. North American Journal of Fisheries 

Management 4:3, 249-256.

Wolanski, E. and Jones, M. (1980) Water 

Circulation around Britomart Reef, Great 

Barrier Reef, during July 1979. Australian 

Journal of Marine and Freshwater Research 31, 

415-430.

116
COUNTRY REPORTS

Destructive Fishing Practices in Vietnam:

Current Status with a Focus on Cyanide Fishing

Ho Thi Yen Thu, Vice-Director

Simone M. Retif, Advisor

Minh Hoang, Acting Head, Coastal Resources Management Department

Tran Thi Hoa, Vice-Head, Community Development Department

Introduction

Vietnam has a coastline of over 3260 km 

with an Exclusive Economic Zone of over 1 

million square kilometers. The fi sheries sector 

plays a vital role in the economic and social 

development of Vietnam. Of a population 

of 84 million the livelihood of 8 million 

people depends on fi sheries resources as the 

household primary income source and an 

additional 12 million get part of their income 

or subsistence from fi sheries (MOFI and 

WB, 2005). Although over the past decade, 

the fi sheries sector has achieved considerable 

growth this has not been without concerns 

of sustainable development. Such problems 

include overfi shing in the coastal area, 

degradation of the marine environment 

and coastal resources, including the use of 

destructive fi shing methods, and the lack of 

effective resource management. The use of 

destructive fi shing methods yields short-term 

economic benefi ts for fi shers but endangers 

the long-term sustainability of fi shing and 

other reef-dependent industries such as 

ecotourism.

A report released in 2002 documented that 

destructive fi shing practices, including the use 

of poison and dynamite, threaten as much as 

85 percent of Vietnam’s reefs (WRI, 2002). 

The leading threats to Vietnam’s reefs in 2004 

continued to be over-fi shing and destructive 

fi shing. Based on 2003-2004 data, most reefs 

surveyed in Vietnam have less than 25% live 

coral cover (Tun et al., 2004).

Vietnam ranks among the top fi ve countries 

most impacted by climate change (Dasgupta 

et al., 2007). Eliminating destructive fi shing 

in Vietnam, including cyanide fishing, will 

assist to enhance resistance and resilience to 

climate change of Vietnam’s coastal resources 

by alleviating the overall pressures on the 

system, giving it more fl exibility to mobilize 

its natural defenses.

This report details the current status of 

management and use of destructive fi shing 

practises in Vietnam with a particular focus 

on cyanide fi shing. To alleviate ecological 

impacts of destructive fishing practises 

particularly cyanide use, a series of urgent 

measures is suggested covering the legislative 

framework, improving law enforcement and 

awareness raising.

Overview of destructive fishing 

practises

In Vietnam four common methods of 

destructive fi shing, which have been illegal in 

Vietnam since 1989, are:

1. Destructive fi shing gear (e.g. illegal 

mesh size)

2. Explosive fi shing (also referred to as 

blast or dynamite fi shing)

3. Electro fi shing 

4. Poison fi shing (mainly cyanide)

119
The first three of these destructive fishing 

methods are discussed below. A detailed 

assessment of cyanide fi shing (a frequently 

used poison for fi shing), one of the most 

commonly used destructive fi shing methods 

used in Vietnam, is provided in section 3. 

Destructive fi shing gear

The majority of fi shing gear used by fi shers 

in Vietnam do not conform to mesh size 

regulations (Nguyen Chu Hoi, 2008). For 

example the sizes of trawl cod end, purse 

seine’s bunt net and trammel net are so small 

that a large proportion of juveniles are caught. 

A number of harmful fi shing gear such as 

estuary set nets and scoop nets are still used 

(RIMF (2006). There is scant information on 

the extent and impact of destructive fi shing 

gear on fi sheries resources as well as impacts 

on bycatch and habitats. 

Dynamite/blast fi shing

Dynamite or ‘‘blast’’ fi shing is one of the 

most immediate and destructive threats to 

coral reefs worldwide. Dynamite fi shing 

mainly occurs in the marine environment 

in Vietnam. As with other methods of 

destructive fi shing in Vietnam there is little 

quantitative information available on the 

extent of use of dynamite fi shing. A survey in 

Nha Trang Bay showed that around 10% of 

manta-tows showed evidence of blast fi shing 

in the form of craters and or other obvious 

physical damage to reef areas (Vo Si Tuan 

et al., 2002, cited in Tuan V.S. et al 2005). 

Based on the fi eldwork of the study team and 

available data a Ministry of Fisheries (MOFI) 

and World Bank report in 2005 concluded 

that dynamite fi shing is still widespread in 

some areas in Vietnam, but in areas where the 

resource has already been severely depleted it 

has ended (MOFI and WB, 2005). Dynamite 

has also been recorded as being used in 

conjunction with push nets where the push 

net is used to herd the fi sh and dynamite is 

thrown into the herded school of fi sh (Smith 

et al., 2003).

Electro fi shing

Electro fi shing occurs in freshwater and salt 

water areas in Vietnam. Several kinds of 

electro fi shing are used in Vietnam a number 

of which are detailed below.

Xiec Dien – electro fi shing equipment made 

with a large Y-shaped wooden scoop net at 

the end of which are two electric poles that 

transmit the voltage into the water stunning 

small shrimp and fi sh.

Gia nui – electric fi shing equipment is attached 

to a dragnet to enhance its effi ciency

Gia tiep – fi shers use electric poles between 

two boats and close to the sea fl oor affecting 

a large area.

Incentives to engage in illegal electro fi shing 

have been documented as a low initial 

investment cost and compared to traditional 

fi shing methods it is less labour intensive and 

no specifi c knowledge is needed. This is in 

contrast to traditional fi shing methods where 

nets are more costly and require specifi c 

fi shing knowledge of where and when to fi sh 

(Strehlow, 2006). The level of enforcement 

and the fi ne levels are also not considered a 

suffi cient a deterrent. 2005 MOFI and World 

Bank report concluded that the use of electric 

fi shing in Vietnam is increasing, destructive 

and diffi cult to prevent (MOFI and WB, 

2005).

Cyanide fi shing

Poisons (Chat doc) are used widely in 

Vietnam as a method of fi shing. One of the 

most commonly used poisons is Cyanide. 

Marine aquarium species that are diffi cult to 

catch by mesh and scoop nets because they 

hide in holes and burrows such as Angel fi sh 

(Serranidae) and eels (Anguilla) are caught by 

cyanide or anaesthetics (Truong Si Ky, 2006). 

120
The detrimental effects of cyanide fi shing 

on coral reef fi sh as well as coral are well 

documented (Rubec et al., 2001, Cervino et 

al., 2003). 

Current status of cyanide fi shing

Cyanide fi shing in Vietnam is largely driven 

by two key fi sheries:

1. The international trade in live reef food 

fi sh

2. The marine aquarium fi shery

A report on the status of Vietnam’s reefs in 

2005 noted that although dynamite fi shing 

had decreased, cyanide fi shing had become 

more popular due to the increased demand of 

the live fi sh trade (Tuan et al., 2005). Vietnam 

is located near to the two biggest live reef 

fi sh markets Hong Kong and China - a nation 

importing up to 90% of food fi sh worldwide 

(Tuan et al., 2005). 

The distribution of the use of cyanide 

fi shing in Vietnam has not been surveyed 

in a systematic way. Areas of use of cyanide 

have only been identifi ed in scatted reports. 

The use of cyanide is common in reef sites in 

the north (Co To, Bach Long Vy) and central 

provincial islands (Ly Son, Cu Lao Cham) 

due to the availability of cheap cyanide pills 

(8000 VND each - USD 0.5). Exploitation of 

coral reef fi sh using cyanide in Co To Island 

(Quang Ninh province), has been identifi ed as 

the major cause of a critical decline in marine 

aquarium fi shery resources (Nguyen Van 

Quan et al., 2007). Overfi shing and use of 

destructive methods (cyanide, blast and fi ne 

mesh nets) for a range of coral reef fi sheries 

has resulted in widespread destruction of 

reefs in the following provinces in Vietnam: 

Quang Ninh, Nghe An, Quang Binh, Thua 

Thien-Hue, Quang Nam, Da Nang, Quang 

Ngai and Khanh Hoa (See Figure 1) (Chou, 

2000).

Hon Mun MPA island communities (Khanh 

Hoa Province) are home to many poor 

fisher families whose subsistence includes 

a decreasing catch supplying the local and 

international aquarium fi sh trade. They are 

responsible for a large proportion of the 

unsustainable fi shing activity near Hon Mun 

including the use of cyanide and dynamite 

(GEF Project documentation http://

www.gefweb.org/COUNCIL/GEF_C14/

vietnam/viethon.pdf).According to local 

sources cyanide fi shing was only introduced 

into Khanh Hoa between 1994 and 1996. 

Local government offi cials believe that Hong 

Kong-based middlemen and traders supply 

cyanide to fi shermen to guarantee the supply 

of certain volumes and species (McCullough 

and Hai, 2001).

In Bach Long Vy Island, the level of cyanide 

in the sea was on average 0.65mg/l, more 

than 13 times the permitted limit; 300mg/

kg in sediment, 3 times more than Canada’s 

standard; 40mg/kg in seaweed, 20 times more 

than America’s standard; 550mg/kg in abalone, 

more than 2.5 times America’s standard. The 

coral “jungle” in north-east Bach Long Vy has 

been signifi cantly destroyed due to the use of 

cyanide to fi sh for grouper. The environment 

surrounding Bach Long Vy Island has been 

polluted by poisons for a long time, one of 

the reasons leading to exhausted abalone 

resources(Nguyen Huy Yet et al., 2004). 

This scattered reporting of the use of cyanide 

use highlights the need for developing a 

structured project to determine the areas 

and extent of use in order to inform suitable 

management responses.

In Vietnam, prices paid to fi shers for live 

coral groupers are three times higher than for 

frozen (McCullough and Hai, 2001), making 

the use of illegal cyanide a viable option 

particularly when fi nes are relatively low and 

121
enforcement is minimal. There is anecdotal 

information that middlemen that buy live reef 

food fi sh also supply fi shers with the cyanide 

(Strehlow, 2006). 

In 2002 it was reported that 80% of the 

live marine aquarium fi sh exported from 

Vietnam die during transport to consuming 

nations from the detrimental effects of 

cyanide, ammonia and stress (Rubec and 

Palacol, 2002). The current percentage of 

mortalities in live marine aquarium fi sh from 

Vietnam is unknown. High mortality rates, 

due to cyanide capture and poor collection, 

transport, and handling procedures is a major 

problem impacting the viability of the marine 

aquarium and live reef food fi sh export trade 

from Vietnam.

In Vietnam little attention is paid to the 

health of fi shers using diving equipment, 

despite warnings in the media by scientists 

regarding the harmful effect of using cyanide 

for fi shing and using directly compressed air 

without fi ltering while diving (Barber and 

Pratt, 1997, 1998). Divers in Vietnam have 

reported feeling drunk for a short time after 

using too much cyanide (Strehlow, 2006). 

Little is known about the long term health 

impacts of cyanide on fi shers.

Although there seems to be a good 

understanding of the negative impacts of 

cyanide fi shing, the economic returns surpass 

those of traditional fi shing, and with little 

enforcement, this illegal activity persists. 

Researchers have worked with local 

communities in Indonesia and the Philippines 

to develop cyanide-free net capturing 

techniques for the marine aquarium trade 

(Rubec et al., (2001). In order to reduce the 

impact of cyanide fishing on coral reefs, 

as well as on diver health, alternatives for 

cyanide fi shing need to be developed for local 

Vietnamese fi shing communities.

An assessment of the legislative landscape for cyanide 

management

The Vietnamese government has made 

various commitments internationally and 

promulgated legislation domestically to 

eliminate destructive fishing practises. 

