Cyanide Testing
- Thread starter PeterIMA
- Start date
The first thing I would like to discuss is a report prepared by the Marine Aquarium Council that I believe was submitted to the World Bank Market Transformation Initiative in 1999. The title of the report is "Peer Review of Cyanide Detection Testing Methods Employed In The Philippines" Holthus (1999) .
This was supposed to be a confidential report. However, after a decade it is still relevant, since it forms the basis of defamatory claims made by the MAC about IMA's Standard Operating Procedure (SOP) for CDT. I feel compelled to discuss it and IMA's response to correct false statements still being made by MAC about IMA's CDT.
Peter Rubec, Ph.D.
This was supposed to be a confidential report. However, after a decade it is still relevant, since it forms the basis of defamatory claims made by the MAC about IMA's Standard Operating Procedure (SOP) for CDT. I feel compelled to discuss it and IMA's response to correct false statements still being made by MAC about IMA's CDT.
Peter Rubec, Ph.D.
The following are exerpts from IMA's response to the MAC report cited in the previous posting submitted to MAC on November 28, 1999.
Response to the Review of the IMA Cyanide Testing Standard Operating Procedure Prepared By The Marine Aquarium Council
Evaluation on CDT SOP
Rising concern of the Philippine Bureau of Fisheries and Aquatic Resources (BFAR) and the International Marinelife Alliance (IMA) about the detrimental effects of the widespread use of sodium cyanide for collection of live reef fishes, led to the creation of a cyanide detection test (CDT) laboratory at the BFAR headquarter in Quezon City in 1992. Additional CDT laboratories were created in Puerto Princesa (Island of Palawan) in 1993, near the Manila airport in 1994, in Zamboanga City (Island of Mindanao) 1995, Palo, Leyte (Island of Leyte) in 1996 Davao (Island of Mindanao) and Cebu City (Island of Cebu) in 1997. IMA also operates four monitoring, inspection and sampling (MIS) stations located in Coron (Northern Palawan), Subic, Zambales (Island of Luzon), Virac (Island of Catanduanes) and Batangas (Island of Luzon) staffed by MIS Officers who collect samples from fishing boats and forward the samples to the Central CDT laboratory. The first laboratory located at the BFAR office was closed after the new one opened at the airport. IMA still maintains and operates the remaining six CDT laboratories and four MIS stations under contract with BFAR.
Generally, a distillation apparatus in combination with the addition of certain chemicals are needed to remove interfering substances prior to cyanide testing. The IMA solved these problems by adopting the Standard Operating Procedure (SOP) published in the 1996 Annual Book Of Standards Vol. 11.02, (D2036-91) by the American Society of Testing and Materials (ASTM 1996) and Standard Methods For The Examination of Water and Wastewater, 19th Edition, published by the American Public Health Association (APHA), American Water Works Association (AWWA), and the Water Pollution Control Federation (WPCF) published in 1991. It is somewhat surprising that the review document (Holthus 1999) of the SOP (Manipula et al. 1995) for BFAR/IMA CDT laboratories by the Marine Aquarium Council (MAC) does not emphasize the fact that the IMA testing methods are based on ASTM procedures in general use in the United States and other countries (ASTM 1996).
There is overwhelming evidence that cyanide is toxic to most life forms including fish, wildlife, and invertebrates (Duodoroff 1976, 1980; Leduc et al. 1982, Leduc 1984, Eisler 1991, Hanawa et al. 1998) as well as corals (Jones 1997, Jones and Steven 1997, Jones and Hoegh-Guldberg 1998, Jones et al. In Press). The detrimental effects of cyanide use in marine fish and coral reefs are very apparent to small-scale fishermen. This was reported by IMA in numerous articles in aquarium magazines and scientific publications (Rubec and Pratt 1984; Robinson 1985a,b; Rubec 1986, 1987, 1988; Pratt 1996; Barber and Pratt 1997, 1998). The CDT laboratories over the past four years have tested over 25,000 fish and invertebrates sampled from local markets, seized by law enforcement officials, sampled from exporters' facilities, and intercepted on boats or at airports intended either for export to the world aquarium trade or for sale for human consumption in Chinese restaurants. The laboratories have confirmed the widespread presence of cyanide in the tissues of the specimens tested.
The cyanide problem is an eye-opener to the private sector, government and live fish trades. It has widespread implications concerning the sustainability of coral reefs that provide fish protein in the Philippines and other SE Asian countries (Johannes and Riepen 1995; Pratt 1996; Barber and Pratt 1997, 1998). To deal with the problem, IMA-Philippines developed the Cyanide Fishing Reform Program (CFRP). This involves cyanide testing and village-based education programs to provide alternatives to destructive fishing methods. IMA has recently obtained funding from various sources to expand its Destructive Fishing Reform Initiative (DFRI) to other countries throughout SE Asia and into the South Pacific. Hence, the CDT is only one component of a much larger program to provide environmentally safe alternative livelihoods, while protecting the long-term sustainability of coral reefs and other marine resources for the benefit of local people.
