Biosensors and Bioelectronics 20 (2005) 2581–2593

Review

Cyanide fishing and cyanide detection in coral reef fish using

chemical tests and biosensors
Karen K.W. Maka,∗ , Hideshi Yanaseb , Reinhard Renneberga
a Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
b Department of Biotechnology, Tottori University, Tottori 680-8552, Japan

Received 31 July 2004; received in revised form 14 September 2004; accepted 20 September 2004

Abstract

Sodium cyanide has been used in the Philippines to collect tropical marine fish for aquarium and food trades since the early 1960s. Cyanide
fishing is a fast method to stun and collect fish. This practice is damaging the coral reefs irreversibly. In most countries cyanide fishing is
illegal, but most of the exporting and importing countries do not have test and certificate systems. Many analytical methods are available
for the detection of cyanide in environmental and biological samples. However, most of the techniques are time consuming, and some lack
specificity or sensitivity. Besides, an ultra sensitive cyanide detection method is needed due to the rapid detoxification mechanisms in fish.
The aim of this review is to give an overview of cyanide fishing problem in the south-east Asia and current strategies to combat this destructive
practice, summarise some of the methods for cyanide detection in biological samples and their disadvantages. A novel approach to detect
cyanide in marine fish tissues is briefly discussed.
© 2004 Elsevier B.V. All rights reserved.

Keywords: Biosensor; Coral reef fish; Cyanide degrading enzyme; Cyanide fishing; Optical test

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2582
1.1. Cyanide fisheries—when and where did they start? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2582
1.2. Threats to coral reefs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2582
1.3. Control and monitoring in south-east Asia countries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2582
1.4. The Hong Kong trade in live reef fish for food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2583
1.5. Toxicity of cyanide to fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2583
2. Standard cyanide detection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2583
2.1. Titrimetric methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2584
2.2. Colorimetric method based on König reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2584
2.3. Ion selective electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2584
3. Other methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2585
3.1. Chromatographic methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2585
3.2. Spectrophotofluorometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2585
3.3. Indirect atomic absorption spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2585

∗ Corresponding author. Tel.: +852 2358 7410; fax: +852 2358 1594.
E-mail address: karen@ust.hk (K.K.W. Mak).

0956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bios.2004.09.015
2582 K.K.W. Mak et al. / Biosensors and Bioelectronics 20 (2005) 2581–2593

4. Biosensors for cyanide detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2586

4.1. Microbial cyanide sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2586
4.2. Cyanide biosensors based on enzyme inhibition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2586
4.3. Cyanide biosensors based on cyanide degrading enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2586
4.4. Advantages and disadvantages of biosensors for cyanide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2587
5. Strategies to fight cyanide fishing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2587
6. Novel biotest and biosensor for cyanide detection in marine fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2588
6.1. Microbial cyanide degrading enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2588
6.2. Optical biotest for determination of cyanide in marine fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2589
6.3. Biosensor system based on cyanidase reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2590
6.4. Advantages of the novel biotest for cyanide detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2590
7. Summary and conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2591
8. Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2591
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2591