International Commitments

In the international arena Vietnam has made 

numerous commitments to phasing out 

destructive fi shing practices as detailed in 

Appendix 1. Key commitments include: 

• FAO Code of Conduct for Responsible 

Fisheries (1995) which contains a 

commitment to end destructive fi shing 

practices 

• APEC Fisheries Working Group (1997) 

- Endorsed a resolution urging “all 

members of the international community 

to cease the practise of cyanide fi shing”

• WSSD (2002) - Contains a commitment to 

phasing out destructive fi shing practices 

in the marine environment by the year 

2012

Current domestic legislation and policies

A range of domestic legislation has been 

promulgated relating to destructive fi shing in 

Vietnam (see Appendix 2). A decree strictly 

forbidding the use of dynamite, poison 

(cyanide) and electric fi shing was fi rst made 

in 1989 (Chapter II, Article 8 (1) Ordinance 

on the protection and development of 

aquatic resources 1989). Despite enactment 

of this legislation the use of destructive 

fishing practises still persisted 9 years later, 

particularly in provinces in central Vietnam. In 

response to this situation the Prime Minister in 

1998 produced a directive “strictly banning the 

use of explosives, electric impulses and toxics 

to exploit aquatic resources”, also known as 

Directive 01 (Directive 01/1998/CT-TTg 2 

122
January 1998). The directive prescribed action 

on compliance, enforcement and awareness 

raising. Additionally it directed District 

Peoples Committees to make plans to survey 

and classify poor fi shers and take measures 

to assist them in developing aquaculture 

or alternative sustainable fi shing methods 

(Article 6 Directive 01/1998/CT-TTg).

The use of dynamite, electro and poison 

fi shing declined during the period 1999-2003 

following the implementation of Directive 

01. However, since 2006, due to declining 

coastal fi sheries resources and higher petrol/

oil prices, the use of dynamite, electro and 

poison fi shing has increased. In response to 

this increase the government of Vietnam, 

since early 2007, has set up a permanent 

Steering Committee (also called the Steering 

Committee 01) to prevent dynamite, electric 

and poison fi shing. The Steering Committee 

has identifi ed the following important tasks 

that they have accomplished: 

Figure 1: Key provinces with wide spread destruction of reefs due to destructive fi shing practices (Source: Chou, 

2000)

123
1. Management/guidance to the Ministry of 

Fisheries (MOFI) (consulted by Steering 

Committee 01) concerning continued 

strengthening and implementation 

of the Directive 01 (promulgated 

the document 1679/BTS-BVNLTS 

26/7/2007). MOFI specifi cally requests 

Provincial Peoples Committees to:

• Implement widely and frequently 

Directive 01 to district and commune 

levels and people unions.

• Strengthen the Steering Committee 

from provincial to district and 

commune levels. The Steering 

Committees should include some 

institutions: fisheries, public 

security/police, military, culture 

and information. Commune 

people’s committee chairmen 

should have the main responsibility 

for implementation. 

• Strengthen communications of 

Directive 01.

• Undertake regular inspections,

• Investigate and propose alternative 

livelihoods for communities living 

near coastal, water bodies, lagoons.

• Report to the Steering Committee 

every 3, 6 and 12 months.

2. Communication, education and training

3. Inspection of implementation of 

Directive 01 in some local areas

4. Cooperation with DOFI inspectors: in 

inspecting in the rivers and the sea

The 01 Steering Committee detailed the 

following solutions for implementing 

Directive 01 in 2008 including:

• Development of new regulations for the 

01 Steering Committee activities

• Strengthening local Steering committees 

and establishing Steering committees 

in areas where they have not yet been 

established

• Review and summary by localities of 10 

years of implementation of Directive 01 

(1998-2008) to prepare for a National 

conference expected to be held in the 

third quarter of 2008 (MARD, 2007)

In order to develop Directive 01 effectively, 

the Steering Committee 01 was strengthened 

with the membership of four Ministries: 

MOFI (recently integrated to MARD), 

Ministry of National Defence, Ministry of 

Public Security and Ministry of Information 

and Communications. This combination 

of ministries highlighted the important role 

of information to strengthen community 

awareness and understanding of banned 

fi shery activities. However this role has not yet 

been adequately considered at the local level 

where the illegal fi shers are active because of 

the ineffective collaboration between local 

stakeholders and communication system.

Although cyanide fi shing is illegal, this 

chemical is still circulated and traded quite 

openly on the market in Vietnam. Cyanide 

is not listed in the government decision on 

chemicals banned from export and import 

(Decision 05/2006/QD-BCN Article 1 lists 

chemicals banned from import and export), as 

cyanide is used widely in industrial production 

and only referred to as a deleterious waste 

(Decision 23 QD-BTNMT Article 2 Declare 

the list of deleterious waste matters). The lack 

of government regulations on limiting and 

defi ning authorized institutions/individuals 

in the use and trade of poisons has led to the 

situation where the management of cyanide is 

diffi cult to control.

Fish harvested with cyanide are more likely 

to have a higher mortality rate (Baquero, 

1999) and as a result experienced aquarium 

owners are willing to pay more for higher 

quality fi sh (Tsang, 2001). A number of 

countries have developed facilities that can 

test for cyanide in fi sh caught for the marine 

124
aquarium trade. The Bureau of Fisheries and 

Aquatic Resources (BFAR) in the Philippines 

have established a Cyanide Detection Test 

(CDT) to analyze cyanide content in fi sh and 

in water. The laboratory issues certifi cations 

on analysis result to support regulatory 

and enforcement laws as well as export 

requirements (http://www.bfar.da.gov.

ph/infocorner/cyanide_detectiontest.htm 

(accessed 6/03/07); Rubec et al., 2003). 

There have been a number of calls for the 

development of a CDT in Vietnam both for 

the Live Reef Food Fish fi shery and the marine 

aquarium fi shery to assist in monitoring the 

use of cyanide and enforce national laws and 

regulations (Nguyen Duc Cu, 2001). To date 

such a system has not been developed.

The USA Lacey Act prohibits the import of 

any wildlife that has been traded in violation 

of foreign law. This Act was adopted to assist 

in controlling the destructive wildlife trade 

in settings where the environmental laws 

of originating states could not be enforced 

in those nations. Thus it is illegal to import 

into the USA marine aquarium species taken 

in Vietnam with cyanide. The USA Coral 

Reef Task Force are investigating cyanide 

detection tests that can both be used for local 

management and enforcement authorities in 

source countries and also a test that could 

reliably be used several weeks after exposure 

in order to allow the USA to apply the Lacey 

Act to suspected illegal imports (CRTF, 

2006).

Compliance and enforcement

Despite the existence of clear legislation 

regarding destructive fishing, the use of 

destructive fi shing gear and methods has 

not been effectively prevented in Vietnam 

(Nguyen Long RIMF, ftp://ftp.fao.org/

docrep/fao/007/ad939e/ad939e00.pdf). The 

challenges in Vietnam for enforcement of the 

destructive fi shing legislation are:

• The vast geographic area with a coastline 

of more than 3260km

• A lack of management coordination 

between National, Provincial and District 

levels

• A lack of capacity and human resources

Government agencies with the power to 

enforcement destructive fishing legislation 

are (Articles 25, 26 & 27 of Decree 128/2005/

ND-CP):

• Fishery Inspectors (Administrative and 

Enforcement)

• The Chairs of Provincial/District People’s 

Committees

• Other Functional Departments (Force of 

Border Military, Force of People Police, 

Marine Police)

The administrative structure for management 

of fisheries enforcement is provided in 

Figure 2. At a national level, NADAREP 

is the state authority responsible for 

implementing law/regulations relating to 

protecting fi shery resources. At the provincial 

level, the Department of Aquatic Resources 

Exploitation and Protection (DAREP) is the 

representative offi ce of NADAREP but is 

under the Department of Fisheries (DOFI) 

which has a consultation role to the Provincial 

Peoples Committee within the fi shery domain 

and concerned policies instead of authority 

of enforcement. Although NADAREP 

directly manage the local fi shery enforcement 

inspectorate in terms of line management, 

key “additives” such as human resources and 

fi nance integral to effective enforcement are 

decided by the local People’s Committee. 

Due to the above limitation, local fi shery 

enforcement has to work in cooperation 

with other functional departments (Force 

of Border Military, Force of People Police, 

Marine Police), however, the “integrated 

management” issue is a signifi cant challenge 

125
in Vietnam now and requires more effort and 

resources to resolve.

At the district/local level, sub-offices of 

DAREP placed in local areas can be active in 

the fi eld. However, these offi ces have limited 

human resources and enforcement capacity.

At the provincial level, fi shery inspectors are 

the key law enforcers, however most of them 

are educated in terms of technical domain but 

lack an understanding of law enforcement. 

Additionally human resources are limited, 

with each province commonly allocated 5-10 

fi sheries inspectors. The equipment/vehicles 

supporting enforcement process are poor and 

less invested (only 1-2 boats per province for 

fi shery enforcement). Patrol boats are mostly 

improved fi shing vessels with weak engine 

meanwhile violators’ boats are quite speedy 

and mobile. The fi eld allowance for fi sheries 

inspectors, paid on top of salary for work in 

the fi eld is quite low (range 10,000-15,000 

VND/inspector/day (less than $1 USD)).

Fines associated with the illegal use of 

destructive fi shing methods, declared in 2005, 

are detailed in Table 1. Despite these fi nes, 

cyanide fi shing remains a widespread problem 

in Vietnam, where the laws have been diffi cult 

to enforce (Burke et al., 2002), as is the case 

elsewhere is south-east Asia.

The fi nes related to destructive fi shing are not 

in line with the authority of inspectors and are 

impractical to apply in the fi eld. The lowest 

fine likely to be applied to cyanide fi shing 

is 3 million VND but at the local/district 

level fishery inspectors are limited to issue 

a maximum fi ne of 200,000 VND (Article 

38 of Ordinance 44/2002/PL-UBTVQH10 

). That means that only the provincial level 

inspectors, engaged specifically in 

management and political fi eld, are capable 

of issuing the full scope of fi nes. Inspectors 

in some cases do not follow the correct rate 

of fi nes or procedures in fi ning. The fi nes and 

cases may also not be reported.

There is a lack of awareness of fi shers who 

largely have a poor education, pressures from 

increasing subsistence and poor access to 

alternative livelihoods.

During dialogues with community members 

in MCD’s project sites in the Red River Delta 

Violation Fine (VND) Fine (USD) Article*

Use of a net with mesh smaller than 

regulation 200,000 - 300,000 6 – 20 10.2(b)

Chemical fi shing (or storing chemicals 

onboard a fi shing vessel) 3,000,000 - 5,000,000 200 - 300 10.5(a)

Electro fi shing 1,000,000 - 6,000,000

depending on engine capacity 60 - 375 10.6(a) – (c)

Explosive fi shing (or storing explosives on 

board a fi shing vessel) 5,000,000 - 10,000,000 300 - 625 10.9

Additional sanctions include the confi scation of gear, catch and the destruction of equipment. 11

Table 1: Fines associated with illegal destructive fi shing practises in Vietnam promulgated in 2005

* Decree No. 128/20051ND-CP of October 11, 2005, Providing for sanctioning of administrative violations in 

the fi sheries domain.

126
and Central Vietnam it was revealed that 

deterrents to using illegal destructive fi shing 

methods where insuffi cient due to:

1. The fi nes being so low that even if the 

equipment was confi scated it was still 

profi table for the fi shers to buy new 

equipment and continue fi shing.

2. Enforcement patrols where very rare.

During 1998-2001, the National Directorate 

of Aquatic Resources Exploitation and 

Protection (NADAREP) dealt with 355 

cases of blast fi shing, 106 cases of chemical 

fi shing and confi scated 846 liters of chemical 

solution (Vu Huy Thu, 2001). Particularly in 2007, 

dynamite fi shing reduced, but still occurred in 

some areas. In 2007, 53 cases were fi ned and 

treated (19 cases: dynamite fi shing, 34: storing, 

sale/trade dynamite), keep 361 fuse, 258.2 kg 

dynamite, 137.8 m slow fi red wire. Electro 

fi shing still occurred in some places, fi ne 991 

cases, treat 510 cases, keep 555 battery, 1017 

electrify set and 226 illegal tools, fi ne over 627 

million VND (40,000 USD).

Conclusions and practical steps for a way 

forward

This report highlights the current lack of 

information on the extent and impact of 

destructive fi shing practises in Vietnam, in 

particular the use of cyanide. This information 

could be made available through more 

dedicated monitoring and research efforts 

of the government, fi sheries managers and 

research institutes. 