The BFAR/IMA's CDT SOP (Manipula et al. 1995) was developed to screen-harvested aquarium and food fishes for the presence of cyanide. Certified professional chemists, laboratory technicians, fish biologists, and fishery officers staff the six test laboratories. The timely reporting of test results to the Philippine government by IMA led to the expansion of funding for the laboratories through Fisheries Resource Management Program (FRMP) funded by the Asian Development Bank. It is somewhat surprising that during the period that the MAC reviewed the CDT SOP, no members of the council or any of the scientists listed as reviewers visited any of the CDT laboratories to directly observe the equipment and testing procedures. Some of the scientists listed in the report are associated with local Philippine universities. It is surprising that the MAC can find funds to send people to conferences (including one in Manila last year) but did not pay for any scientific experts to visit the BFAR/IMA laboratories.
Response to the Review of the IMA Cyanide Testing Standard Operating Procedure Prepared By The Marine Aquarium Council
Evaluation on CDT SOP
Rising concern of the Philippine Bureau of Fisheries and Aquatic Resources (BFAR) and the International Marinelife Alliance (IMA) about the detrimental effects of the widespread use of sodium cyanide for collection of live reef fishes, led to the creation of a cyanide detection test (CDT) laboratory at the BFAR headquarter in Quezon City in 1992. Additional CDT laboratories were created in Puerto Princesa (Island of Palawan) in 1993, near the Manila airport in 1994, in Zamboanga City (Island of Mindanao) 1995, Palo, Leyte (Island of Leyte) in 1996 Davao (Island of Mindanao) and Cebu City (Island of Cebu) in 1997. IMA also operates four monitoring, inspection and sampling (MIS) stations located in Coron (Northern Palawan), Subic, Zambales (Island of Luzon), Virac (Island of Catanduanes) and Batangas (Island of Luzon) staffed by MIS Officers who collect samples from fishing boats and forward the samples to the Central CDT laboratory. The first laboratory located at the BFAR office was closed after the new one opened at the airport. IMA still maintains and operates the remaining six CDT laboratories and four MIS stations under contract with BFAR.
Generally, a distillation apparatus in combination with the addition of certain chemicals are needed to remove interfering substances prior to cyanide testing. The IMA solved these problems by adopting the Standard Operating Procedure (SOP) published in the 1996 Annual Book Of Standards Vol. 11.02, (D2036-91) by the American Society of Testing and Materials (ASTM 1996) and Standard Methods For The Examination of Water and Wastewater, 19th Edition, published by the American Public Health Association (APHA), American Water Works Association (AWWA), and the Water Pollution Control Federation (WPCF) published in 1991. It is somewhat surprising that the review document (Holthus 1999) of the SOP (Manipula et al. 1995) for BFAR/IMA CDT laboratories by the Marine Aquarium Council (MAC) does not emphasize the fact that the IMA testing methods are based on ASTM procedures in general use in the United States and other countries (ASTM 1996).
There is overwhelming evidence that cyanide is toxic to most life forms including fish, wildlife, and invertebrates (Duodoroff 1976, 1980; Leduc et al. 1982, Leduc 1984, Eisler 1991, Hanawa et al. 1998) as well as corals (Jones 1997, Jones and Steven 1997, Jones and Hoegh-Guldberg 1998, Jones et al. In Press). The detrimental effects of cyanide use in marine fish and coral reefs are very apparent to small-scale fishermen. This was reported by IMA in numerous articles in aquarium magazines and scientific publications (Rubec and Pratt 1984; Robinson 1985a,b; Rubec 1986, 1987, 1988; Pratt 1996; Barber and Pratt 1997, 1998). The CDT laboratories over the past four years have tested over 25,000 fish and invertebrates sampled from local markets, seized by law enforcement officials, sampled from exporters' facilities, and intercepted on boats or at airports intended either for export to the world aquarium trade or for sale for human consumption in Chinese restaurants. The laboratories have confirmed the widespread presence of cyanide in the tissues of the specimens tested.
The cyanide problem is an eye-opener to the private sector, government and live fish trades. It has widespread implications concerning the sustainability of coral reefs that provide fish protein in the Philippines and other SE Asian countries (Johannes and Riepen 1995; Pratt 1996; Barber and Pratt 1997, 1998). To deal with the problem, IMA-Philippines developed the Cyanide Fishing Reform Program (CFRP). This involves cyanide testing and village-based education programs to provide alternatives to destructive fishing methods. IMA has recently obtained funding from various sources to expand its Destructive Fishing Reform Initiative (DFRI) to other countries throughout SE Asia and into the South Pacific. Hence, the CDT is only one component of a much larger program to provide environmentally safe alternative livelihoods, while protecting the long-term sustainability of coral reefs and other marine resources for the benefit of local people.