1. Introduction 1.2. Threats to coral reefs

1.1. Cyanide fisheries—when and where did they start? Cyanide fishing causes the destruction of thousands of
hectares of coral reefs every year. Cyanide fishing is consid-
The marine aquarium or ornamental industry began in ered one of the most destructive to the coral reef ecosystem.
1957 when Earl Kennedy began exporting fish from the Cyanide leaves the physical structure of the reef intact, but in
Philippines to the pet shops and aquariums in Europe and fact, kills the coral polyps. It destroys the coral reefs by dis-
North America. Fish were caught with cotton nets and traps rupting the zooxanthellae, symbiotic algae that live in their
placed on coral reefs (Rubec, 1986). Cyanide fishing was coral hosts. The coral polyps are unable to survive because
first practiced in 1962 in the Philippines (Robinson, 1985). lacking nutrients, resulting in discoloration or “bleaching”.
The process is simple: fishermen first crush sodium cyanide Cyanide is also fatal to various forms of marine life near the
(NaCN) pellets into squirt bottles filled with seawater. Then sprayed area (Jones and Steven, 1997; Jones, 1977).
they dive down to coral reef areas and squirt the concen- In many cases while cyanide solution is squeezed,
trated cyanide solution into the crevices where fish hide. The nonetheless, the fish escape into reef crevices. Fishermen
cyanide stuns the fish temporarily, making them easier to have to remove and hammer the reefs apart to retrieve the
capture (Rubec, 1988). Live fish are brought to the ship and fish. This practice causes irreversible damage to the reefs
put into fresh seawater. Cyanide fishing is considered a fast (Barber and Pratt, 1998b). Healthy reefs can produce 35 t of
method to stun and collect ornamental fish. fish per square kilometer each year, while degraded reefs pro-
The use of cyanide for collecting large live reef food duce significantly less (White et al., 2000). According to two
fish, mainly groupers and Humphead Wrasse from Palawan regional surveys published in 2000, only 4.3% of the Philip-
(Philippines) destined for Hong Kong’s expanding live reef pine reefs and only 6.7% of those in Indonesia are still in
fish industry, was first documented in the early 1970s. With excellent conditions (Simpson, 2001).
the expansion of the trade, the use of cyanide to capture live
reef fish has spread to many other places including Indonesia, 1.3. Control and monitoring in south-east Asia countries
Cambodia, the Maldives, Thailand and Vietnam (Barber and
Pratt, 1997). Cyanide fishing is technically illegal in most of the coun-
Since the 1960s, it is estimated that over 1 million kilo- tries, however, it is abetted by corrupt civilian and military
grams of cyanide have been used on the Philippine reefs. officials, and it takes advantage of poor rural workers for
Cyanide is used also in approximately 70% of the fish cap- whom the profits from the trade are irresistible. Many export
tured for the aquarium industry. Although the deleterious as well as import countries do not have testing and certi-
effects of cyanide are well documented, its use continues, fication systems. Only few countries have taken additional
albeit illegally, in many countries (Barber and Pratt, 1997). actions. For instance, the export of Napoleon wrasse is re-
Cyanide is extremely destructive since it can kill both target stricted in Indonesia since 1995. The Philippines, where the
fish and non-target organisms such as small non-target fish, birthplace of cyanide fishing was, has a program aimed at
corals, and invertebrates along with their eggs and larvae and eliminating cyanide fishing (Barber and Pratt, 1997).
microorganisms and destroys important coral reef habitats In 1991 the non-profit organization, International
(Johannes and Riepen, 1995). Marinelife Alliance-Philippines (IMA), and the Philippine
 K.K.W. Mak et al. / Biosensors and Bioelectronics 20 (2005) 2581–2593
Bureau of Fisheries and Aquatic Resources (BFAR) have de-
veloped the cyanide detection test (CDT) to determine the
presence of cyanide in live-caught fish with the help of an
American analytical testing laboratory, basing on the Amer-
ican Society of Testing Materials (ASTM) standards. The
first CDT laboratory was established at BFAR headquarters
in Manila in 1992. Since then, the CDT network has been
expanded to include six laboratories and three monitoring-
inspection stations at all major collection and shipment points
in the country. Each CDT test takes about 1.5 h, including
sample preparation, distillation, and reading of the results.
The IMA CDT uses ion selective electrodes (ISEs) to detect
the concentration of cyanide in the distillate. The statistical
data collected from all the laboratories and monitoring sta-
tions are routinely submitted to the BFAR.
The IMA has been testing random samples for a num-
ber of years. In 1996, 739 out of 3950 samples (19%) were
tested positive for cyanide (Barber and Pratt, 1997). In the
light of our findings about the insensitivity of the test, this
was an amazing number (see Section 5). In addition, the ac-
tual percentage of fish caught with cyanide was likely much
higher: the number of samples taken was quite low in com-
parison to the volume of the trade because the capacities of
the laboratory network. It was found the more than 90% of
live fish vessels boarded at sea by enforcement authorities
were found to be using cyanide. Little is known about the
rates at which cyanide is excreted from the fish, although it is
thought to happen relatively quickly (see Section 6). Where
fish have been kept in sea cages for some period of time,
cyanide-caught specimens may not test positive for cyanide.
In short, cyanide fishing is still a widespread problem. The
details of cyanide detection test by IMA will be discussed
later in this review.
1.4. The Hong Kong trade in live reef fish for food
Fish plays a very important role in the culture and cui-
sine. In modern China, especially in Hong Kong and south-
ern coastal regions, live marine fish plays a very significant
cultural and social role particularly for business dinners and
banquets. Keeping fish alive until minutes before cooking has
been a popular Chinese custom for centuries. Hong Kong is
the largest consumer of live reef food fish, accounting for
60% of the world trade (Johannes and Riepen, 1995).
Only a small number of species are sought in high volumes
for the food fish trade. Common reef fish eaten are groupers
(Serranidae), wrasses (Labridae), and snappers (Lutjanidae).
The Humphead (Napoleon) wrasse (Cheilinus undulatus),
High-finned Grouper (Cromileptes altivelis), Giant Grouper
(Epinephelus lanceolatus) are traded only in small volumes
but are particularly valued (Johannes and Riepen, 1995;
Sadovy and Vincent, 2002). Hong Kong imports live reef
food fish from over 10 different countries and regions. About
45% of the fish are imported from Indonesia, closely followed
by the Philippines, Australia, Maldives, Vietnam, Malaysia
and Thailand (Chan, 2000). Hong Kong was importing about
2583
1000–2000 t live reef food fish per annum in the late 1980s
and 1990s, which had increased from 30,000 to 35,000 t by
the later 1990s (Johannes and Riepen, 1995; Lau and Parry-
Jones, 1999).
Live reef food fish are very valuable in Hong Kong. The
average wholesale price was U.S. $17–22 per kilogram in the
later 1990s, whereas dead fish fetched less than one-tenth of
that value over the same period (Sadovy and Vincent, 2002).
Total wholesale value of the trade was about U.S. $500 mil-
lion in Hong Kong alone in 1998, giving a global trade worth
about U.S. $830 million wholesale and assuming that Hong
Kong accounts for 60% of the world trade.
1.5. Toxicity of cyanide to fish
Rubec and Pratt (1984) have acknowledged that very lit-
tle scientific research has been published on the effects of
cyanide on marine fish. They have summarized numerous
papers that confirm cyanide is very harmful to freshwater