As one village leader for central Vietnam 

was quoted as saying “illegal fishing only 

benefi ts one family but destroys the living 

of 10 others”. Understanding of the extent 

and impact of destructive fi shing practices 

is critically needed as the basis for issuing 

legislation to control the situation as well as 

for complying with and enforcing the rules. 

In addition, it also enables the fi shers to make 

sensible choice and trade-off amongst the 

fi shing practices.

The report also confi rms that the Government 

of Vietnam is clearly committed to eliminating 

the use of destructive fi shing methods with 

a clear domestic legal mechanism as well as 

commitments in various international fora. 

Besides, it draws the attention that low 

compliance and week enforcement are the key 

challenges in management of the destructive 

fi sheries. Although it has been almost 20 

years since destructive fi shing methods where 

fi rst made illegal their use is still wide spread. 

It suggests the key to the success of these 

commitments will be an effective compliance 

and enforcement mechanism. The immediate 

need now is to build capacity and enabling 

regulations and policies to ensure effective 

implementation of these national policies and 

legislation.

It is also implied from the facts provided in the 

report that the legislative controlling efforts 

from the government could not be successful 

if the issue of sustainable livelihoods for 

fishers who exercise illegal fishing is not 

effectively handled. Lack of awareness of the 

long term implications of destructive fi shing, 

lack of knowledge of actual damage caused by 

cyanide in particular, and no alternative fi shing 

methods or enabling policy for it, are among 

the problems that need to be addressed. 

Having highlighted the above, in this report, 

key issues and recommendations are also 

provided (see table 2) focusing on assessing 

the status of the use of cyanide, evaluating 

the impact of harvesting and developing and 

implementing sustainable management. Such 

issue and recommendations mainly target 

the attention of the government authorities, 

where the Centre of Marinelife Conservation 

and Community Development as the hosting 

127
organization of the report authors is also 

recommended to take part in the process, 

in line with its vision, mission and fi elds of 

expertise and interest.

Figure 2: Administrative structure for fi sheries enforcement in Vietnam

128
ISSUES RECOMMENDATIONS MCD ACTION

1. Status of cyanide fi shing

Lack of information on the status 

of destructive fi shing in Vietnam, 

especially cyanide fi shing

Determine the extent of cyanide fi shing 

in Vietnam

Support and assist 

researchers to carry out 

related studies

2. Enforcement

Fines not strong enough to prevent 

violators in the future

Strengthen the awareness of 

enforcement forces

Support organisation 

of training useful 

to enforcement 

strengthening

Insuffi cient enforcement resources

Call for international support for 

capacity building and equipment 

(vehicles; devices)

More investment required from local 

authority

Punishment mechanism inadequate Improve the authority of fi eld 

enforcement inspectors

Confusing institutional arrangements 

Build a clear mechanism to allocate 

rights and responsibilities for involved 

institutions

A clear understanding of the 

livelihoods of people fi shing 

destructively is essential to the design 

of enforcement and patrol

A socio –economic assessment of 

fi shers using destructive methods to 

be undertaken to inform appropriate 

management responses

3. Awareness raising

Lack of awareness of the long term 

implications of destructive fi shing 

Awareness raising of the long term 

implications of destructive fi shing 

supported by strong scientifi c proof

Support organisation of 

community dialogues 

of destructive fi shing 

impacts

No alternate fi shing methods 

available

Development of alternative sustainable 

fi shing methods

Support poor fi shermen 

to alter their livelihoods 

Lack of knowledge of actual damage 

caused by cyanide

Promote the understanding of serious 

damage of cyanide to fi shermen and 

managers

Support to organise the 

trainings and awareness 

campaigns

Limited cooperation among fi sheries 

inspectors, border military, marine 

police/security and informationculture force

Awareness raising for dangers of use of 

cyanide

Communication via public media about 

fi sheries law/regulations

Develop a cooperative mechanism 

between stakeholders to strengthen 

effective enforcement

4. Improvements for legal framework

Lack of subsidy policy for 

enforcement inspectorate

Assess the risk facing the enforcement 

inspectorate and build an adequate 

support mechanism

No institutions, policy for poor 

fi shermen to alter livelihood

Investigate development of local 

regulations for destructive fi shing 

Advocate the alternative 

livelihood needs of 

small-scale fi shers

Low regime for fi sheries conservation 

inspectors

Assess the adequacy of the level of fi nes 

for illegal destructive fi shing

Table 2: Issues and recommendations

129
References

Baquero, J. (1999) Marine ornamentals trade: 

Quality and sustainability for the Pacifi c Region 

May 1999. South Pacifi c Forum Secretariat, 

Fiji, www.aquariumcouncil.org/pdf/

publications.PDF (accessed March 2007).

Barber, C.V. and Pratt, V.R. (1998) Poison and 

profi ts: cyanide fi shing in the Indo-Pacifi c. 

Environment 40:8, 5–34.

Barber, C.V. and Pratt, V.R. (1997) Sullied 

seas: Strategies for combating cyanide fi shing in 

Southeast Asia and beyond. World Resources 

Institute, Washington, D.C. 

Burke, L., Selig, E. and Spalding, M. (2002) 

Reefs at Risk in Southeast Asia. World 

Resources Institute, Washington, D.C.

Chou, L.M. (2000) Southeast Asian reefs 

- status update: Cambodia, Indonesia, 

Malaysia, Philippines, Singapore, Thailand, 

Vietnam. In Status of Coral Reefs of the 

World: 2000 (Wilkinson, C.R., Ed.) 117-129, 

Australian Institute of Marine Science, 

Townsville, Australia.

Coral Reef Task Force (2006) Final CRTF 

Decision 5/04/06. U.S. Coral Reef Task 

Force Meeting May 4, 2006, Washington, 

DC.

Dasgupta, S., Laplante, B., Meisner, C., 

Wheeler, D. and Yan, J. (2007) The Impact 

of Sea Level Rise on Developing Countries: A 

Comparative Analysis. World Bank Policy 

Research Working Paper 4136.

MARD (2007) NADAREP report on 

implementation of the Primer Minister’s 

Directive 01/1998/CT-TTG forbidding 

the use of dynamite, electro and poison 

fi shing in 2007.

McCullough, B. and Hai, P.G. (2001) The live 

reef fi sh trade in Vietnam: a preliminary 

report from the fi eld. SPC Live Reef 

Fish Information Bulletin 8, 12-16. http://

www.spc.int/coastfish/News/LRF/8/

LRF8-A4.pdf

MOFI and WB (2005) Vietnam: Fisheries 

and Aquaculture Sector Study, Final Report. 

http://siteresources.worldbank.org/

INTVIETNAM/Resources/vn_fi sheriesreport-fi nal.pdf 

Nguyen, Chu Hoi (2008) Challenges in fi sheries 

management in Vietnam. Presentation at 4th 

Global Conference on Oceans, Coasts 

and Islands: Advancing Ecosystem 

Management and Integrated Coastal and 

Oceans Management in the Context of 

Climate Change. April 7-11, 2008, Hanoi, 

Vietnam.

Nguyen, Duc Cu (2001) The Impacts of 

Cyanide Fishing on Coral Resources in 

Vietnam. Proceedings of the Live Reef Fish 

Trade Workshop, 23rd April 2001, 14-15.

International Marinelife Alliance Vietnam 

and Institute of Fisheries, Economics and 

Planning.

Nguyen, Huy Yet, Lang, Van Ken and 

Nguyen, Dang Ngai (2004) Species diversity 

and distribution of hard corals (Scleractinia) in the 

northern coastal marine area (in Vietnamese).

Workshop on cooperative science VietnamItaly, Biodiversity conservation in Vietnam 

coastal zone, Hanoi National University 

Publishing House.

Nguyen, Nhat Thi and Nguyen, Van Quan 

(2005) Biodiversity and living resources of 

the coral reef fi shes in Vietnam marine waters

(monograph book, in Vietnamese). Science 

and Technology Publishing House, Hanoi, 

Vietnam.

130
Nguyen, Van Quan, Nguyen, Nhat Thi 

and Chung, Bui Dinh (2007) Status of 

Marine Ornamental Fish Resources in 

Vietnam. Vietnam Marine Science and 

Technology Association, Marine Vietnam 3 

(in Vietnamese), 24-26.

RIMF (2006) Current State of Resources 

and Fisheries Development Trends. 

Research Institute for Marine Fisheries (Hai 

Phong), www.fi stenet.gov.vn/details_e.

asp?Object=2112463&news_ID=7263407 

(updated 7/2/2006). 

Rubec, P.J and Palacol, J. (2002) Farming 

Coral Reef Invertebrates for Reef 

Rehabilitation and the Aquarium Trade. 

In Marine Geography: GIS for the Oceans and 

Seas (Breman, J., Ed.) 95–101, ESRI Press, 

Redlands, CA. 

Rubec, P.J., Cruz, F., Pratt, V.R., Oellers, 

R., McCullough, B. and Lallo, F. (2001) 

Cyanide-free net-caught fi sh for the marine 

aquarium trade. Aquarium Sciences and 

Conservation 3, 37–51.

Rubec, P.J., Pratt, V.R., McCullough, B., 

Manipula, B., Alban, J., Espero, T. and 

Suplido, E.R. (2003) Trends determined 

by cyanide testing on marine aquarium fi sh 

in the Philippines. In Marine Ornamental 

Species: Collection, Culture & Cultivation (Cato, 

J.C. and Brown, C.L., Eds.) 327-340, Iowa 

State Press, Ames, IA.

Smith, B D., Braulik, G., Jefferson, T.A., Bui 

Dinh Chung, Chu Tien Vinh, Doan Van 

Du, Bach Van Hanh, Pham Dinh Trong, 

Dao Tan Ho and Vo Van Quang (2003) 

Notes on two cetacean surveys in the Gulf 

of Tonkin, Vietnam. The Raffl es Bulletin of 

Zoology 51:1, 165-171.

Strehlow, H.V. (2006) Integrated natural resources 

management of coastal fi sheries – the case of 

Nha Phu Lagoon, Vietnam. 286 pp, Doctoral 

dissertation, Humboldt University, Berlin, 

Germany. 

Truong, Si Ky (2006) Current Status of Vietnam 

Marine Ornamental Fishery (unpublished 

report to MCD). 4 pp, Nha Trang Institute 

of Oceanography.

Tsang, A.C.W. (2001) A glance at the marine 

aquarium fi sh trade in Hong Kong, 

Porcupine 24:December 2001, www.hku.hk/

ecology/porcupine/por24/24-vert-fish.

htm (accessed March 2007).

Tuan, V.S., Ben, H.X., Long, N.V. and Hoang. 

P.K (2005) Status Of Coral Reefs In 

Southeast Asia: Vietnam. In Status of Coral 

Reefs in East Asian Seas Region: 2004 (Japan 

Wildlife Research Center, Ed.) 95-112, 

Ministry of Environment, Tokyo, Japan.

Tun, K., Chou, L. M., Cabanban, A., Tuan, 

V. S., Philreefs, Yeemin, T., Suharsono, 

Sour, K. and Lane, D. (2004) Status of 

Coral Reefs, Coral Reef Monitoring and 

Management in Southeast Asia, 2004. In 

Status of coral reefs of the world: 2004, Volume 

1 (Wilkinson, C., Ed.) 235-276, Australian 

Institute of Marine Science, Townsville, 

Queensland, Australia. 

Vu Huy Thu (2001) Various active solutions 

to promote the implementation of Decree 

01/1998 CT-TTg by Prime Minister. Fishery 

Magazine 2/2001 (in Vietnamese), 32-34.

WRI (2002) Reefs at Risk in Southeast Asia 2002. 

World Resources Institute, Washington, 

D.C., www.wri.org

131
Agreement/Convention Detail Date 

joined

United Nations Convention on the 

Law of the Sea (UNCLOS) (1982)

Articles 61 through 64 ,116-119 and 123 – promoting 

responsible fi shing practices 1994

Convention on Biological Diversity 

(CBD)

Contains articles concerning conservation and sustainable 

use of biodiversity components as well as the equitable 

sharing of the benefi ts and relevant technologies application.