The BFAR/IMA's CDT SOP (Manipula et al. 1995) was developed to screen-harvested aquarium and food fishes for the presence of cyanide. Certified professional chemists, laboratory technicians, fish biologists, and fishery officers staff the six test laboratories. The timely reporting of test results to the Philippine government by IMA led to the expansion of funding for the laboratories through Fisheries Resource Management Program (FRMP) funded by the Asian Development Bank. It is somewhat surprising that during the period that the MAC reviewed the CDT SOP, no members of the council or any of the scientists listed as reviewers visited any of the CDT laboratories to directly observe the equipment and testing procedures. Some of the scientists listed in the report are associated with local Philippine universities. It is surprising that the MAC can find funds to send people to conferences (including one in Manila last year) but did not pay for any scientific experts to visit the BFAR/IMA laboratories.
It is encouraging that the MAC concluded that the SOP was "reliable and effective in detecting cyanide in those fishes that have been subjected to the chemical in the capture" (MAC Report, page 18). Independent laboratory tests by an unspecified MAC consultant confirmed that the method used is in fact reliable and effective in detecting cyanide in fish that have been subjected to the chemical in the capture process (MAC Report, Sec. 4.1, page 18). Tests using spiked homogenized fish samples were in the range of the recovery of the Laboratory Controlled Samples (LCS). Comparative tests done using colorimetric methods and an Ion Selective Electrode (ISE) had recoveries ranging from 51%-60% and 36% -71% as shown in Tables 2 & 3 (pages 14 &15), respectively. Both methods indicated no cyanide present with blank samples. This is indicates that the BFAR/IMA ISE test is in fact effective in determining the presence of cyanide in the tissues of fish that
been exposed to cyanide.
The evaluation done by the independent laboratory on the Standard Operating Procedures (SOP) is a great help for IMA. The review panel reviewed the appropriateness of the test adopted, suggested some things that need to be added to the SOP manual, offered suggestions for the improvement and detailed discussion of the step-by-step practices undertaken at the laboratory, and provided comments concerning means to validate the test. However, the draft document raised questions, which need to be answered-and conclusions, which appear to be incorrect.
There were criticisms that the present CDT SOP does not clearly explain all of its methods of calibration and the testing of standard cyanide concentrations. This indicates that the SOP needs some modification to elaborate practices actually being performed, which were not clearly stated in the manual. Hence, it is necessary for us to reply to these comments and correct some misleading statements in the MAC report.
Section 1. Scope of the Analysis
The analysis herewith described covers the determination of cyanide on fish tissues (and water samples). The test involves the liberation of hydrogen cyanide (HCN) from acidified samples during reflux-distillation. The hydrogen cyanide (HCN) vapor liberated by digesting fish tissues with sulfuric acid is then collected in an absorber tube containing sodium hydroxide solution. The concentration of cyanide ion (CN-) is determined using an ISE electrode attached to a digital pH/ISE meter. Feedback from the expert group generally commented that the SOP looks good and is technically satisfactory. This independent confirmation by the independent laboratory test further validates the reliability of the test for detecting cyanide in fish tissues.
Section 2. Concerns on the Comments Made by the Peer Review
2.1 Appropriateness of the Method
The IMA chemistry department disagrees with the following unsubstantiated MAC assertions "Cyanide, once incorporated into the fish is rapidly converted to thiocyanate and excreted. The half-life of cyanide is extremely short, so that the detection in fish tissues will be dependent not only on the exposure but the length of time that it has taken to get the fish from the site of exposure (i.e. the reef) to the laboratory. It may be that the detection of total cyanide in fish would occur only under "optimal conditions"; i.e., extreme exposure and then rapid transport to the laboratory (Holthus 1999, Section 2.3, p. 4).
The dissociation of cyanide in water is pH dependent with most being in the form of HCN below pH 8.5 (Leduc 1984). The HCN is rapidly absorbed across membranes, such as the gills and the stomach of fish into the blood stream. The HCN in the blood is converted to thiocyanate ion (SCN-) by an enzyme called rhodanese mainly situated in the liver. The SCN- is then excreted from the fish through urine.
The concentration of cyanide present in the living fish 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, post-collection treatment (changes of water etc.). The conversion rate of HCN to SCN-appears to be limited on the availability of sulfur (Leduc 1984). Hence, both HCN and SCN- occur in the blood. Thiocyanate levels increased gradually over a 20-day period (Raymond et al. 1986, Brown et al. 1995). Raymond et al. (1986) found that there was 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. The excretion of SCN- was found to take several weeks in rainbow trout (Brown, et. al. 1995).