fish. The early research found cyanide to be acutely toxic to
aerobic organisms at concentrations generally greater than
0.1–0.3 mg L−1 (3.8 × 10−6 to 1.1 × 10−5 M) causing death
within 96 h (Doudoroff, 1980). Chronic toxicity also occurs
when fish were exposed to cyanide do not die within 96 h, but
suffer damage and stress that leads to their subsequent death.
Low concentrations in the range from 0.005 to 0.01 mg L−1
(1.9 × 10−7 to 3.8 × 10−7 M) of hydrocyanic acid were found
on prolonged exposure to have many adverse effects on fish
eggs, young and adult fish, such as reduced growth, impaired
swimming performance, increased metabolism, inhibition of
reproduction due to alteration of lipid metabolism and in-
creased respiratory rates (Leduc, 1984).
HCN enters a fish’s bloodstream through the gills and in-
testine and is rapidly distributed to other body tissues. The
toxic effects can be traced to the fact that cyanide interferes
with oxygen metabolism by blocking the key enzyme sys-
tems, cytochrome oxidase (Metzler, 2001), and blocks enzy-
matic pathways in the liver (Solomonson, 1981). Some of the
effects, such as blocking enzyme functions, are irreversible
and hence lead to the death of the fish (Way et al., 1988).
2. Standard cyanide detection methods
Many standard methods have been established for the
determination of cyanide compounds in both environmen-
tal water and wastewater (APHA, 1998; U.S. EPA, 1983).
These methods make use of for example titrimetry, colorime-
try (spectrophotometry), and cyanide ion selective electrodes
(ISEs). However, all are subject to interference problems.
There are certain inherent difficulties in the detection and
determination of cyanide especially in biological samples.
Pretreatment procedures for extracting cyanide from aque-
ous matrix usually involve acidifying the sample followed
by heating and refluxing to evolve hydrogen cyanide (HCN)
gas, which is then trapped in an alkaline absorption media
2584 K.K.W. Mak et al. / Biosensors and Bioelectronics 20 (2005) 2581–2593
Fig. 1. Reaction scheme of the titrimetric method for cyanide
determination.
(e.g. sodium hydroxide solution) to form free cyanide ions
(CN− ). Interferences are eliminated or reduced to a minimum
by the sample pretreatment.
2.1. Titrimetric methods
Bark and Higson (1963) have reviewed on visual titrimet-
ric methods for determination of cyanide. The earliest method
for determining cyanide was reported as early in 1850s by
Liebig. In a sample solution cyanide ion is titrated with a
silver nitrate (AgNO3 ) solution to form a soluble cyanide
complex, Ag[Ag(CN)2 ] (Liebig, 1852). After a small excess
of silver ion is added, the excess of Ag− ions is indicated
by p-dimethylaminobenzalrhodanine, which turns from yel-
low to red (Fig. 1). Titrimetric procedures are usually em-
ployed for the determination of large quantities of cyanide
such as higher than 1 ppm (>3.85 × 10−5 M). One disadvan-
tage of titrimetric methods is the sample should be without
any weakly complexed metal cyanides or other interferences
(Pohlandt et al., 1983).
2.2. Colorimetric method based on König reaction
For the detection of small amounts of ionic cyanide, col-
orimetric methods based on the König synthesis are most
commonly used (König, 1904, 1905; Ludzack et al., 1954). In
these methods cyanogen bromide or chloride reacts with pyri-
dine and an aromatic amine to form a dye. Aldridge’s method
is a modification of the König synthesis, in which cyanide
reacts with excess of bromine, removing the excess bromine
with arsenous acid solution and allowing the cyanogen bro-
mide to react with a pyridine-benzidine reagent (Aldridge,
1944). An intense orange color is formed, which changes to
red during 6 min, and is stable for 6–24 min at 20 ◦ C. How-
ever, a disadvantage of this method is the use of hazardous
chemicals such as benzidine which is carcinogenic.
A similar method also based on the König reaction was
proposed by Epstein (1947), which involves the oxidation of
cyanide to cyanogen chloride with chloramine-T. Cyanogen
Fig. 2. The chemistry of the colorimetry determination of cyanide. Cyanide
is converted to cyanogen chloride (ClCN) by chloramine-T. ClCN is then
reacted with pyridine to form N-cyanopyridinium chloride (König reaction),
which undergoes hydrolysis to glutaconic aldehyde. The aldehyde is couple
with barbituric acid to form a colored product.
chloride is then reacted with pyridine containing pyrazolones,
and then the produced blue color is measured at 630 nm. This
sensitive method can be used in acid, neutral, or slightly al-
kaline media. A modification of Epstein’s method that em-
ploys a more stable reagent, pyridine-barbituric acid, was
recommended by the American Public Health Association
(APHA, 1985; Burtis and Ashwood, 1994). This method is
preferable for cyanide between 0.1 and 1 ppm (3.8 × 10−6 to
3.8 × 10−5 M). The chemistry of the pyridine-barbituric acid
method is shown in Fig. 2.
2.3. Ion selective electrodes
The use of ion selective electrodes (ISEs) has become very
popular, primarily because it is a convenient and rapid method
of analysis. An ISE is basically a potentiometric application.
Of the halide-membrane electrodes, cyanide-selective mem-
branes based on silver iodide (AgI) or a mixture of AgI and
silver sulfide (AgS) are most commonly used (Clysters and
Adams, 1976). For both of these, some form of ion-exchange
reaction takes place at the boundary phases of the membrane
 K.K.W. Mak et al. / Biosensors and Bioelectronics 20 (2005) 2581–2593
layer. The electrode responds primary to the liberated iodide
ion:
2CN− + AgIsolid
Ag(CN)2 − + I− (1)
CN− + AgIsolid
AgCN + I− (2)
As the electrode responds only to the dissociated ionic
cyanide, i.e. CN− ions, the effect of pH on the electrode
response can be predicted from a log diagram of the hydro-
cyanic acid-ionic cyanide dissociation system. Theoretically,
the optimum range of pH for the electrode is between 12 and
13, but it can be used at a pH value as low as 7 (provided that
the solution is carefully buffered), probably as a result of the
reaction:
2HCN + AgIsolid
Ag(CN)2 − + 2H+ + I− (3)
In the presence of metal-cyano complexes, those com-
plexes with stability constants lower than that of silver (I)
cyanide, e.g. the complexes of zinc and cadmium, also give
an electrode response and report as ionic cyanide.
The cyanide ISE can be used in the direct determination
of ionic cyanide in solution, where it gives a true Nernstain
response over the concentration ranges from 10−1 to 10−5 M
(2600–0.26 ppm) (Pohlandt et al., 1983). However, same as
most of other ISEs, the cyanide ISEs respond to numer-
ous interferences such as sulfur, chlorine, iodine, bromine,
cadmium, silver, zinc, copper, nickel and mercury (pHoenix
Electrode Co., 2004; ATSDR, 1997).
3. Other methods
In recent years, many methods for cyanide determination
have been developed using chromatography, spectrophotoflu-
orometry, and indirect atomic absorption spectrometry.
3.1. Chromatographic methods
The measurement of hydrogen cyanide directly by gas
chromatography (GC) has been reported (Valentour et al.,
1974), but this method lacks sensitivity with most detec-
tors. However, the use of chloramine-T to oxidize cyanide
to cyanogen chloride for subsequent extraction with hexane
has led to a sensitive method. The sensitivity of this method
which employs an electron capture detector and as little as
0.25 ␮g mL−1 (9.5 × 10−6 M). Gas chromatographic tech-
niques are not widely used for determining cyanide primarily
Table 1
Analytical methods for cyanide determination
Chemical method Range of detection
Colorimetric 3.8 × 10−6 to 3.8 × 10−5 M
ISE 1 × 10−1 to 1 × 10−5 M
Titrimetric >3.8 × 10−5 M
Gas chromatography >9.5 × 10−6 M
Spectrophotofluorometry >1 × 10−6 M
Indirect atomic absorption spectrometry 1.14 × 10−5 to 1.14 × 10−4 M
2585
because other methods are more convenient. However, the
inherent sensitivity and selectivity of this method insure its
application to specialized samples, particularly those requir-