1994

FAO Code of Conduct for 

Responsible Fisheries (1995) Contains a commitment to end destructive fi shing practices 1995

Basel convention

Control of transboundary movements of hazardous wastes 

and their disposal Cyanide can be consider a hazardous waste 

from industrial production

1995

APEC Fisheries Working Group

Endorsed a resolution urging “all members of the 

international community to cease the practise of cyanide 

fi shing”

1997

World Summit on Sustainable 

Development (2002)

Contains a commitment to phasing out destructive fi shing 

practices in the marine environment by the year 2012 in 

accordance with chapter 17 of Agenda 21

2002

Putrajaya Declaration of Regional 

Cooperation for the Sustainable 

Development of the Seas of East 

Asia

Signed among 12 littoral states, recognizing the importance 

of sustainable development and management of coastal 

and marine resources within the region, and committing 

individual and collective efforts of the countries to the 

implementation of the Sustainable Development Strategy 

of the Seas of East Asia (SDS-SEA). It includes developing 

measures against destructive fi shing practises Objective 3 

s.3(b). www.pemsea.org/pdf-documents/sds-sea/SDSSEAFull.pdf 

2003

• CDT • Cyanide Detection Test

• DAREP • Department of Aquatic Resources Exploitation and Protection

• DOFI • Department of Fisheries (Provincial Level)

• GoV • Government of Vietnam

• IMER • Institute of Marine Environment and Resources

• MAF • Marine Aquarium Fishery

• MARD • Ministry of Agriculture and Rural Development

• MCD • Centre for Marinelife Conservation and Community Development

• MOFI • Ministry of Fisheries (merged into MARD in 2007)

• MPA • Marine Protected Area

• NADAREP • National Directorate of Aquatic Resources Exploitation and Protection

• PPC • Provincial People’s Committee

• RIMF • Research Institute of Marine Fisheries

• VIFEP • Vietnam Institute of Fisheries and Economic Policy

• WRI • World Resources Institute

Appendix 1: Key international agreements relating to destructive fi shing in Vietnam

Acronyms

132
Law/Ordinance/Decree

/Decision

Title Detail Date 

Signed 

Ordinance 18-HDNN

Ordinance on the protection 

and development of aquatic 

resources

Promulgator: Government 

council, signed by VN 

president

Chapter II, Article 8 (1) Bans 

the use of poison, dynamite, 

electro which destroy, kill aquatic 

resource for aquatic resource 

exploitation

1989

Directive 01/1998/CT-TTg

Directive strictly banning the 

use of explosives, electric 

impulses and toxic to exploit 

aquatic resources

Promulgator: The 

Government

Prime Minister’s instruction on 

prohibiting the use of dynamite, 

electric shock and poisons to 

extract fi shery resources

1998

Decision 1971/1999/QDBCNMT

Decision promulgating 

technology process to 

demolish or re-use cyanide

Promulgator: Ministry of 

Science, Technology and 

Environment

Technology process of cyanide 

demolishment or re-use. The 

Decision declares that cyanhydric 

acid and its cyanide salts are 

critically powerful poisons, one 

person can be killed with only 

about 50 mg.

1999

7/2003/QH11

Fishery Law 

Promulgator: National 

Assembly

Article 6 (6) Prohibits fi shing 

activities include the production, 

circulation and use of prohibited 

fi shing gear; the use of prohibited 

fi shery activities and methods; 

the use of explosives, poisons, 

electric and other destructive 

fi shing methods.

2003

Decree 109/2003/ND-CP

Conservation and 

sustainable development 

of wetlands

Promulgator: The 

Government

Article 7 (4) Prohibits activities 

including the use of serially 

destructive catching methods 

such as electricity shock, blast, 

chemical, toxic matters, various 

nets with mesh size against 

regulation to fi shing in wetlands.

2003

Decree 121/2004/ND-CP

Providing for sanctioning of 

administrative violations in 

the environment protection 

domain

Promulgator: The 

Government

Article 21 (3) Fines of 20 to 

30 million VND are applied 

to activities letting out and 

excrete into environment 

pollutants excessive to allowable 

environment standard. Cyanide 

is not mentioned specifi cally but 

pollutants generally.

2004

Appendix 2: Key domestic legislation relating to destructive fi shing in Vietnam

133
Law/Ordinance/Decree

/Decision

Title Detail Date 

Signed 

52/2005/QH11

Environment Protection 

Law

Promulgator: National 

Assembly

Article 7 (2) Strictly forbids 

activities including exploitation 

and capture of biological 

resources by destructive tools 

and methods, and opposed to 

season and production allowed by 

regulations.

2005

Decree 128/2005/ND-CP

Providing for sanctioning of 

administrative violations in 

the fi sheries domain.

Promulgator: The 

Government

Provides fi nes for electro, dynamite 

and chemical fi shing (see table 1 

for details) 2005

Decree 12/2006/ND-CP

Detail rules to implement 

Trade Law in international 

goods business and agency 

practices on trading, 

processing and transiting to 

foreign countries.

Promulgator: The 

Government

Appendix 03 (VII) Ministry of 

Industrial manages the export and 

import of toxic chemicals and 

products in the list of allocated 

goods.

2006

06/2007/QH12

Chemical Law

Promulgator: National 

Assembly

Article 7 (4) Prohibited chemical 

practices include the use of toxic 

matters to hunt animals and the 

implementation of activities 

detrimental to human health, 

property and environment 

Cyanide is not mentioned 

specifi cally.

2007

Decision 79/2007/QD-TTg

The National Action Plan 

on Biodiversity up to 2010 

and orientations towards 

2020 for implementation 

of the Convention on 

Biological Diversity and 

the Cartagena protocol on 

Biosafety

Promulgator: Prime 

Minister

Article 4 (c) Prevention, control 

and strict handling of illegal 

exploitation, trading and use of 

biological natural resources to 

eliminate destructive methods 

of exploiting biological natural 

resources and the destruction of 

sensitive ecosystems.

2007

134
Background

Live reef fi sh trade started as a hobby in 

early 1950s, based on BFAR studies and 

notes. Trade picked up after 10 years as other 

business men began investing. Importance of 

the LRF trade was noticed only in 1968 when 

export reached 3,931kg amounting to PhP 

153, 329.00 (quite substantial at that time). 

In 1970, export value reached a million pesos 

and increased twenty-fold in a span of ten 

years. In the subsequent years, the live reef 

fi shes are within the top ten fi shery products 

exported by the country. 

Traditional methods include fi ne mesh nets, 

modified spear guns and hookah diving. 

Due to the lucrative nature of this business, 

catchers opted for easy, less expensive and 

environmentally destructive techniques such 

as the use of cyanide to stun the fi sh.

Under Philippine laws, the trade of cyanide 

tainted live reef fi sh is not allowed. Such 

laws are Republic Act 8550, “Fisheries Act 

of 1998”; and Republic Act 6969, “An Act 

to Control Toxic Substances, Hazardous 

and Nuclear Wastes”. RA 8550 - Section 88 

prohibits the use of obnoxious substances 

e.g. cyanide in fi shing. Under this law, the 

Bureau of Fisheries and Aquatic Resources is 

mandated to develop, improve, manage and 

conserve the country’s fi sheries and aquatic 

resources. BFAR monitors and regulates 

the importation and exportation of fish 

and marine fi shery and aquatic products. 

 

RA 6969 specifi es the requirements pertaining 

to the importation, manufacturing and use 

of cyanide and cyanide compounds and 

its storage, transport and disposal of its 

generated wastes, and provides penalties 

for violations and for other purposes. The 

Department of Environment and Natural 

Resources (DENR) is the lead agency in the 

regulation of registration and importation 

of chemicals. BFAR as a cooperating agency 

issues endorsement letter provided that the 

client met the requirements.

Cyanide Detection Test

Before the 1990’s, cyanide analyses were 

conducted at the Chemistry Section of 

BFAR’s Post Harvest Division. The method 

used then, Picrate Method, can only give 

qualitative results, e.g. positive or negative. In 

the 1990s the distillation and ISE analytical 

method was introduced. 

CDT labs serve two major functions. The fi rst 

is support to law enforcement activities. Such 

activities may include evidences that require 

laboratory tests like fish and substances 

suspected to contain cyanide like the tablets 

and water in squirt bottles. When needed the 

laboratory chemist may be called to court 

hearings as an expert witness. The second is 

required by law that is analysis of samples for 

trading purposes, i.e. laboratory testing for 

live fi sh prior to shipment. CDT laboratories 

analyzed 2800 (on the average) samples in 

a year with the majority coming from the 

Palawan area.

BFAR contracted the operation of its 

CDT labs with IMA since the agency was 

undermanned to attend to the large volume 

Summary of the Use of Cyanide in the Philippines

Gil Adora and Aqualino Alvarez

Philippines Bureau of Fisheries and Aquatic Resources

135
of samples required for analysis. Under this 

contract several of the laboratories were 

established. There were six laboratories 

established and these were located in the 

strategic areas of Palawan, Leyte, Davao, 

Cebu, Zamboanga and Manila. 

In 2001, BFAR regular employees operated 

the CDT labs when contract with IMA 

terminated. Due to logistics constraints, 

only four laboratories remained operational 

and these are the Puerto Prinsesa, Cebu, 

Zamboaga and Manila. However, at a later 

date, BFAR was able to acquire patrol vessels 

equipped with several laboratory equipment 

including CDT that can be relocated to 

land-based laboratories.

Methods. Cyanide is recovered from fi sh 

samples through distillation. ISE method 

is used to quantify the cyanide in the sample. 

Results are reported as mg CN/ Kg meat. To 

ensure the reliability of results, the laboratory 

protocols adhere to standard operating 

procedures (SOPs). These SOPs are:

• Calibration – using standard 

cyanide solutions, being the basis of 

quantifying cyanide content is done at 

the start of every analysis

• Checking of linear response – 

conducted together with calibration 

since quantitative determination is 

done using a linear calibration curve

• Distillation of water and fi sh tissue 

matrices spiked with cyanide is 

conducted to check cyanide recovery

• Electrode efficiency is regularly 

checked according to manufacturer 

specifi cations

Procedures. There are several collection 

points wherein samples can be collected. 

These are fi shing boats, holding tanks, and 

cages. The required number of samples are 

collected at random based on the number 

of fi shes to be shipped out. Submission of 

samples require accompanying documents, 

which are:

• Collection and submission form which 

contain information directly relating 

to the sample. This information 

includes the names and addresses 

of the fi shermen, date and place 

of collection etc. Information that 

accompanies the sample will enable 

authorities to trace and link it to the 

fi shermen/trader, especially for legal 

purposes

• For Palawan in particular, there is the 

Certifi cate Receipt of Live Fish Taken 

for Examination which contained 

information on the origin of the 

sample as certified by designated 

authorities

• If applicable, apprehension reports 

are also provided to the laboratory for 

added information 

Issues:

Accessibility. The first issue is the 

Archipelagic nature of the Philippines. Some 

areas have diffi cult access to the laboratories 

due to the travel time involved, especially 

those coming from island areas.

From the laboratory point of view, this 

archipelagic nature poses problems fi rst in the 

purchase of supplies, particularly chemicals, 

which have specifi c requirements during 

transport. The second is the need for repair 

and preventive maintenance conducted by 

product engineers. In both cases, suppliers 

and their respective engineers are located in 

key cities like Manila and Cebu. One way to 

mitigate this problem is to have spare sets 

of equipment and a relatively larger stock 

of supplies which will require more fi nancial 

expenses.

136
Due to the island nature of the Philippines, 

some samples are sent via couriers or 

representatives which poses sample security 

concerns. With commercial couriers, signs 

of tampering can be easily checked; however, 

with samples sent via representatives (in 

kind), which could be unauthorized, there is a 

need to come up with procedures to prevent 

tampering.

Lack of Manpower. When BFAR took 

over the operation of the CDT labs and 

its associated responsibilities and tasks, these 

were to be performed on top of the existing 

responsibilities. These activities include 

inspection and sampling at the start of supply 

chain, laboratory and administrative work. With 

all the other activities, BFAR personnel are 

faced with multi-tasking. Laboratory analysts 

are tasked not only with cyanide analysis 

but also red tides, chemical, microbiological 

and antibiotic residue analyses. In addition 

there are no suffi cient vacant positions in the 

government to absorb those that came from 

the IMA-run laboratories.