Marine fishes internally conserve freshwater to osmoregulate (Smith 1982). Hence, the urinary excretion rate in marine fish is slower and cyanide is retained in the fish for a longer period. While more research is needed, concerning uptake and release rates of cyanide by marine fish; the IMA believes that the experiment by the MAC consultant, involving dosing five test fish with about 2 mg/L of sodium cyanide (NaCN) for an unspecified period, is invalid in predicting the concentration in the fish or how long it would be retained under the real world situation.
IMA can detect cyanide ion (CN-) obtained from acid digested fish tissues as HCN several weeks after the live fish were collected and transported to Manila. The IMA CDT testing laboratories routinely detect cyanide ion concentrations in fishes killed and tested two to three weeks after they were collected.
Experiments by Dempster and Donaldson (1974), Hall and Bellwood (1996) and others (review Rubec 1987) indicate that most marine fishes are immobilized with cyanide concentrations of 1-5 mg/L in less than 90 seconds. Some fish species (such as gobies) can survive concentrations as high as 50 mg/L for up to several minutes (Dempster and Donaldson 1974). A review by Ireland and Robertson (1974) similarly concluded that a concentration of 5 mg/L of sodium cyanide was effective in immobilizing marine fishes. One might assume from this literature, that the range of cyanide concentrations used by aquarium fish collectors are in the range described above. However, this assumption is not correct.
IMA is able to measure cyanide in fish after two weeks because the initial concentration used by the collectors is much higher than that used by the independent consultant. One 20 gram tablet of sodium cyanide dissolved in a one-liter squirt bottle would be about 20,000 mg/L (ppm) NaCN. Many aquarium fish collectors use 1-2 tablets in the squirt bottle. Collectors gathering live groupers for the food-fish trade commonly use 3-5 cyanide tablets (J. Pet and F. Cruz, personal communications 1999).
Not all the cyanide dissolves initially. Both dissolved HCN and particulate NaCN is squirted on the coral heads as a whitish plume. Testing done by the Nature Conservancy indicates that the dissolved HCN concentration leaving the squirt bottle is about 1,500-2,000 mg/L (Pet and Djohani 1999). Fishes in the cyanide plume die quickly (acute toxicity). Fishes on the side of the plume may survive for a longer period, if rapidly moved to clean seawater. Hence, it appears that the concentrations being used by the collectors are far higher than what has been found to immobilize fish under laboratory conditions.
Attached below are some specific comments concerning the appropriateness of various cyanide detection methods such as the ISE, colorimetric, and ion chromatography.
been exposed to cyanide.
The evaluation done by the independent laboratory on the Standard Operating Procedures (SOP) is a great help for IMA. The review panel reviewed the appropriateness of the test adopted, suggested some things that need to be added to the SOP manual, offered suggestions for the improvement and detailed discussion of the step-by-step practices undertaken at the laboratory, and provided comments concerning means to validate the test. However, the draft document raised questions, which need to be answered-and conclusions, which appear to be incorrect.
There were criticisms that the present CDT SOP does not clearly explain all of its methods of calibration and the testing of standard cyanide concentrations. This indicates that the SOP needs some modification to elaborate practices actually being performed, which were not clearly stated in the manual. Hence, it is necessary for us to reply to these comments and correct some misleading statements in the MAC report.
Section 1. Scope of the Analysis
The analysis herewith described covers the determination of cyanide on fish tissues (and water samples). The test involves the liberation of hydrogen cyanide (HCN) from acidified samples during reflux-distillation. The hydrogen cyanide (HCN) vapor liberated by digesting fish tissues with sulfuric acid is then collected in an absorber tube containing sodium hydroxide solution. The concentration of cyanide ion (CN-) is determined using an ISE electrode attached to a digital pH/ISE meter. Feedback from the expert group generally commented that the SOP looks good and is technically satisfactory. This independent confirmation by the independent laboratory test further validates the reliability of the test for detecting cyanide in fish tissues.
Section 2. Concerns on the Comments Made by the Peer Review
2.1 Appropriateness of the Method
The IMA chemistry department disagrees with the following unsubstantiated MAC assertions "Cyanide, once incorporated into the fish is rapidly converted to thiocyanate and excreted. The half-life of cyanide is extremely short, so that the detection in fish tissues will be dependent not only on the exposure but the length of time that it has taken to get the fish from the site of exposure (i.e. the reef) to the laboratory. It may be that the detection of total cyanide in fish would occur only under "optimal conditions"; i.e., extreme exposure and then rapid transport to the laboratory (Holthus 1999, Section 2.3, p. 4).
The dissociation of cyanide in water is pH dependent with most being in the form of HCN below pH 8.5 (Leduc 1984). The HCN is rapidly absorbed across membranes, such as the gills and the stomach of fish into the blood stream. The HCN in the blood is converted to thiocyanate ion (SCN-) by an enzyme called rhodanese mainly situated in the liver. The SCN- is then excreted from the fish through urine.