ing the differentiation of various cyanide species (Way, 1984;
Kage et al., 1996).
Other chromatographic methods include high-
performance liquid chromatography (HPLC) and ion
chromatography (IC). These methods employ electron cap-
ture detectors, electrochemical detectors, UV–vis detectors,
fluorescence detectors, conductivity detectors, or ampero-
metric detectors (Kutseva and Kashin, 1995; Nonomura,
1992, 1994).
3.2. Spectrophotofluorometry
Spectrophotofluorometry is a convenient, sensitive
method for measuring cyanide in biological fluids, provided
that prior microdiffusion is employed to isolate and con-
centrate the cyanide (Sano et al., 1989). Two fluorometric
methods have been adapted to measure cyanide in biologic
fluids (Morgan and Way, 1980; Guilbault, 1990; Guilbault
and Kramer, 1965). Approximately 1 × 10−6 M (0.026 ppm)
of cyanide can be detected by this method. However, like
many fluorometric methods, the disadvantage of the above
methods is, its interference by various small amounts of
extraneous fluorescent materials in the laboratory.
3.3. Indirect atomic absorption spectrometry
There are two general methods for the determination of
cyanide by indirect atomic absorption spectrometry (Danchik
and Boltz, 1970; Gómez and Calatayud, 1998). One proce-
dure involves the formation of an insoluble metal cyanide
compound; then the metal in the precipitate or the excess
metal in the supernatant is measured by atomic absorption
spectrometry. In another method, a relatively stable metal-
cyanide complex is formed which is then extracted into an
organic solvent and the metal content of the extract is de-
termined. The sensitivity range for this type of determina-
tion is approximately from 0.3 to 3.0 ppm (1.14 × 10−5 to
1.14 × 10−4 M). Although very sensitive, indirect atomic ab-
sorption spectrometric methods are not widely used, probably
because these methods are indirect and comparatively slow
due to the complicated separation steps involved and require
expensive equipments.
Table 1 summarizes analytical methods for cyanide deter-
mination.
Reference
APHA (1985), Burtis and Ashwood (1994)
Pohlandt et al. (1983)
Bark and Higson (1963)
Way (1984)
Morgan and Way (1980), Guilbault (1990)
Danchik and Boltz (1970), Gómez and Calatayud (1998)
2586 K.K.W. Mak et al. / Biosensors and Bioelectronics 20 (2005) 2581–2593
4. Biosensors for cyanide detection
Recently, many biosensors have been developed for envi-
ronmental monitoring, such as those for the determination of
ammonia, phosphate and biological oxygen demand. Biosen-
sors are suitable for environmental monitoring because of
their rapid response, high selectivity, pollution-free, inexpen-
sive and convenient to use. A number of microbial and enzy-
matic biosensor systems for cyanide determination have been
developed, which are discussed in this section.
4.1. Microbial cyanide sensors
Many microorganisms have the abilities to degrade
cyanide have been reported (Knowles and Bunch, 1986;
Raybuck, 1992). Some bacterial strains that aerobically
biodegrade cyanide have been used to develop microbial
cyanide sensors. Cyanide oxidase in these bacteria converts
cyanide into cyanate consuming oxygen:
(4)
Therefore, the oxygen consumption by the immobilized
bacteria due to the oxidative cyanide degradation could be
monitored with a Clark oxygen electrode. Lee and Karube
have developed the first microbial sensor for the determina-
tion of cyanide in 1995. A bacterial strain, Pseudomonas flu-
orescens NCIMB 11764, utilizes cyanide as a sole source of
nitrogen. The decrease of dissolved oxygen by cyanide degra-
dation of immobilized bacteria was directly determined by a
Clark-electrode. This microbial sensor was capable to de-
tect potassium cyanide (KCN) in rivers in the range from 0.1
to 1 ppm (3.8 × 10−6 to 3.8 × 10−5 M). Since then Karube’s
group has developed several devices based on immobilized
cyanide degrading bacteria such as a biosensor for gaseous
cyanide in solution and a reactor type system for river water
(Lee and Karube, 1996a, 1996b).
On the other hand, several cyanide biosensors have
been developed based on inhibition of microbial respira-
tion. Cyanide inhibits cytochrome oxidase in the respira-
tory chain of aerobic cells. Subsequently, the decrease in
oxygen consumption by the microbial cells was monitored
with the Clark-electrode. The yeast, Saccharomyces cere-
visiae, was used by Ikebukuro et al. (1996a, 1996b) to de-
velop biosensors for cyanide detection. Recently, a practical
device for determining cyanide in fruit brandies was devel-
oped by Filipović-Kovačević (2002) also using S. cerevisiae.
The detection limit of this amperometric biosensor was up to
0.8 ppm (3.04 × 10−5 M) cyanide.
4.2. Cyanide biosensors based on enzyme inhibition
The catalytic activity of enzymes is not only very spe-
cific for their substrates but also can be sensitive to the in-
hibitors. Since the cyanide toxicity is by its binding specifi-
cally to cytochrome oxidase, Albery et al. (1990a) have de-
scribed and characterized a membrane enclosed cytochrome
oxidase-based amperometric sensor for cyanide. In this sys-
tem oxygen is reduced through successive electron transfers
from a modified gold electrode to cytochrome c and then
to cytochrome oxidase. At ambient oxygen concentration the
current is proportional to the cytochrome oxidase activity and
inhibition of the current occurs in the presence of cyanide.
The sensor was able to measure cyanide concentration down
to 0.4 ␮mol dm−3 (0.01 ppm) in the solution phase (Albery
et al., 1990b). Five years later, Amine et al. (1995) have
developed an inhibition-based sensor for cyanide using cy-
tochrome oxidase immobilized in a carbon paste/lipid matrix
with the detection limit of 0.5 ␮M (0.013 ppm) cyanide.
Smit and Cass (1990) have demonstrated an enzyme elec-
trode containing horseradish peroxidase (HRP) was also sen-
sitive to cyanide. Their study utilized a dual electrode strategy
for the electrochemical generation of substrate (H2 O2 ) at one
electrode and detection of the solution mediator, ferrocene, at
the second electrode. Inhibition of tyrosinase has also been
used for the sensing of cyanide (Smit and Rechnitz, 1993;
Besombes et al., 1995; Robinson et al., 1995; Hu and Leng,
1995).
4.3. Cyanide biosensors based on cyanide degrading
enzymes
One disadvantage of microbial cyanide biosensors is their
selectivity. Therefore, a few biosensors based on cyanide
degrading enzymes that showed better selectivities have
been developed. An enzyme thermistor using immobilized
rhodanese and injectase, respectively, was developed by
Mattiasson and Mosbach (1977) for cyanide detection in blast
furnace water. Their linear ranges were obtained from 20
to 600 ␮M cyanide for injectase and from 20 to 1000 ␮M
cyanide for rhodanese. The measurement of heat evaluation
during the enzymatic reactions, however, is a complicated
procedure and requires an expensive thermistor unit.
An amperometric biosensor for cyanide determination in
brass plating and rinsing solutions was developed by Groom
and Luong (1991). This system consisted of rhodanese im-
mobilized on aminopropyl glass beads to form an enzyme
column and sulfite oxidase immobilized on a pre-activated
nylon membrane and attached to the sensing surface of an
amperometric electrode. Fig. 3 shows the bi-enzyme reac-
tion scheme. This system exhibited a linear response from 5
to 1000 ␮M cyanide. However, the data obtained were not in
agreement to those of the commercially available cyanide-
selective electrodes as the authors state.
The above biosensors lack sensitivity for cyanide detec-
tion. Based on the rhodanese-sulfite oxidase reactions and
subsequent luminol chemiluminescence reaction, a highly
sensitive biosensor system was developed and applied to river
water analysis (Ikebukuro et al., 1996c). Hydrogen peroxide
produced by the sulfate oxidase was reacted with luminol
 K.K.W. Mak et al. / Biosensors and Bioelectronics 20 (2005) 2581–2593 2587