Unlike when the labs were under the 

management of IMA, there are personnel 

for each specifi c task. These tasks are for 

chemists and biologists, and hiring personnel 

with these qualifi cations is also hampered 

by the lack of vacant positions, or, as in the 

case of services charged to maintenance and 

operations cost, the salary allowed is not 

commensurate. Sometimes there are those 

who accept the position but do not stay for 

very long. This results in a high turn over rate, 

which is not benefi cial for the laboratory.

Most regulatory offi cers are not properly 

informed on how to collect samples. Copies 

of protocols or “how to’s” were sent to the 

regional labs. Most often the protocols were 

followed on samples sent to the labs early 

on. But later, there were instances wherein 

the proper protocols were not followed, 

compromising the integrity of the samples 

and leaving the lab with no alternative but to 

issue rejection. The regional offi cers/staff had 

to be regularly reminded of the protocols.

Final inspection of live fi sh is at the port of 

exit. Fishery quarantine personnel may not 

be properly informed when to look for CDT 

results in shipments of fi shery products.

What Is Needed:

Expansion/Additional labs. With the 

above mentioned issues, if BFAR wants to 

expand its CDT activities several things will 

be needed. There is a need for additional 

laboratories in several areas in order to have 

easier access to its services.

It is suggested that several labs be established 

in Palawan due to the live fish trade 

present in almost all of its municipalities. 

Other areas were chosen so that the regional 

counterparts will have a more direct access to 

the lab.

Capacity building. Training of personnel, 

especially for law enforcers (PNP Maritime, 

Coast Guard, Local Government Units, 

BFAR Regional Offi ces), is required especially 

for sample collection and sample handling 

in order to assure that samples will arrive at 

the laboratory at a viable state. Moreover, 

management training is necessary for people 

at the local level to further enhance capabilities 

policy formulation.

Environmental awareness. There is a need 

to conduct environmental awareness training 

for local fi sher folk to emphasize the negative 

long term effect of destructive fishing 

techniques to the environment including fi sh 

habitats.

137
Field test kit. BFAR recognizes the 

inadequacies of the current method employed 

and more importantly the need for cyanide 

testing for regulatory purposes. BFAR is 

open to collaborative work in particularly in 

the investigations of methods for cyanide 

detection. In the past, BFAR, MAC and 

Merck jointly conducted a comparative study 

between a methods developed by Merck. The 

development of a method particularly ideal 

for fi eld use will be benefi cial to BFAR. 

Alternative livelihood for displaced 

fishermen. Possible alternative sources of 

income are necessary to wean fi shers from 

cyanide fi shing. Fishermen resort to this 

destructive fi shing method due to a complex 

set of issues, i.e. poverty, easy cash returns at 

less capital, etc. 

Sustainable Financing

Maintenance & operation per lab per annum

 Chemicals 200,000.00

 Laboratory supplies 100,000.00

 Offi ce supplies 30,000.00 

 Labor Services (1 chemist, 2 lab assistants) 500,000.00

 Utility bills/expenses 125,000.00

 Total: PhP 955,000.00

 US$ 23,507.11* 

(*US$1.00 = PhP 40.626 01 Feb 08 exchange rate, www.bsp.gov.ph)

Finances

In the establishment of the laboratory:

 New structure: 

 Per Laboratory 900,000.00 

 If structure is to be rented

 Renovation (fi t for lab) 100,000.00

 Rent (Manila rate) per month 75,000.00

 Conduct of nationwide training (regulatory and fi sherfolk) 1, 000, 000.00

 Production of IEC materials 500, 000.00

 Total PhP 1, 500, 000.00

138
Background

While the misuse of cyanide in live fish 

collection started in the Philippines in the 

early 1950s, the country was also the fi rst to 

implement a comprehensive Cyanide Fishing 

Reform Program anchored on the adoption 

of the world’s fi rst-ever technology for the 

detection of the presence of cyanide in fi sh 

tissues and organs.

Between 1995 and 2001, the Philippine 

Bureau of Fisheries and Aquatic Resources, 

using funds sourced from a Fisheries Sector 

Program loan, set up a nationwide network of 

laboratories, acquired the necessary equipment 

and chemicals, hired staff, organized mobile 

monitoring, control and surveillance (MCS) 

teams and set in motion an integrated reformoriented program dedicated to combating the 

cyanide fi shing menace.

During that period, thousands of fi sh were 

tested for the presence or absence of cyanide, 

several live fi sh shipments were confi scated, 

hundreds of fi shermen were arrested, a few 

dozens of whom were successfully prosecuted 

in court.

The original CDT laboratories were located 

in: Metro Manila; Puerto Princesa in Palawan; 

Cebu; Davao; Tacloban; and Zamboanga.

They served two major functions: 

First was to respond to request from law 

enforcement agencies for evidentiary support 

against cyanide fi shing violators. 

The second was to satisfy the requirements of 

a local law in the Province of Palawan which 

required CDT laboratory testing for live fi sh 

shipments. In Palawan, the only live fi sh that 

could be sold and transshipped were those 

that had been certifi ed cyanide-free.

Unfortunately, shortage of funds compelled 

BFAR to scale down the operation of the 

laboratories. Faced with a budget that had 

remained stagnant for years and instructed 

by the Department of Agriculture to use 

limited resources in production-enhancement 

activities, BFAR was compelled to integrate 

the functions of the CDT laboratories with the 

activities of BFAR’s central offi ce laboratory 

whose many responsibilities include red tide 

monitoring; chemical, microbiological and 

antibiotic residue analyses; fi sh health, and 

fi shery product quality control management. 

To date, only four of the six laboratories 

are functioning. These are the ones located 

in Manila, Puerto Princesa, Cebu and 

Zamboanga. However, only the laboratory 

in Puerto Princesa is operating full-time and 

this is largely because the city government is 

subsidizing its operation. 

Request for Assistance in the Operation of an Expanded Cyanide 

Detection Test (CDT) Laboratories in the Philippines

Gil Adora and Aqualino Alvarez

Philippines Bureau of Fisheries and Aquatic Resources

139
Political Will

Having planted the seed of reform in the live 

coral reef fi shery, BFAR fi nds it frustrating 

that a prolonged financial drought has 

stymied the momentum it has painstakingly 

built towards combating cyanide fi shing. 

Resourcefulness has enabled our agency to 

acquire complete laboratory equipment for 

seven more laboratories. Unfortunately, our 

optimism about being able to raise funds to 

set up and operate at full capacity an expanded 

CDT network has faded with the onset of 

discouraging developments in the fi nancial 

front.

It is extremely disappointing that while 

BFAR’s resolve to stamp out cyanide fi shing 

remains strong and unwavering, there is not 

enough money to get our agenda of reform 

off and running. 

Conviction

BFAR is convinced that no effort to regulate, 

manage and reform the marine ornamental 

trade will succeed unless it is supported 

by a solid enforcement tool in the form of a 

credible cyanide detection testing technology. 

We believe that the ISE method we have been 

using since 1995 works. We have no doubt that 

this technology will remain a strong deterrent 

to cyanide misuse in live fish collection 

especially in provinces and municipalities 

where the problem is prevalent.

Needs and Wants

BFAR is hoping for financial assistance 

in the form of a long-term grant that will 

enable us to re-invigorate our anti-cyanide 

fi shing campaign. As we already have seven 

brand-new complete CDT equipment on 

top of the six existing ones that may need 

minor refurbishing, BFAR has the potential 

to set in motion an expanded network of 

13 laboratories strategically located in major 

centers of ornamental fi sh collection. But we 

need operating funds to be able to hire project 

management staff and consultants, pay rent, 

procure chemicals and supplies, acquire 

on-shore and off-shore mobility, conduct 

monitoring, control and surveillance (MCS), 

support enforcement activities, undertake 

information and education activities and 

provide skills enhancement trainings to 

project staff.

BFAR is also open to collaborative work in the 

development of a simpler – and, if possible, 

portable - CDT test. The development of 

a method particularly ideal for fi eld use will 

be benefi cial to BFAR given the country’s 

archipelagic character.

140
Legal and Management Measures towards Sustainable and Community-based 

Marine Ornamental and Food Fishery in Indonesia

Agus Dermawan

Directorate General for Marine, Coast and Small Islands 

Ministry of Marine Affairs and Fisheries of Indonesia

Ambrosius Ruwindrijarto

President of Telapak

Indonesia

Ferdinand Cruz

Executive Director of East Asia Seas and Terrestrial Initiative

Philippines

Introduction

The past 15-20 years have witnessed a 

progressive deterioration in Indonesia’s 

coral reefs. A regional survey by Indonesia’s 

Academy of Sciences (LIPI) found that only 

6.7% of Indonesian reefs are still in excellent 

condition. Overfishing and destructive 

harvest techniques negatively influence 

biodiversity in all coastal and marine 

ecosystems, including mangrove forests, 

inter-tidal areas, sea grass beds, coral reef 

systems, and the pelagic and abyssal zones. 

The widespread use of cyanide fishing 

to collect marine ornamental fi shes and live 

reef food fi shes are well-documented in the 

Indonesian archipelago, as are the resulting 

lethal effects upon the marine environment.

The global trade in marine ornamental fi sh 

and associated accessories is an industry in 

excess of USD 70 million per year. Indonesia 

is one of the world’s leading exporters of 

products derived from coral reefs, and the 

overwhelming majority of these are collected 

in unsustainable ways. Millions of people 

worldwide, in Asia and in Indonesia depend 

on coral reefs, and unsustainable harvesting 

techniques jeopardize livelihoods of coastal 

communities.

The trade in marine ornamental fi shes and 

accessories fi rst took root in Indonesia in 

the 1960s. In the early 1990s Bali became the 

major base for the marine aquarium industry 

in Indonesia, replacing Jakarta and Surabaya. 

From 1990-1999 total export volume of reef 

fi shes from Bali grew from 2.14-3.94 million 

reef fi sh per year; with peak levels reached 

in 1996 when 9.78 million reef fi sh were 

exported.

It is interesting to note that though export 

volume peaked in 1996 and thereafter slowed, 

it declined further with the onset of the Asian 

fi nancial crisis in 1998. The price setting power 

in the marine ornamental and live reef food 

fi sh industry lays solely in the hands of the 

exporters who ship internationally from Bali, 

Surabaya, Manado, and Jakarta. Yet the crisis 

was not the only factor which infl uenced 

export volume from Bali after 1996. Internal 

political chaos in Indonesia starting from 

1998 led to an unhealthy business situation 

whose repercussions were experienced most 

141
gravely by fi shing communities. The fi nancial 

and political crisis is not the only factor that 

contributed to a decrease in export; reef 

degradation caused by blast fi shing and 

cyanide fi shing may also have contributed to 

a decrease in export of aquarium fi sh from 

Indonesia.

Action Plan Against Cyanide Fishing

The Ministry of Marine Affairs and Fisheries 

of the Republic of Indonesia (MMAF), in 

collaboration with Telapak and other national 

and international organizations have been 

developing an action plan to abate cyanide 

fi shing. The action plan includes the following 

components:

Institutional Set Up and Capacity Building

MMAF is the National Management Authority 

for fi sheries, coastal and marine affairs. Some 

of the authorities related to coral reef and 

protection of endangered species are now 

being transferred from the previous authority, 

the Ministry of Forestry, to MMAF. When the 

transfer of authority is completed, MMAF 

will have full authority and responsibility 

to enforce CITES for coral reef organisms 

and other aquatic species in the list. The 

ornamental fi sh industry has so far been very 

loosely regulated by MMAF. For instance, to 

export ornamental fi sh the exporter will only 

need two documents: a Health Certifi cate 

issued by the Immigration and Customs 

Agency, and an Export Notifi cation issued 

by the Immigration and Customs Agency in 

coordination with the Ministry of Trade. For 

fi shes and coral reef species under CITES, it 

was the Ministry of Forestry which had been 

the agency to issue regulations and permits. 

It is envisaged that a stronger MMAF will 

be able to regulate ornamental fi sh and coral 

reef exports as the National Management 

Authority.

The MMAF plans to establish Technical 

Managements Unit at the regional level, and 

MMAF will also explore the establishment 

of district-level Technical Management Units 

at districts that participate in COREMAP 

Phase II. These units will be tasked with 

implementation of regulations related to 

fi sheries, coastal and marine affairs. Initially, 

these units will be established at main fi sheries 

centers, including Bali, Jakarta, Sorong, and 

Manado. It is envisioned that Coral Detection 

Testing Labs will be under these regional 

Technical Management Units, and MMAF 

will explore the possibility to contract out the 

establishment and management of the labs 

to NGOs or outside contractors. Under the 

World Bank / ADB Coral Reef Rehabilitation 

and Management Program Phase II, tools 

and technologies for cyanide testing will be 

explored.