The concentration of cyanide present in the living fish 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, post-collection treatment (changes of water etc.). The conversion rate of HCN to SCN-appears to be limited on the availability of sulfur (Leduc 1984). Hence, both HCN and SCN- occur in the blood. Thiocyanate levels increased gradually over a 20-day period (Raymond et al. 1986, Brown et al. 1995). Raymond et al. (1986) found that there was 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. The excretion of SCN- was found to take several weeks in rainbow trout (Brown, et. al. 1995).
Marine fishes internally conserve freshwater to osmoregulate (Smith 1982). Hence, the urinary excretion rate in marine fish is slower and cyanide is retained in the fish for a longer period. While more research is needed, concerning uptake and release rates of cyanide by marine fish; the IMA believes that the experiment by the MAC consultant, involving dosing five test fish with about 2 mg/L of sodium cyanide (NaCN) for an unspecified period, is invalid in predicting the concentration in the fish or how long it would be retained under the real world situation.
IMA can detect cyanide ion (CN-) obtained from acid digested fish tissues as HCN several weeks after the live fish were collected and transported to Manila. The IMA CDT testing laboratories routinely detect cyanide ion concentrations in fishes killed and tested two to three weeks after they were collected.
Experiments by Dempster and Donaldson (1974), Hall and Bellwood (1996) and others (review Rubec 1987) indicate that most marine fishes are immobilized with cyanide concentrations of 1-5 mg/L in less than 90 seconds. Some fish species (such as gobies) can survive concentrations as high as 50 mg/L for up to several minutes (Dempster and Donaldson 1974). A review by Ireland and Robertson (1974) similarly concluded that a concentration of 5 mg/L of sodium cyanide was effective in immobilizing marine fishes. One might assume from this literature, that the range of cyanide concentrations used by aquarium fish collectors are in the range described above. However, this assumption is not correct.
IMA is able to measure cyanide in fish after two weeks because the initial concentration used by the collectors is much higher than that used by the independent consultant. One 20 gram tablet of sodium cyanide dissolved in a one-liter squirt bottle would be about 20,000 mg/L (ppm) NaCN. Many aquarium fish collectors use 1-2 tablets in the squirt bottle. Collectors gathering live groupers for the food-fish trade commonly use 3-5 cyanide tablets (J. Pet and F. Cruz, personal communications 1999).
Not all the cyanide dissolves initially. Both dissolved HCN and particulate NaCN is squirted on the coral heads as a whitish plume. Testing done by the Nature Conservancy indicates that the dissolved HCN concentration leaving the squirt bottle is about 1,500-2,000 mg/L (Pet and Djohani 1999). Fishes in the cyanide plume die quickly (acute toxicity). Fishes on the side of the plume may survive for a longer period, if rapidly moved to clean seawater. Hence, it appears that the concentrations being used by the collectors are far higher than what has been found to immobilize fish under laboratory conditions.
Attached below are some specific comments concerning the appropriateness of various cyanide detection methods such as the ISE, colorimetric, and ion chromatography.
Question-How does this method compare with those colorimetric methods, ion chromatography, or other methods commercially available, i.e. why was this method chosen?
-The CDT Lab collects no fees for the test; operating expenses are funded with small grants. Hence, IMA believes it is more economical to use the ISE method than to adopt more expensive methods that serve the same purpose. There is no need to adopt more expensive methods, if the ISE method gives similar results, as indicated in the MAC report.
-The ISE method is more convenient and easy to maintain not compromising the reliability of the result. It does not require highly toxic/dangerous reagents like barbituric acid and pyridine.
-The Environment Management Bureau of the Philippine Department of Environment and Natural Resources (DENR) preferred the use of ISE for cyanide analysis.
Question-What is the sensitivity of the method, e.g., limits of detection for total tissue cyanide concentrations, precision of the analysis, range of concentrations at which the response of the electrode is linear; precision among replicates; accuracy of the method (% recovery in the distillation procedure)?
-The ISE gives reliable readings down to 0.01 mg/L. It is possible to obtain ISE readings to lower levels that may not be reliable. The apparatus is calibrated daily over a range of CN- concentrations from 0 to 10 mg/L. The calibration curve is linear over the range 0.01-10mg/L.
-The precision and accuracy found by IMA chemists are similar to that found by U.S.-EPA. Spiking of control samples was done to establish the baseline level for the declaration of positive results. The process is being repeated now using a certified reference material (CRM). The range of concentrations at which the electrode responds linearly is within the concentrations of the calibration standards. The slope of the calibration should range only from -57±4 mV.
Question-How does the detection limit/sensitivity relate to the biological damage caused by the cyanide?