Table 2
Recently developed cyanide biosensors
Biosensor type Range of detection and limit of Reference
detection (LOD)
Microbial sensor
Oxygen uptake by bacteria 3.8 × 10−6 to 3.8 × 10−5 M Lee and Karube (1995, 1996a)
1.9 × 10−6 to 3.8 × 10−5 M Lee and Karube (1996b)
Inhibition on yeast respiration LOD = 1.5 × 10−7 M; 0–1.5 × 10−5 M Ikebukuro et al. (1996a, 1996b)
LOD = 3.04 × 10−5 M Filipović-Kovačević (2002)
Enzyme sensor
Cytochrome oxidase inhibition LOD = 3.8 × 10−7 M Albery et al. (1990a, 1990b)
LOD = 5 × 10−7 M Amine et al. (1995)
Peroxidase inhibition LOD = 1.9 × 10−7 M Smit and Cass (1990)
Tyrosinase inhibition 1 × 10−5 to 2.5 × 10−5 M, Smit and Rechnitz (1993), Besombes et al. (1995),
LOD = 2 × 10−8 M, 2 × 10−7 to Hu and Leng (1995)
4 × 10−5 M
Heat evaluation by rhodanese 2 × 10−5 to 1 × 10−3 M Mattiasson and Mosbach (1977)
Rhodanese and sulfite oxidase reactions 5 × 10−6 to 1 × 10−3 M Groom and Luong (1991)