The MMAF will soon have the capacity to 

enforce law on fi sheries. The DirectorateGeneral of Surveillance and Control of 

Marine Resources and Fisheries, MMAF 

has authority to conduct investigation and 

prosecution of violations of fi sheries- related 

laws. The capacity development plan of this 

Agency includes recruitment of personnel, 

trainings, and procurement of equipment. 

Trained staff of this Agency will have a the 

right to bear arms.

Under the existing law, violations against 

fi sheries related laws are to be prosecuted 

at Fisheries Tribunal, which initially are 

present in Jakarta, Medan in North Sumatra, 

Pontianak in West Kalimantan, Bitung in 

North Sulawesi, and Tual in Molucca.

Capacity building for MMAF to be 

able to take on these new authority and 

responsibilities will include strengthening of 

human resources, both in terms of number 

of personnel and quality of those personnels, 

142
and securing suffi cient operational funds for 

the establishment and running of labs for 

detection of cyanide as well as other chemicals 

that are prohibited for catching fi sh.

The physical infrastructure needed for these 

policy direction of MMAF will include 

establishment of labs at regional/export 

centers, and provision of fast, easy, cheap, 

and portable Cyanide Detection System at 

the District Level, to be operated by a District 

agency.

Legal and Policy Framework

The MMAF will employ a two layer approach 

in reforming cyanide fi shing in Indonesia. The 

fi rst layer will be setting up a legality standard, 

which will be the fi rst step towards the second 

layer: ecolabel certifi cation. The legality 

standard will be applied through licensing 

and permits based on existing regulations, 

including the Fisheries Act No. 31/2004 

which explicitly prohibits the industry from 

using chemical substances and pose sanction 

up to 10 years imprisonment and a fi ne of 

maximum Rp 2 billion. The Fishery Resources 

Conservation Decree No. 60/2007 regulates 

that “...fi sh harvest must hold a license...” 

and “...licensing considers fi shing techniques 

and gear...”. It is envisioned that MMAF will 

issue a Ministerial Regulation and Decrees 

which will include a cyanide-free certifi cate 

as a requirement for the legality of marine 

ornamental business and export. For coral 

reef trade, the existing regulations call for 

culture permit, trade permit, and a quota 

system for export. This regime is currently 

under the Ministry of Forestry, soon to be 

transferred to MMAF. This will also become 

the legality requirement for companies to 

meet the legality standard regarding coral reef 

trade.

When the legality standard has been developed 

and implemented, the development of a 

certifi cation system for marine products will 

be possible. Learning from the success of 

certifi cation system in forestry, and failure of 

existing certifi cation system for marine product 

as developed by the Marine Aquarium Council 

(MAC), the development of an ecolabel 

certifi cation system for marine product should 

consider the following: 1. Development of 

economic, social, and environmental criteria, 

2. A national processes which shall be based 

on multi-stakeholder constituency, involving 

meaningful participation and ownership of 

MMAF, the industry, NGOs, and fi sherfolk 

community, and 3. Market education and 

reform. 

Telapak’s Integrated Approach to 

Destructive Fishing Reform

While MMAF has been developing an 

institutional arrangement and legal and policy 

framework in abating cyanide fi shing, Telapak 

is contributing to the goals of sustainability 

and people-centered marine ornamental 

industry through an integrated approach to 

destructive fi shing reform. This calls for an 

integration of conservation purposes and 

improvement of local livelihoods. As have 

been implemented in the villages in Bali, the 

intervention into destructive fi shing includes: 

trainings the fi sherfolk in environmentally 

fi shing techniques, transfer of science and 

technology from universities and the high 

end industry to be adapted into local cost 

and material, building local organizations and 

institutions, and developing micro-enterprises 

to refl ect the “from fi sher to exporter” 

changes, and public education.

Reforms of the marine ornamental industry 

in Indonesia will call for transparent and 

independent monitoring system based on the 

networks of civil society as its constituent. 

Under this direction, Telapak is working with 

other NGOs and research institutions to 

develop Marine Watch Indonesia. This should 

143
become the partner and counterpart of the 

CDT labs network especially, the marine 

ornamental industry reform in general.

In 1997, Telapak collaborated with the IMA 

to implement the Destructive Fishing Reform 

Program (DFRP) in Indonesia. Cyanide 

testing was one component of the DFRP; a 

broader community-based program involving 

rapid resource appraisal, net-training and 

other alternative livelihood training programs, 

enterprise trainings, marine education of 

school children, instruction of fi sherfolk 

in coastal conservation practices, farming 

corals and giant clams, and mapping using 

geographic information systems (GIS). Plans 

by Telapak to implement CDT in Indonesia 

in collaboration with the IMA (discussed at 

the International Coral Reef Symposium held 

in Denpasar in 2000) did not happen because 

of the lack of funding and other problems.

Telapak’s main achievement with destructive 

fi shing reform was the establishment of 

two villages, which are presently completely 

cyanide-free. Through education, economic 

incentives, and peer pressure, the villages 

now capture and export totally cyanide-free 

MAF from the village of Les (situated in 

northern Bali) and farm artifi cial live rock 

and corals grown from fragments in the 

village of Serangan (situated near Denpasar 

in southern Bali). Telapak in collaboration 

with the Philippines based East Asia Seas 

and Terrestrial Initiatives have succeeded 

in changing the behavior of fi sherfolk for 

sustainable use of marine resources and 

the implementation of reef restoration 

programs. This was achieved after a number 

of setbacks. Community organizing, technical 

intervention, management support, market 

reform, and educational components were 

integrated into a holistic community-based 

program supporting conservation of marine 

ecosystems and sustainable use by the MAF 

fi shery.

144
WORKSHOP ABSTRACTS

Ion-Selective Electrodes for Cyanide Detection: 

Present Status and Future Possible Improvements

Dr. Martin S. Frant

Chemotics Consulting

chemotics@gmail.com

We all want a cyanide analytical technique that 

is as simple and fast as possible, inexpensive, 

capable of being done by minimally-trained 

personnel; that will withstand judicial scrutiny. 

Ultimately, it may be possible to meet all 

of these requirements, but not within any 

short-term time frame. Cyanide in fi sh, as in 

other animals, is rarely present in the unbound 

form, and requires pretreatment to free it 

up for measurement. After digestion and 

distillation, the sample consists of unbound 

cyanide ion in an alkaline background and, 

from the analyst’s point of view, is the 

same whether it originated in fi sh tissue or 

wastewater.

Round robin tests on cyanide “unknowns” 

were conducted at different times by the 

two recognized volunteer U.S. standards 

organizations, using a method with a carefully 

spelled-out distillation step and cyanide 

ion-selective electrode (ISE) measurements. 

Both tests had extremely good results, 

including lower limits of detection on a linear 

calibration plot to cyanide levels of 0.03 and 

0.06 ppm. A more recent EPA-funded third 

party evaluation had similar results. The 

cyanide ion detection method, involving acid 

digestion, distillation, and ISE measurement, 

is approved by the U.S. Environmental 

Protection Agency (EPA). This information 

is all in the literature, and should give strong 

backing against any legal challenges.

There are a number of interesting options 

for modifying the present method, and these 

will be discussed. They include techniques for 

electrode measurements down to 0.003 ppm, 

several possible changes in the distillation step, 

and the use of other electrodes, such as the 

silver ISE or an ISE for HCN. The problem 

is that validating any changes will require 

extensive testing, independent collaborative 

data, and fi eld experience before they can be 

used for enforcement.

147
Cyanide Detection Testing (CDT)

Used by the BFAR/IMA Laboratories in the Philippines

Benita Manipula

Former Chief Chemist

International Marinelife Alliance

dragon9262002@yahoo.com

The use of cyanide to capture marine aquarium 

fi sh and food fi sh alive is widespread in the 

Asia-Pacifi c region and not only leads to high 

mortality rates of the captured fi sh, but also 

damages and kills corals and other organisms 

on the reefs. The use of cyanide is illegal in all 

countries where it is used, but enforcement is 

diffi cult. 

A cyanide detection test (CDT) developed by 

the American Society of Testing and Materials 

(ASTM) was adopted by the International 

Marinelife Alliance (IMA) and applied to 

test marine organisms (mostly aquarium 

fi sh and food fi sh species) in the Philippines 

under contract with the Philippine Bureau 

of Fisheries and Aquatic Resources (BFAR) 

(APHA 1992, ASTM 1997). A network 

of six BFAR/IMA CDT laboratories was 

established throughout the Philippines 

staffed by chemists. Sampling was conducted 

by biologists deputized by BFAR at various 

locations including the boats of fi shers, home 

ports, distribution points such as airports, 

and at export facilities in Manila. Over 48,000 

fi sh and invertebrate specimens were tested 

for the presence of cyanide by IMA chemists 

from 1993 to 2001. The method involves 

the digestion of fi sh tissues in sulfuric acid 

to liberate hydrogen cyanide gas, and capture 

of cyanide ions in sodium hydroxide solution 

after refl ux distillation. The chemicals added 

to the apparatus facilitate the extraction 

of cyanide from fi sh tissues and help to 

eliminate interfering substances. Cyanide ion 

concentrations were determined using an ion 

selective electrode (ISE) linked to an ISE 

meter manufactured by Thermo-Orion. 

The ASTM ISE method for cyanide ion 

detection is internationally recognized. It 

has been in use since the early 1980s and is 

periodically updated in separate publications 

by the ASTM, the American Public 

Health Association (APHA), and the U.S. 

Environmental Protection Agency (US-EPA). 

The ASTM ISE method has been used by 

a variety of organizations. Cyanide testing 

should be used to enforce laws against illegal 

fi shing and to accredit marine ornamentals 

(MO) as being cyanide-free to support a 

sustainable MO trade.

148
References

Alban, J., Manipula, B.E. and Rubec, P.J. 

(2001) Standard Operating Procedures For 

Sampling Marine Fish And Invertebrates Used 

By The Philippines Cyanide Detection Test (CDT) 

Laboratory Network. Philippine Department 

of Agriculture-Bureau of Fisheries and 

Aquatic Resources/International Marinelife 

Alliance.

APHA, AWWA, WPCF (1992) Standard 

Methods for the Examination of Water 

and Wastewater. American Public Health 

Association 19.

ASTM (1997) Annual Book Of Standards Vol. 

11.02, (D2036-91), American Society of 

Testing and Materials.

Manipula, B., Suplido, E.R. and Astillero, N.M. 

(2001a) Standard Operating Procedures For 

Preparation Of Marine Fish And Invertebrate 

Samples For Cyanide Testing, Grouper Research, 

And Assessment Of Blast Fishing. Philippine 

Department of Agriculture-Bureau of 

Fisheries and Aquatic Resources and 

International Marinelife Alliance.

Manipula, B.E., Suplido, E.R. and Astillero, 

N.M. (2001b) Standard Operating Procedures 

For Cyanide Testing Used By The Philippines 

Cyanide Detection Test (CDT) Network.

Philippine Department of AgricultureBureau of Fisheries and Aquatic Resources 

and International Marinelife Alliance.

Manipula, B.E., Rubec, P.J. and Frant, M. 

(2001c) Standard Operating Procedures For 

Use Of The Thermo Orion Ion-Selective 

Electrode (ISE) And The ISE/pH Meter To 

Assess Cyanide Concentrations. Philippine 

Department of Agriculture-Bureau of 

Fisheries and Aquatic Resources and 

International Marinelife Alliance.

149
Search for a Simplifi ed Field Test: the Soundararajan Digestion

Combined with ISE or Colorimetric Methods for Detection 

of Cyanide Ion Concentrations in Marine Fish

Peter J. Rubec1

 and Benita Manipula2

International Marinelife Alliance

1. peterrubec@cs.com

2. dragon9262002@yahoo.com

A new method for testing fi sh samples for 

the presence of cyanide was developed by Dr. 