-The CDT laboratories presently do not have histopathology equipment (the cost exceeds $25,000 for paraffin embedding and staining apparatus). IMA is now finalizing plans to collaborate with BFAR and use histology equipment owned by the Bureau.
Question-Does cyanide at any level indicates that it has been used to collect the fish being tested?
-Review of the scientific literature indicates that cyanide ion is not found in marine fish as a natural background. This is substantiated by the fact that marine aquarium fishes caught with nets exhibit zero cyanide concentrations when tested by the BFAR/IMA CDT laboratories. Hence, the presence of cyanide ion (at any concentration) in marine aquarium fish can be interpreted as resulting from the anthropogenic release of cyanide to the marine environment.
Question-The SOP must state the acceptance criterion, i.e., what levels of cyanide are acceptable and why?
-All cyanide ion levels within the calibration range are scientifically acceptable and could be used to support prosecution of collectors for violation of Philippine fishery laws (e.g., Presidential Decree 704). But, for benefit of the doubt and fairness to the collectors and fishermen a threshold level of 0.2 mg/kg (ppm) wet weight was adopted for legal purposes.
Question-Can cyanide be present in the fish from any other source, if it has not been used to collect the fish?
-The CDT test will reveal whatever cyanide is in the tissues of the fish.
-The test is not selective with respect to whether cyanide was intentionally used during collection or was already present in the area of collection (i.e. fishing in cyanide contaminated water). For example, a cyanide spill did occur from a vessel in Manila Bay several years ago. It is also possible that traces of cyanide excreted from cyanide-caught fish could be absorbed by net-caught fish in holding tanks (if the fish are kept together).
Question- Can the method detect the reaction products of cyanide and the protein of any electropholic substances in the fish?
-The ISE for cyanide ion does not measure other cyanide reaction products. The ISE test is not influenced by organic compounds such as proteins of electropholic substances. Organic compounds are broken down by sulfuric acid in the distillation flask. Cyanide is captured in the sodium hydroxide absorber tube after the reflux distillation.
Question- Are there results available from testing seawater contaminated with cyanide?
-Seawater samples have been analyzed by the CDT laboratories. There were zero readings from cyanide-free water and there were some very low levels of cyanide (close to 0) detected in seawater samples collected from fish holding tanks at a facility situated near field collection sites.
2.2 Quality Assurance/Quality Control
Quality Assurance/Quality Control measures are being done at the CDT laboratory, but were not laid out in the SOP manual. Of the seven items listed by the MAC group, all are being performed except the last one because we do not have access to an independent laboratory that can prepare known concentrations of cyanide derived from a fish-tissue matrix for comparative analyses. This will be done soon. The Department of Environment and Natural Resources (DENR) Environmental Management Bureau, IMA and other testing agencies plan to form a group for inter-laboratory cyanide comparisons and analyses.
Distillation and Analysis of Blanks Solutions
-The CDT laboratory prepares reagent blanks every week, i.e. the distillation using distilled water + the reagents used during the distillation of samples.
Use of Standard Reference Material relevant to the Test
-Besides the standard solutions prepared by the laboratory, we are already are using certified reference materials (matrix is water) to check for the accuracy of the analysis. However, there are no standard cyanide solutions prepared from a fish-tissue matrix available from an independent laboratory.
Use of Blank Fish Samples
-As a baseline or reference level, the CDT laboratory has prepared samples using specimens representative of a variety of marine fish species. The CDT laboratories routinely obtain uncontaminated fish samples from the IMA Net-Training teams, who collect fishes with barrier nets.
Use of Unknowns
-Though intermittently done, the CDT lab conducts parallel testing among the six CDT labs nationwide. This is also our way of counter-checking the test results among the CDT laboratories.
Use of Spikes
-The addition of known amounts of cyanide to homogenized fish tissues prior to distillation is regularly undertaken by the CDT Laboratory to: a) determine the percent recovery of the distillation process and b) to assess electrode response to cyanide recovered in the distillates. The percent recovery is generally greater than 90%.
Distillation Efficiency Check
-The IMA does not see the need to use potassium ferricyanide to check the efficiency of the distillation. It is not a primary standard. The ASTM method (D2036-98) describes the titration of a 25.0 ml aliquot of the 1000 mg/L NaCN solution. This will give the purity of the NaCN and relate it directly to a NIST Primary Standard. Low recoveries of CN- from distilling aliquots of this standard solution would indicate that the NaCN might have formed hydrates (Williams 1948, The Merck Index, 11th Edition) upon storage or HCN is lost during distillation. If hydrates are formed with NaCN, potassium cyanide (KCN) should be used for the distillation efficiency check.
2.3 Equipment, Water, Reagents
When the CDT SOP is revised, we will incorporate this portion in the manual. Information about the points stressed in the MAC Report (Holthus 1999) are available to the IMA/BFAR laboratories. The last point mentioned on the use of volumetric pipette does not literally mean that 0.10 ml volumetric pipette is used, but rather the measurements of the standard solution is made through a 1 ml graduated pipette.