catalyzed by peroxidase. This highly sensitive method had a
linear detection range from 0.12 to 3.8 ␮M cyanide.
Table 2 summarizes the cyanide biosensors have been de-
veloped in recent years.
4.4. Advantages and disadvantages of biosensors for
cyanide
Enzyme biosensors based on cyanide degradation are ideal
since the enzymes are simply acting as the selective catalysts,
theoretically enable such biosensors to run continuously. Un-
fortunately, these sensors lack sensitivity for cyanide detec-
tion. Cyanide biosensors based on enzyme inhibition, on the
other hand, are non-ideal because of the inhibition of catalytic
activity and the need to regenerate or replace the enzymes,
thus making measurements intermittent. Moreover, the num-
ber of natural enzymes that can be isolated and used in both
ways is very limited.
Microbial sensors are suitable for environmental monitor-
ing and on-line process control since they are relatively in-
expensive and simple to produce and operate. Unfortunately,
they were less selective and the microbial respirations were
rather easily affected by nutrients in samples like glucose as
well as glutamic acid (Lee and Karube, 1995) and other ma-
terials assimilated by the microorganisms. Toxic compounds
Fig. 3. Rhodanese/sulfate oxidase bi-enzyme scheme. The hydrogen perox-
ide produced in the coupled enzymatic reactions is determined electrochem-
ically by a platinum anode vs. Ag/AgCl cathode polarized at +0.7 V.
such as pesticides and herbicides would also affect the mi-
crobial respiration.
5. Strategies to fight cyanide fishing
For over a couple of decades now, sodium cyanide is
widely used in the Philippines’ live fish trade on the highly
diverse coral reefs. Every year, it is estimated that at least
150,000 kg of this broad spectrum poison are sprayed on coral
reefs by some 4000 Filipino fish collectors hunting for aquar-
ium and live food fish. As the use of cyanide for fishing is
illegal in most countries, governments are increasingly seek-
ing improved means to enforce this, such as legislation and
regulations like cyanide detection testing (Rubec, 1986).
In response to the need for an effective tool to monitor the
live reef fish trade and regulate the use of destructive fishing
with cyanide, the cyanide detection test (CDT) method has
been developed in the Philippines in 1991 (Barber and Pratt,
1997). The test has been employed since 1992 in a network
of labs jointly operated by IMA and the Philippines Govern-
ment Bureau of Fisheries and Aquatic Resources (BFAR).
On a daily basis, fisheries officers of CDT/BFAR submit fish
samples taken randomly from fishers, local buyers (middle-
men) and exporters to the nearest CDT lab for testing. In
brief, internal organs of a fish, where cyanide is rapidly ab-
sorbed, were homogenized with water in a blender. Then the
homogenate was strongly acidified in a 1-h reflux distilla-
tion. If cyanide is present in the sample, it is liberated as
hydrogen cyanide (HCN) and absorbed into a sodium hy-
droxide solution. Finally, the presence of cyanide in absorb-
ing solution was detected using ISE. Each CDT lab is ca-
pable of running at least 20 tests per day. Each test takes
about 1.5 h including the procedures described as above. Af-
ter 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).
2588 K.K.W. Mak et al. / Biosensors and Bioelectronics 20 (2005) 2581–2593
In our laboratory in Hong Kong, the cyanide distillation
procedures applied in the Philippines was simulated and re-
viewed in accordance to the Standard Operating Procedures
(SOP) for Cyanide Analysis in Fish Tissues (International
Marinelife Alliance, 2000). The aim was to answer the fol-
lowing questions: (1) is the cyanide distillation method com-
bining with the ISE appropriate and effective? and (2) what
kinds of improvements can be done to achieve a higher sen-
sitivity and accuracy? To check any improvement possible,
modification of the distillation procedures were set up in our
laboratory for comparison.
The ISE was fully calibrated each day of use. A cyanide
calibration curve was constructed on a semi-logarithmic pa-
per. The measured electrode potential in mV (linear axis) was
plotted against the cyanide concentration (log axis). As de-
scribed in the instruction manual (pHoenix Electrode Co.),
a linear range from 1 × 10−5 to 1 × 10−2 M (0.26–260 ppm)
cyanide was established. Calibration of zero was not possible
as the electrode responded erratically. The lowest detectable
cyanide concentration by the ISE was 1 × 10−5 M. This con-
centration is the lower limit of Nernstian response (commonly
quoted as the lower limit for useful measurement) of the ISE
(Evans, 1995). The plotting of an accurate curved calibration
line below the limit of Nernstian response requires a large
number of measuring points and would be time consuming.
In addition, it is known the ISEs tend to malfunction sud-
denly, rather than slowly over time, so a daily generation of
the calibration curve and monitoring of electrode slope were
the best preventative maintenance procedures available for
the electrode.
Another cyanide calibration curve was obtained using
the pyridine-barbituric acid colorimetric method. A linear
range from 1 × 10−6 to 4 × 10−5 M (0.026–1.04 ppm) was
established. To obtain the blank signal, cyanide standard
was replaced by sodium hydroxide solution, which was then
mixed with the reagents. The detection limit was calculated
to be 6.25 × 10−8 M cyanide. Due to the higher sensitivity,
the colorimetric method was used for determination of low
cyanide concentrations instead of ISE. Besides, the colori-
metric method was preferred when only very small volume of
sample was available for cyanide determination. Up to 10 mL
of sample was required for ISE because of technical reasons,
while the colorimetric method only required some hundreds
micro-liter. Therefore, for measurements of cyanide below
1 × 10−5 M, the colorimetric method was suggested to use.
To study the efficiency of cyanide distillation method,
standard potassium cyanide solutions were distilled for 1 h to
recover any cyanide with ISE. The cyanide recovery percent-
ages were from 85 to 111% that hardly satisfy. It was found
that the distillation process is the source of the biggest errors
in the analysis. An acceptable cyanide recovery is suggested
to be between 90 and 110%, i.e. with less errors. Particular
causes for the errors lie in the vigorous distillation and the
flow rate of the vapors through the sodium hydroxide (ab-
sorbing) solution. In addition, using ISE to detect cyanide
was insufficiently sensitive. With the ISE one could not mea-
sure cyanide concentration below 1 × 10−5 M, while much
lower cyanide concentration in seawater was enough to suf-
focate a 500 g marine food fish (Mak, 2003). Therefore, the
cyanide reflux distillation method together with an ISE was
not sensitive enough for the determination of cyanide traces
in post cyanide exposed fish.
In the subsequent set of experiments, individual Rus-
sell’s Snappers (≈500 g each) were poisoned separately by
spiking concentrated potassium cyanide solution into sea-
water until the fish was obviously immobilized and no gill
motion was observed. The final cyanide concentration in
seawater was calculated based on the volume of the stock
solution added and the final seawater volume in the con-
tainer. Then the cyanide concentrations in the homogenate
of fish tissues and seawater were determined after distilla-
tions. However, since very low concentration (in average
6.9 × 10−8 M) of cyanide in seawater was toxic enough to kill
a fish, cyanide was hardly determined with the colorimetric
method because the concentrations were very closed to the
detection limit. On the other hand, cyanide was not detectable
from distillate of fish homogenate. These results indicated
the amount of cyanide necessary to kill fish is well below
the amounts that can reliably determined using the studied
procedures.
The detection of cyanide, cyanide compounds, or cyanide
metabolites in fish tissues is quite difficult. Cyanide, once
incorporated by the fish is rapidly converted into thiocyanate
(SCN− ) and than excreted. The half-life of the cyanide is
extremely short (see the following section), so that detection
in the fish tissues will be dependent not only on exposure but
the length of time that it has taken to get the fish from the site
of exposure (i.e. the reefs) to the testing laboratory. It may be
that detection of total cyanide in fish would occur only under
“optimal conditions”; that is, extreme exposure and then rapid
transport to the laboratory. That is, the cyanide distillation
method combines with ISE described in the SOP manual
and employed in the IMA laboratories requires considerable
modification and elaboration. A reliable and ultra sensitive
system for cyanide detection in fish is urgently needed.
6. Novel biotest and biosensor for cyanide detection
in marine fish
6.1. Microbial cyanide degrading enzymes
Despite its toxicity, a wide range of microorganisms are
able to either produce (cyanogenesis), or degrade cyanide.
The microbes degrade cyanide either to detoxify it, or utilize
it as a source of nitrogen for growth. Various aspects of mi-
crobial cyanide metabolism have been reviewed previously
(Knowles and Bunch, 1986; Barclay and Knowles, 2001).
Cyanogenesis in microorganisms typically happens at the
end of the growth phase. Cyanide is probably the most com-
mon as well as the simplest secondary metabolite (Knowles,
1988). Cyanogenesis is also widespread amongst plants, but
 K.K.W. Mak et al. / Biosensors and Bioelectronics 20 (2005) 2581–2593 2589