Rengarajan Soundararajan in 1990 (Rubec and 

Soundararajan 1991). The method involves 

the digestion of fi sh tissues in concentrated 

sodium hydroxide (NaOH). The method 

is appealing since it is relatively simple and 

quick to conduct. The cyanide ion is released 

from the tissues into a basic, high pH solution 

preventing the cyanide from being lost to 

the atmosphere. Cyanide ion concentrations 

can then be measured with an ion-selective 

electrode (ISE). 

Five fi sh species obtained from Indonesia tested 

in the USA had cyanide ion concentrations 

ranging from 5.8 to 23 mg/kg (ppm) (Rubec 

and Soundararajan 1991). A Clown Triggerfi sh 

obtained from the Philippines exhibited a 

cyanide ion measurement of 1120 mg/kg. No 

cyanide was detected in two Flame Angelfi sh 

obtained from the Marshall Islands, for two 

French Angelfi sh from the Caribbean, and 

two species of surgeonfi sh obtained from 

Hawaii. Hence, cyanide was detected in fi sh 

from countries known to have collectors 

using cyanide and no cyanide was detected in 

fi sh obtained from countries where cyanide is 

not used for collecting marine aquarium fi sh.

The International Marinelife Alliance (IMA) 

evaluated the technique in 1991, but abandoned 

it after it was found to give anomalously high 

cyanide readings. It was suspected that the 

anomalously high readings found by the IMA 

might have occurred because of false-positive 

readings caused by sulfi de interference with 

the ISE electrode.

Further research was conducted by Aquarium 

Systems with a small grant obtained by the 

IMA from the Columbus Zoo (Frakes and 

Studt 1996). The basic procedure was to 

expose the fi sh to known concentration 

of cyanide ion, kill the fi sh, then puree the 

samples in 5M-NaOH. The NaOH volume 

was calculated to yield a 10% by weight fi sh 

slurry. This slurry was allowed to settle and a 

clear aliquot was diluted with distilled water 

to produce another 10% by weight dilution. 

Cyanide ion (CN-

) concentrations in the fi nal 

solution, 1% fi sh tissue, and approximately 

0.5M-NaOH were measured in millivolts 

(mV) recorded with an Orion CN-

 ISE linked 

to an Orion ISE meter. 

These readings were compared with a 

semi-log plot produced with known cyanide 

concentrations of 0.1, 1.0, and 10 mg/L 

150
levels with mV readings recorded as each 

level (Frakes and Studt 1996). Lead carbonate 

was added to the supernatant solution to 

precipitate sulfi des from solution. The 

fi rst trial compared readings with Atlantic 

Blennies, which were net-caught and exposed 

to cyanide. Both the test and the control 

fi sh (not exposed to cyanide) were found to 

exhibit cyanide levels of about 300 mg/L 

(ppm). Hence, the addition of lead carbonate 

to the solution prior to ISE testing did not 

eliminate the anomalously high readings. A 

second experiment evaluated whether the high 

readings might be due to iodide interference 

with the ISE. Again, the experiment was 

inconclusive. In a third experiment, test fi sh 

(4 Caribbean Blue Chromis, 1 Atlantic Wrasse, 

and 3 goldfi sh) were exposed to cyanide 

concentrations ranging from 1.3 to 3.9 ppm 

CN-

 for times ranging from one minute (for 

the 4 chromis) to 14 minutes (for a goldfi sh). 

Readings for control fi sh (1 wrasse and 2 

goldfi sh) not exposed to cyanide produced 

higher mV readings than fi sh exposed to 

cyanide. 

Recent analyses by Ms Benita Manipula using 

the Soundararajan procedure and cyanide 

measurements obtained using an Industrial 

Test Systems (ITS) Inc. colorimetric Cyanide 

Reagent StripTM (Cyanide Test Kit 484003) have 

also experienced diffi culty in obtaining reliable 

measurements of cyanide concentrations 

in comparison to known concentrations of 

cyanide ion in sodium hydroxide solution 

(lacking slurry). The problem does not 

appear to be related to the reliability of the 

ITS cyanide test strips, since they have been 

demonstrated to be sensitive (down to 0.02 

mg/L) and reliable for measuring cyanide ion 

concentrations in water (Battelle 2005).

The most likely explanation for these results 

is that organics in the digested tissue solutions 

containing sodium hydroxide produced the 

anomalous readings both with the ISE and the 

ITS test strips. Further research is needed to 

see whether it is possible to measure cyanide 

ion concentrations in solutions obtained from 

marine fi sh using the Soundararajan tissue 

digestion method. 

References

Battelle (2005) Industrial Test Systems, Inc. Cyanide 

ReagentStripTM Test Kit. Environmental 

Technolgy Verifi cation Report, 42 pp, 

under contract to U.S. Environmental 

Protection Agency.

Frakes, T. A. and Studt, R. (1996) Evaluation 

of Methods for the Cyanide Detection Test for 

Fish Utilizing Alkaline Digestion and Cyanide 

Specifi c Electrode. Report by Aquarium 

Systems associated with IMA grant from 

the Columbus Zoo, 8 pp, Columbus, OH.

Rubec, P.J. and Soundararajan, R. (1991)

Chronic toxic effects of cyanide on 

tropical marine fi sh. Proceedings of the 

Seventeenth Annual Toxicity Workshop: 

November 5-7, 1990 (Vancouver, B.C.). 

Canadian Technical Report Fisheries and Aquatic 

Sciences 1:1774, 243-251.

151
Battling the Silent Killer of Coral Reefs

Gil Adora 

Assistant Director 

Philippines Bureau of Fisheries and Aquatic Resources

gi_adora@yahoo.com

The misuse of sodium cyanide - the silent 

killer of coral reefs - in the live reef fi sh trade 

remains a major and fundamental challenge 

to fi sheries resource management and 

conservation in the Philippines. Although 

the practice is illegal and fraught with health 

risks, live fi sh collectors continue to take their 

chances, emboldened by the knowledge that 

government has very limited capability to 

apprehend and prosecute them.

The country’s response to the cyanide fi shing 

menace was originally focused on training 

programs that encourage collectors to shift 

to coral-friendly harvesting techniques. 

However, the failure of the market to reward 

them with better returns for their enterprise 

has been cited as a major disincentive that 

eventually compels reformed fi shermen to 

revert to cyanide use.

The Philippine campaign against cyanide 

fi shing reached a major turning point when 

our agency adopted a cyanide detection test 

that uses Ion Selective Electrodes to detect the 

presence of cyanide in fi sh tissues and organs. 

The new test was regarded as a signifi cant 

breakthrough, inspiring law enforcers to 

make arrests and enabling BFAR to fi le cases 

and successfully prosecute a number of them 

in court.

Unfortunately, a shortage of funds compelled 

BFAR to scale down the operation of its 

six CDT laboratories. To date, only four of 

the six laboratories are functioning: Manila, 

Puerto Princesa, Cebu and Zamboanga. 

The good news is that BFAR’s political will 

to stamp our cyanide use remains strong and 

has only been put on hold due to budgetary 

to limitations. In fact, BFAR has a standing 

plan to expand the existing CDT laboratory 

network by establishing new laboratories 

in other centers of live fi sh collection. The 

better news is that the agency been able to 

source from recent grants and loans a total 

of seven complete CDT equipment which, 

funds permitting, would be used in strategic 

priority areas. 

BFAR, however, needs help in running 

and maintaining these labs and other 

complementary activities. It is our hope that 

this workshop will result - well, eventually 

-in providing us with the wherewithal to put 

in operation a serious and effective cyanide 

fi shing reform program.

152
Trends Determined by Cyanide Testing 

on Marine Aquarium Fish in the Philippines

Peter J. Rubec1

 and Benita Manipula2

International Marinelife Alliance

1. peterrubec@cs.com

2. dragon9262002@yahoo.com

Cyanide has been demonstrated to kill 

corals and there is scientifi c evidence that 

it contributes to the high delayed mortality 

of marine fi sh in the aquarium trade. The 

International Marinelife Alliance (IMA) 

conducted cyanide detection testing (CDT) 

on marine aquarium fi sh and food fi sh as well 

as marine invertebrates in the Philippines. 

The testing was conducted under contract 

with the Philippine Bureau of Fisheries 

and Aquatic Resources (BFAR). A network 

consisting of six Cyanide Detecting Testing 

(CDT) laboratories and three regional Marine 

Inspection and Sampling (MIS) offi ces were 

established. The CDT network conducted 

random sampling of fi sh from collectors, 

middlemen, and exporters throughout the 

Philippines. Samples were also collected by 

law enforcement personnel and voluntarily 

submitted by fi sh exporters to determine 

whether or not the fi sh were cyanidefree. Over 48,000 aquarium and food fi sh 

specimens were tested from 1993 to 2001. 

Since then, the ASTM method involving acid 

digestion, distillation, and determination of 

cyanide concentrations using ion selective 

electrodes (ISE) has been conducted by staff 

associated with BFAR. The CDT laboratories 

in conjunction with law enforcement efforts 

have served to deter cyanide fi shing in the 

Philippines. The proportion of marine 

aquarium fi sh tested with cyanide present 

dropped from 43% in 1996 to 8% in 1999, 

then rose to 29% in 2000. The data are broken 

down by family to indicate the proportion of 

specimens with cyanide present or absent. 

Cyanide testing needs to be expanded in the 

Philippines and implemented in Indonesia, 

Vietnam, and Malaysia to support law 

enforcement and conserve coral reefs and 

their associated fi sheries. It should be tied to 

an accreditation system of marine ornamental 

species exported to other countries including 

the U.S.A. 

Rubec, P.J., Pratt, V.R., McCullough, B., 

Manipula, B., Alban, J., Espero, T. and 

Suplido, E. (2003) Trends determined by 

cyanide testing on marine aquarium fi sh in 

the Philippines. In Marine Ornamental Species: 

Collection, Culture and Conservation (Cato, J.C. 

and Brown, C.L., Eds.) 327-340, Iowa State 

Press, Ames, IA.

153
Conservation Policies for Abating

Cyanide Fishing in Indonesia

Agus Dermawan

Deputy Director

Aquatic Conservation Areas and Marine National Parks

Ministry of Marine Affairs and Fisheries

agusder81@yahoo.com

Strategies towards abating cyanide fi shing 

for aquarium fi sh

• Support for development of sustainable 

sources of aquarium fi sh (environmentally 

sound capture methods including nets, 

capture of larval fi sh)

• Certifi cation of aquarium fi sh 

(collaboration with Marine Aquarium fi sh 

Council, and others)

• Marine Protected Area networks

• Enforcement

Enforcement − constraints

Standard gear for cyanide fi shers: cyanide 

“kit” with weights for quick discarding of 

squirt bottles and cyanide tablets if a patrol 

approaches

• Long marketing chains (many smallscale fi shers, various traders, wholesaler, 

exporter)

• Fisheries police and fi sheries court not 

yet fully operational

• Low expertise, low capacity in legal follow 

up and collection of evidence in rural 

areas

• No reliable test for cyanide in live fi sh 

(especially if already several weeks in 

holding facility)

Enforcement − solutions

• Hookah compressor ban (at least two 

districts)

• Further development of Fisheries Police 

and Fisheries Court

• Community-based enforcement 

(COREMAP PokMasWas)

• Mobile mini-CDT lab (in development 

under COREMAP)

Criteria for a test-kit for use in Indonesia

• Must detect cyanide in live fi sh ca. 2 weeks 

after capture

• Simple methodology

• Low cost, Portable

• Test results must be acceptable as evidence 

for Indonesian court

Cooperation towards abatement of 

cyanide fi shing

• Awareness Training in environmentally 

friendly fi shing techniques, handling, and 

marketing

• Support certifi cation to build a trust 

relationship between fi shers, traders, and 

aquarium keepers

• Non-Governmental Organization

• Governmental of Indonesia

1. Enforcement

2. Regulations

154
Implementing agencies:

• Quarantine, Technical Implementation 

Units of Fisheries Service

• Support establishment of MPA networks, 

as recommended by the Convention 

on Biological Diversity and other 

international forums

Coral Reef Rehabilitation and 

Management Program

• US$ 80 million, 2005 - 1010

• De-centralized

• World Bank and Asian Development 

Bank

• Ministry of Marine Affairs and Fisheries, 

Ministry of Forestry, and Indonesia 

Academy of Sciences, local governments

• Seven Districts

• Focus on support for Marine Protected 

Areas (10% of reef in no-take areas)

• Support for Monitoring, Control, and 

Surveillance

• (MCS), community-based enforcement 

(SISWASMAS)

• Support for certifi cation, training in 

sustainable capture methods

• Development of cyanide test-kit

155
Policy and Legislative Measures Needed to Enhance Enforcement 

for Combating Destructive Fishing in Vietnam

 Ho Thi Yen Thu and Simone Retif 

Centre for Marinelife Conservation and Community Development (MCD)

thu@mcdvietnam.org

Although illegal, use of destructive methods 

(cyanide, blast and fi ne mesh nets, etc.) for 

fi shing is still found common in many places 

in Vietnam. The presentation will feature an 

overview of the destructive fi shing practices 

in the country together with an assessment of 

the legal framework currently governing the 

issue, from both international and national 

perspectives. Furthermore, an analysis on the 

compliance and enforcement of these legal 

instruments will be provided. Such analysis 

will set the basis for recommendations to be 

given on practical steps for a way forward. 