-Calibration of the ISE meter is done every day, following the manufacturer?s instructions.
-An analytical balance is used for the preparation of standard solutions. For fish samples two types of balances are used; first, the total body weight is determined using triple beam balance; secondly, by using top-load balance for the fish tissues intended for distillation.
-It is not practical to use the analytical balance for the type of samples being handled.
-The CDT Lab collects no fees for the test; operating expenses are funded with small grants. Hence, IMA believes it is more economical to use the ISE method than to adopt more expensive methods that serve the same purpose. There is no need to adopt more expensive methods, if the ISE method gives similar results, as indicated in the MAC report.
-The ISE method is more convenient and easy to maintain not compromising the reliability of the result. It does not require highly toxic/dangerous reagents like barbituric acid and pyridine.
-The Environment Management Bureau of the Philippine Department of Environment and Natural Resources (DENR) preferred the use of ISE for cyanide analysis.
Question-What is the sensitivity of the method, e.g., limits of detection for total tissue cyanide concentrations, precision of the analysis, range of concentrations at which the response of the electrode is linear; precision among replicates; accuracy of the method (% recovery in the distillation procedure)?
-The ISE gives reliable readings down to 0.01 mg/L. It is possible to obtain ISE readings to lower levels that may not be reliable. The apparatus is calibrated daily over a range of CN- concentrations from 0 to 10 mg/L. The calibration curve is linear over the range 0.01-10mg/L.
-The precision and accuracy found by IMA chemists are similar to that found by U.S.-EPA. Spiking of control samples was done to establish the baseline level for the declaration of positive results. The process is being repeated now using a certified reference material (CRM). The range of concentrations at which the electrode responds linearly is within the concentrations of the calibration standards. The slope of the calibration should range only from -57±4 mV.
Question-How does the detection limit/sensitivity relate to the biological damage caused by the cyanide?
-The CDT laboratories presently do not have histopathology equipment (the cost exceeds $25,000 for paraffin embedding and staining apparatus). IMA is now finalizing plans to collaborate with BFAR and use histology equipment owned by the Bureau.
Question-Does cyanide at any level indicates that it has been used to collect the fish being tested?
-Review of the scientific literature indicates that cyanide ion is not found in marine fish as a natural background. This is substantiated by the fact that marine aquarium fishes caught with nets exhibit zero cyanide concentrations when tested by the BFAR/IMA CDT laboratories. Hence, the presence of cyanide ion (at any concentration) in marine aquarium fish can be interpreted as resulting from the anthropogenic release of cyanide to the marine environment.
Question-The SOP must state the acceptance criterion, i.e., what levels of cyanide are acceptable and why?
-All cyanide ion levels within the calibration range are scientifically acceptable and could be used to support prosecution of collectors for violation of Philippine fishery laws (e.g., Presidential Decree 704). But, for benefit of the doubt and fairness to the collectors and fishermen a threshold level of 0.2 mg/kg (ppm) wet weight was adopted for legal purposes.
Question-Can cyanide be present in the fish from any other source, if it has not been used to collect the fish?
-The CDT test will reveal whatever cyanide is in the tissues of the fish.
-The test is not selective with respect to whether cyanide was intentionally used during collection or was already present in the area of collection (i.e. fishing in cyanide contaminated water). For example, a cyanide spill did occur from a vessel in Manila Bay several years ago. It is also possible that traces of cyanide excreted from cyanide-caught fish could be absorbed by net-caught fish in holding tanks (if the fish are kept together).
Question- Can the method detect the reaction products of cyanide and the protein of any electropholic substances in the fish?
-The ISE for cyanide ion does not measure other cyanide reaction products. The ISE test is not influenced by organic compounds such as proteins of electropholic substances. Organic compounds are broken down by sulfuric acid in the distillation flask. Cyanide is captured in the sodium hydroxide absorber tube after the reflux distillation.
Question- Are there results available from testing seawater contaminated with cyanide?
-Seawater samples have been analyzed by the CDT laboratories. There were zero readings from cyanide-free water and there were some very low levels of cyanide (close to 0) detected in seawater samples collected from fish holding tanks at a facility situated near field collection sites.
2.2 Quality Assurance/Quality Control
Quality Assurance/Quality Control measures are being done at the CDT laboratory, but were not laid out in the SOP manual. Of the seven items listed by the MAC group, all are being performed except the last one because we do not have access to an independent laboratory that can prepare known concentrations of cyanide derived from a fish-tissue matrix for comparative analyses. This will be done soon. The Department of Environment and Natural Resources (DENR) Environmental Management Bureau, IMA and other testing agencies plan to form a group for inter-laboratory cyanide comparisons and analyses.