Table 3
Cyanide hydratase properties from different fungi and a bacterium
Species pH optimum Native molecular Subunit molecular Km (mmol L−1 ) References
mass (kDa) mass (kDa)
Fusarium solani 7.5 >300 45 4.7 Barclay et al., 1998
Fusarium lateritium 8.5 >300 43 42 Cluness et al., 1993
Gloeocerospora sorghi 7–8 >300 45 12 Wang and Van Etten (1992)
Fusarium solani IHEM8026 7–8 – – – Dumestre et al. (1997)
Fusarium oxysporum – – – – Pereira et al. (1996)
Stemplylium loti 7–9 600 – – Fry and Millar (1972)
Pseudomonas flurescens NCIMB 11764 – – – 12 Kunz et al. (1994)
Leptosphaeria maculans – – – – Sexton and Howlett (2000)
–: not determined.

the process is completely different from that in microorgan-
isms (Mansfield, 1983).
In response to the defense mechanism of cyanogenic
plants, a number of phytopathogenic fungi have evolved the
ability to convert cyanide enzymatically to the less toxic for-
mamide by the action of cyanide hydratase (E.C. 4.2.1.66;
CyHT) (Fry and Myers, 1981):
(5)
This enzyme was first isolated and characterized from
Stemphylium loti, a pathogenic fungus of the cyanogenic
plant birdsfoot trefoil (Lotus corniculatus) by Fry and Millar
(1972). Since that the enzyme has been identified in a number
of other fungi including strains of Fusarium solani, Fusar-
ium oxysporum CCMI 876, Leptosphaeria maculans, etc.
(Table 3). In addition to the detoxification of cyanide, some
fungi would degrade formamide to enable growth via the
conversion to formate and ammonia by formamidase (E.C.
3.5.1.49) (Barclay et al., 1998; Pereira et al., 1996):
zyme, cyanidase (E.C. 3.5.5.X; cyanide hydrolase) has also
been announced its commercial potential, which decomposes
cyanide to ammonia and formate in an apparently one-step
reaction (Basheer et al., 1992):
HCN + 2H2 O → HCOOH + NH3 (7)
However, both commercialized enzymes were inactivated
by, or unable to act upon, metal-cyanide complexes, which
are prevalent in many industrial wastes.
Recently, F. oxysporum N-10 isolated by Yanase’s group
(Yanase et al., 2000) has the ability to utilize a metal-cyano
compound, tetracyanonickelate (II) {K2 [Ni(CN)4 ]; TCN}
as its sole source of nitrogen for growth. This isolate was
obtained under a neutral condition (pH 7.2) from cyanide-
contaminated soil and waste sludge.
The enzyme is a critical component in the bio-detection
system for cyanide traces in fish samples because most of
the cyanide in living organisms is metal-bound. In this study,
an optical biotest have been developed for determination of
cyanide in fish tissues based on the incorporation of the fungal
enzyme extract and formate dehydrogenase.

(6) 6.2. Optical biotest for determination of cyanide in

marine fish
Ammonia is presumably utilized as a nitrogen source for
growth while formate is converted possibly by formate de- The enzyme extract of F. oxysporum contained two en-
hydrogenase (E.C. 1.2.1.2) into carbon dioxide (Fig. 4). zymes to make the cyanide detection possible in the biotest;
It was shown that cyanide hydratase has a commercial cyanide hydratase catalyzes the hydrolysis of metal-bound
potential for detoxification of industrial wastes. Dried fun- cyanide to formamide, which is then converted into formate
gal mycelia with high levels of cyanide hydratase activity and ammonia by formamidase:
were on the market (Royse, 1987). Another microbial en-
Cyanide hydratase
M CN + H2 O + H+ −→ HCONH2 + M+ (8)
Formamidase
HCONH2 + H2 O −→ HCOOH + NH3 (9)

The concentration of produced formate in the couple reac-

Fig. 4. Summary of the pathway for the utilization of cyanide by Fusarium
solani (Barclay et al., 1998; Basheer et al., 1992).
tions is proportional to the initial concentration of the bound
cyanide in fish tissues. The quantitative formate determina-
tion was then achieved by addition of appropriate amount of
NAD+ and the subsequent formate dehydrogenase into the
sample mixture. The rate of NADH production was mon-
itored spectrophotometrically at 340 nm (Mak et al., 2004,
2590 K.K.W. Mak et al. / Biosensors and Bioelectronics 20 (2005) 2581–2593

in press):
Formate dehydrogenase
HCOOH + NAD+ −→ CO2 + NADH (10)