156
APPENDIX

APPENDIX I

Cyanide Detection Workshop

Agenda

6-8 February 2008

Orlando, FL, U.S.A.

Wednesday February 6 

5:00 p.m. Workshop Check-in

7:00 Welcome Reception and Dinner

Thursday February 7

8:30 Welcome Keystone

• Introductions

• Meeting goals

• Agenda review

• Discussion guidelines

• Introduce Cyanide Expert Panel

 -Dr. Bob Kobelski, Lead Chemist, CDC 

 -Dr. Brian Logue, Assistant Professor, South Dakota State University

9:00 Fish Collection, Cyanide Use, Management Issues Dr. Andy Bruckner

9:30 Review of Point of Collection/Export Issues

• Philippines (Gil Adora) 

• Indonesia (Agus Dermawan) 

• Vietnam (Thu Ho)

10:15 Discussion All

10:45 Break 

11:00 Review of Point of Import Issues 

• Ken Goddard

• Vicky Vina

• Eddy McKissick

• Roy Torres

11:30 Discussion

 All

12:00 Application of Cyanide Testing in the Philippines Dr. Peter Rubec

12:30 Lunch

159
1:30 Overview of and Potential Cyanide Testing Methods

• Application of the IMA method Benita Manipula

• Ion-selective Electrodes for Cyanide: Present 

Status and Future Possible Improvements Dr. Martin Frant

• Enzymatic biosensor method Dr. Karen Mak

• Use of biomarkers to detect cyanide impacts Dr. Craig Downs

• Rapid fi eld test procedure Dr. Peter Rubec

• The realm of possibilities for cyanide detection Dr. Brian Logue

3:15 Break

3:30 Discussion: Factors to identify appropriate tests for All

 detection of cyanide in marine fi shes 

5:00 Wrap up and adjourn for the day

6:30 Dinner

Friday February 8

8:30 Re-cap of Previous Day and Agenda Review Dr. Bob Kobelski

8:45 Charge to Breakout Groups Keystone

8:50 Convene Breakout Group Discussions All

• Detection Methods 

• Export Country

• Import Country

10:30 Break items available

11:30 Breakout Group Status Report/Interim Summary All

12:00 Lunch

1:00 Resume Breakout Group Discussions 

2:45 Break

3:00 Breakout Group Reports and Recommendations

3:30 Discussion All

4:30 Next Steps and Wrap Up

• Future directions and Closing Thoughts Dr. Andy Bruckner

 

160
Gil Adora

Assistant Director

Philippines Bureau of Fisheries and Aquatic 

Resources

Quezon City, Metro Manila, Philippines

gi_adora@yahoo.com

Andy Bruckner, Ph.D.

Coral Reef Ecologist

Coral Reef Conservation Program

US National Oceanographic and 

Atmospheric Administration

Silver Spring, Maryland, USA

andy.bruckner@noaa.gov

Ferdinand Cruz

Executive Director

East Asian Seas and Terrestrial Initiatives

Los Banos City, Laguna, Philippines

cruzferdinand@gmail.com

Agus Dermawan

Director of Conservation and Marine 

National Park

Directorate General of Coastal and Small 

Islands

Ministry of Marine Affairs and Fisheries, 

Indonesia

Jakarta, Indonesia

agusder81@yahoo.com

Martin Frant, Ph.D.

Research Director

Thermo-Fisher Inc.

Beverly, Massachusetts, USA

ms.frant2@verizon.net

Thu Ho

Vice Director and Program Manager, Coral 

Reefs

Center for Marinelife Conservation

Hanoi, Vietnam

thu@mcdvietnam.org

Aquilino Alvarez

Policy Advisor for the Director

Philippines Bureau of Fisheries and Aquatic 

Resources

Quezon City, Metro Manila, Philippines

linalvar@yahoo.com

Tomas Cabagay

Vice-President

East Asian Seas and Terrestrial Initiatives

Los Banos, Laguna, Philippines

tomcabagay@gmail.com

Patty Debenham, Ph.D.

Director, Coral Program

SeaWeb

San Francisco, California, USA

pdebenham@seaweb.org

Ken Goddard 

Laboratory Director

US Fish and Wildlife Service 

National Forensics Laboratory

Ashland, Oregon, USA

ken_goddard@fws.gov

APPENDIX II

List of Participants

161
Kristine Johnson

Director

Kingfi sher Foundation

San Francisco, California, USA

kmjohnson38@earthlink.net

Brian Logue, Ph.D.

Assistant Professor of Chemistry

South Dakota State University

Brookings, SD, USA

brian.logue@sdstate.edu

Benita Manipula

Former Chief Chemist

International Marinelife Alliance

Tres Martires City, Cavite, Philippines

dragon9262002@yahoo.com

Eddie McKissick

Resident Agent in Charge

US Fish and Wildlife Service

Division of Law Enforcement

Miami, Florida, USA

eddie_mckissick@fws.gov

Glynnis Roberts

US National Oceanographic and 

Atmospheric Administration

Silver Spring, Maryland, USA

glynnis.roberts@noaa.com

Ambrosius Ruwindrijarto

President

Telapak

Bogor, Indonesia

mbajing@indo.net.id

Roy Torres

National Marine Fisheries Service 

US National Oceanographic and 

Atmospheric Administration

Monterey, CA, USA

Roy.Torres@noaa.gov

Bob Kobelski, Ph.D.

Lead Chemist

National Center for Environmental Health

Centers for Disease Control and Prevention

Atlanta, Georgia, USA

rmk9@cdc.gov

Eric Punkay, Ph.D.

Senior Research Associate

SeaWeb/SeaStrategy Network

Silver Spring, Maryland, USA

epunkay@seaweb.org

Peter Rubec, Ph.D.

Research Scientist

Florida Fish and Wildlife Conservation 

Commission

Saint Petersburg, FL, USA

Peterrubec@cs.com

Doug Thompson

Senior Mediator and Director, Environment 

Practice

Keystone Center

Ashland, Massachusetts, USA

dthompson@keystone.org

Vicky Vina

Supervisory Wildlife Inspector

US Fish and Wildlife Service

Division of Law Enforcement

Miami, Florida, USA 

vicky_vina@fws.gov

162
APPENDIX III

U.S. Coral Reef Task Force Meeting 

May 4, 2006 

Washington, DC 

Resolution 15-1: Form a Working Group on Enforcement and Utilize Task Force 

Expertise to Address the Illegal Use of Cyanide and Other Poisons in International 

Trade 

Responsible Party: USCRTF Steering Committee, International Working Group and 

Enforcement Representatives. Contacts: 

 Karen Wardzinski, DOJ, Karen.Wardzinski@USDOJ.gov (202) 514-0474 

 Beth Lumsden, NOAA, Beth.Lumsden@NOAA.gov (301) 713-3459 x 107 

 Barbara Best, USAID, bbest@usaid.gov (202)-712-0553 

Issue Statement: There is an urgent need for fast, reliable and convenient detection tests for 

determining whether cyanide and other poisons have been used in the collection of live coral 

reef fi sh entering into international trade. Many Task Force member agencies have expertise 

in toxicology, biomarkers and forensics that could be applied to this issue. Field-based cyanide 

detection tests would be extremely useful for local management and enforcement authorities 

in source countries. Tests that could be reliably used several weeks after exposure would also 

allow the U.S. to apply the Lacey Act to suspected illegal imports. 

Although illegal in most countries, the use of cyanide to capture reef fi sh alive is widespread, 

and is driven by the lucrative, growing and largely unregulated international trade in live reef 

food fi sh and marine aquarium industry. The U.S. is the number one consumer of live coral, 

live rock and coral reef fi sh for the aquarium trade and of coral skeletons and precious corals 

for curios and jewelry. Previous studies have estimated that most live reef fi sh entering into 

international trade and imported into the U.S. are collected with the use of cyanide, and thus 

are illegal. 

 

Background: Provide a brief history of relevant documents pertaining to the proposal being 

presented to the SC. Include relevance of EO 13089, Coral Reef Conservation Act of 2000, 

Coral Reef National Action Plan, Coral Reef Action Strategy, Local Action Strategies, Federal 

regulations or other pertinent guidance as necessary. 

Executive Order #13089 for the Protection of Coral Reefs mandates that the U.S. Coral 

Reef Task Force “...assess the U.S. role in international trade and protection of coral reef 

species and implement appropriate strategies and actions to promote conservation and 

sustainable use of coral reef resources worldwide.” 

The Coral Reef Action Plan calls for efforts to reduce global threats to coral reefs and to 

reduce the impacts of international trade in coral reef resources, through exercising global 

leadership in the international arena, strengthening international conventions, providing 

assistance and strengthening international research and management. 

163
Assessment of U.S. Role in Trade: Under the leadership of the Department of the Interior 

(USFWS) and the Department of Justice, the Coral Trade Subgroup of the International 

Working Group conducted an initial assessment of the role of the U.S. in the international 

trade of coral and coral reef species in 2000. The analysis found that the U.S. is the number 

one consumer of live coral, “live rock” and marine reef fi sh for the aquarium trade and of 

coral skeletons and precious corals for curios and jewelry. Results of that assessment, along 

with a recommended strategy for action, were compiled in a report to the Task Force entitled, 

International Trade in Coral and Coral Reef Species: The Role of the United States.

(The report is available at www.coralreef.gov) 

While it is known that the US is the largest importer of coral reef fi sh for the marine aquarium 

trade, the magnitude of the trade is based upon estimates as almost all reef fi sh are not listed 

on CITES. The NMFS is analyzing the USFWS import data – recently modifi ed to identify 

and separate out freshwater and marine fi sh – to assess the quantity of fi sh entering the U.S. 

The initial analysis indicates that more than twice as many coral reef fi sh are entering the U.S. 

for the marine aquarium hobby than was originally estimated, up to 16 million fi sh a year. 

USCRTF Resolution 14-4 Call for Building Enforcement Capacity: At the 14th Meeting 

of the USCRTF in Palau, the Task Force called on its members to increase efforts to build 

enforcement capacity. The Steering Committee was charged with continuing training and 

other efforts, including the development of an enforcement “toolbox” in cooperation with 

the international Coral Reef Initiative (ICRI), to help coral reef management communities 

build enforcement capacity. 

Statement Decision(s): 

 The USCRTF decides to: 

• Form a working group on enforcement to assist in fulfi lling Resolution 14-4. 

• Identify and recommend specifi c experts in law enforcement, fi eld forensics, and 

toxicology/biomarkers to serve on the working group from Task Force agencies. 

• Utilize expertise from Task Force agencies to identify existing or potential cyanide detection 

methods or tests which could be used to determine if fi sh had been exposed to cyanide 

or other poisons. 

• Explore the usefulness and need to convene a broader expert panel to resolve the issues 

associated with cyanide and other poison detection tests, and if needed, assist in convening 

such a panel. 

• Encourage all coral reef jurisdictions to identify and promote alternative, sustainable, 

non-destructive practices in regard to aquarium fi sheries. 

• Experts and relevant representatives from Task Force Agencies will be identifi ed and the 

Working Group on Enforcement formed within two months. Progress on assessing cyanide 

and other poison detection tests and building enforcement capacity will be presented at 

the next USCRTF Meeting. 

164