A common Hong Kong seawater food fish, Russell’s Snap-

per (Lutjamus russellii), was obtained from fish farm. After
being exposed to different levels of cyanide, the fish were
sacrificed to collect the internal organs including heart, stom-
ach, liver, spleen, intestines, etc. The fish tissues were then
homogenized with 5 mM sodium hydroxide solution. Large
particulates were filtered out with 0.45 ␮m nylon membranes,
while the filtrate was designated fish sample for biotests.
The optimal conditions of temperature, buffer pH,
amounts of NAD+ and enzymes for the biotest were investi-
gated. In potassium phosphate buffer solution, the fish sample
was incubated with the fungal enzyme extract at 30 ◦ C for 2 h.
Then NAD+ and formate dehydrogenase were spiked directly
into the sample mixture. Therefore, the pH value should be
the optimum for all enzymes. Formate dehydrogenase was
very stable in a broad pH range, so the optimum buffer pH
value was determined to be pH 8.2 that is the optimum value
of cyanide hydratase.
Fish were exposed to sub-lethal dosage of (10 ␮M) potas-
sium cyanide in seawater for 15 min and than transferred to
clean seawater (i.e. without cyanide) for recovery. After dif-
ferent time of recovery (0–180 min), one fish was sacrificed
to collect fish tissues which were then examined with the op-
timized biotest. The toxicity test has shown an accumulation
of cyanide at the beginning. Then the decrease of cyanide in
fish samples due to detoxification was observed. The cyanide
levels determined by the biotest in different recovery time
matched the fish activities in clean seawater during the course
of the recovery. Before the fish were transferred into the clean
seawater, most of them lost their balance, had only little gill
movements, and were immobilized. After several minutes of
recovery in the clean seawater some fish started to be awaked.
Then most of the fish became active again after 1 h.
Due to the ability to degrade metal-cyanide compounds
of cyanide hydratase, this biotest has a potential for use in
both biological and industrial samples. This system can be
improved by using further purified fungal enzymes.
6.3. Biosensor system based on cyanidase reaction
Cyanidase (cyanide hydrolase) was extracted by Tokuda et
al. (2003) from a cyanide-assimilating bacterium, Klebsiella
sp., which catalyzes the hydrolysis of free cyanide (HCN or
CN− ) to form formate and ammonia in one step. The en-
zyme was applied as a biocatalyst in the biosensor system
for low-level cyanide detection (Mak et al., 2004, in press).
In the biosensing system cyanidase converts cyanide into
formate and ammonia. Potassium cyanide (initial concentra-
tion = 1 mM) was completely removed by cyanidase in 1 h.
Then the produced formate in the cyanide degradation was
detected with a recently developed bi-enzyme electrode for
formate measurement (Fig. 5), in which formate dehydroge-
Fig. 5. Schematic illustration of cyanide biosensor system. Cyanide hydro-
lase (cyanidase) converts free cyanide into formate, which is determined by
the bi-enzyme electrode. The formate concentration, and hence the cyanide
concentration in sample, is monitored with the Clark-electrode.
nase was co-immobilized with salicylate hydroxylase (SHL,
E.C. 1.14.13.1) on a Clark-electrode (Mak et al., 2003). The
principle of the formate sensor is that formate dehydroge-
nase converts formate into carbon dioxide using NAD+ . The
corresponding NADH produced is then oxidized to NAD+
by SHL using salicylate and dissolved oxygen. The oxygen
consumption is monitored with the Clark-electrode.
The optimum buffer pH and temperature for the enzy-
matic hydrolysis of potassium cyanide were studied. The pre-
liminary experiments including the pretreatment of cyanide
with cyanide hydrolase and then detection by the formate
sensor gave a detection limit at 7.3 ␮mol L−1 cyanide. The
linear range of the calibration curve was between 30 and
300 ␮mol L−1 cyanide.
6.4. Advantages of the novel biotest for cyanide
detection
With the incorporation of cyanide degrading enzymes to
the formate biosensor, both free cyanide and metal-cyanide
complexes can be quantitatively determined. This is a novel
biochemical method for the detection of cyanide. Compare
to those conventional chemical methods the biosensor sys-
tem determines cyanide by converting the toxic chemical into
non-toxic product—formate for analyses.
Secondly, the formate produced in the cyanide degrada-
tion was detected by co-immobilized formate dehydroge-
nase (FDH, E.C. 1.2.1.2), salicylate hydroxylase (SHL, E.C.
1.14.13.1) in a poly(vinyl alcohol) (PVA) matrix in front of
a Clark oxygen electrode. The advantages of this biosensor
approach are the effective oxidation and hence the regener-
ation of NADH, short response time of 1 min. However, the
working stability of the electrode was limited to 7 days due
to the inactivation of the enzymes.
In the above two systems no harmful reagents was needed.
Moreover, those bound cyanide in the fish sample was con-
verted and determined by enzymatic reactions, while most
conventional methods often require vigorous reflux distilla-
tion, which is the source of errors.
 K.K.W. Mak et al. / Biosensors and Bioelectronics 20 (2005) 2581–2593
7. Summary and conclusion
In this thesis research, bioanalytical systems for free
cyanide and metal-cyano complexes were developed. This
approach has the following advantages: (1) the electrochem-
ical signal is proportional to the cyanide concentration, (2) the
toxic cyanide is converted by cyanidase (cyanide hydratase
for metal-cyano complexes) into non-toxic products, (3) af-
ter the degradation of cyanide, the produced formate can be
detected either spectrophotometrically or by a biosensor, (4)
the formate determination is based on NAD+ regeneration in
the biosensor, which is economical to produce, (5) cyanide in
biological matrices can be degraded by enzymes and easily
detected without chemical pretreatment, i.e., no distillation
procedure is needed. Finally, (6) the biotests also reduce the
hazards to one’s health because no harmful chemical is used
during the analyses.
8. Future perspectives
Cyanide fishing is illegal in virtually all Indo-Pacific coun-
tries, yet a large number of fishermen are still using cyanide to
catch fish. The eradication of this unsustainable fishing prac-
tice requires: (1) effective laws and policies to be established,
(2) the improvement of enforcement efforts, (3) the enhance-
ment of public awareness, and (4) offering fishermen prof-
itable alternatives. A highly sophisticated biosensor for ac-
curate detection of cyanide can help very little in these areas.
It is necessary to organize international agreements and poli-
cies. In exporting countries, fish should be tested for traces of
cyanide before exported. Firmer legal framework is needed to
make bans more effective. The governments should regulate
the import, distribution, and use of the cyanide. One should
increase public awareness and teach fishermen cyanide-free
alternatives for fishing and make alternatives more profitable.
In importing countries, governments shall monitor imports of
live fish and share information with exporting countries. If
all imports have to be certified as being cyanide-free and fi-
nancial assistance to live fish exporting countries is provided,
this would help combat cyanide fishing best (Barber and Pratt,
1998a).
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