Distillation and Analysis of Blanks Solutions
-The CDT laboratory prepares reagent blanks every week, i.e. the distillation using distilled water + the reagents used during the distillation of samples.
Use of Standard Reference Material relevant to the Test
-Besides the standard solutions prepared by the laboratory, we are already are using certified reference materials (matrix is water) to check for the accuracy of the analysis. However, there are no standard cyanide solutions prepared from a fish-tissue matrix available from an independent laboratory.
Use of Blank Fish Samples
-As a baseline or reference level, the CDT laboratory has prepared samples using specimens representative of a variety of marine fish species. The CDT laboratories routinely obtain uncontaminated fish samples from the IMA Net-Training teams, who collect fishes with barrier nets.
Use of Unknowns
-Though intermittently done, the CDT lab conducts parallel testing among the six CDT labs nationwide. This is also our way of counter-checking the test results among the CDT laboratories.
Use of Spikes
-The addition of known amounts of cyanide to homogenized fish tissues prior to distillation is regularly undertaken by the CDT Laboratory to: a) determine the percent recovery of the distillation process and b) to assess electrode response to cyanide recovered in the distillates. The percent recovery is generally greater than 90%.
Distillation Efficiency Check
-The IMA does not see the need to use potassium ferricyanide to check the efficiency of the distillation. It is not a primary standard. The ASTM method (D2036-98) describes the titration of a 25.0 ml aliquot of the 1000 mg/L NaCN solution. This will give the purity of the NaCN and relate it directly to a NIST Primary Standard. Low recoveries of CN- from distilling aliquots of this standard solution would indicate that the NaCN might have formed hydrates (Williams 1948, The Merck Index, 11th Edition) upon storage or HCN is lost during distillation. If hydrates are formed with NaCN, potassium cyanide (KCN) should be used for the distillation efficiency check.
2.3 Equipment, Water, Reagents
When the CDT SOP is revised, we will incorporate this portion in the manual. Information about the points stressed in the MAC Report (Holthus 1999) are available to the IMA/BFAR laboratories. The last point mentioned on the use of volumetric pipette does not literally mean that 0.10 ml volumetric pipette is used, but rather the measurements of the standard solution is made through a 1 ml graduated pipette.
-Calibration of the ISE meter is done every day, following the manufacturer?s instructions.
-An analytical balance is used for the preparation of standard solutions. For fish samples two types of balances are used; first, the total body weight is determined using triple beam balance; secondly, by using top-load balance for the fish tissues intended for distillation.
-It is not practical to use the analytical balance for the type of samples being handled.
2.4 Sample Selection and Collection
The IMA Sampling Officers (we call them MIS Officers) were given orientation as to what species should be taken from the source. They also have guidelines on how many fish samples have to be taken, depending on whether they are aquarium or food fishes. Samples are collected on boats, fish buying stations, airports, piers and other transshipment points.
-Besides the receipt issued for the samples taken from the facility/owner, another information sheet is filled out. This latter form can be used to record the time of the samples were taken at the holding facility or fish cages. Information is also obtained concerning approximately when and where the fish were caught (in the case of food fish).
-As to the selection of what species to pick up, the MIS Officers are familiar with the species commonly captured for the aquarium or the food fish trades. For example, most species tested in Puerto Princesa are groupers or snappers, obtained from local fishermen/collectors from the nearby municipalities, species mostly coming from NE coast of the Island of Palawan (Coron & Calamian Islands) are groupers. Where possible, aquarium fishes are sampled at random from collectors and from exporters’ holding facilities. Some resistance has been encountered with exporters, who refuse to supply higher priced species such as angelfish for CDT analyses. Despite these difficulties, the samples are generally representative of what is present in shipments within the country or destined for export.
-Further research is needed with the CDT database to determine whether certain species are being targeted with cyanide, or whether smaller fish species exhibit greater sensitivity to cyanide. These questions are best answered from a data set based on random sampling.
-It does not appear that certain tropical fish species are more likely to be cyanide collected. The data indicate that almost all the species exported by the aquarium trade are collected with cyanide. While the frequency of fishes found to contain cyanide has declined over the past three years, no species-specific patterns are apparent from visual inspection of the data. From a sampling perspective, it does not make sense to target certain species for CDT testing without a prior knowledge of which ones are contaminated with cyanide.
-Ice packs in styrofoam coolers are used to transport fish specimens to the CDT laboratories.
-For the water samples, keeping it at 4°C is not always possible. Sampling is done sometimes in far-flung areas like islands, where there is no electricity or ice available. But, when there is ice or a refrigerator available, the water samples are subjected to lower temperature. We will try transporting water samples from field sites with “Blue-Ice” packs and/or by adding sodium hydroxide to the water samples, following the U.S.EPA methods discussed in the MAC Report .
2.5 Sample Preparation·
Keeping the solution alkaline during the crushing/blending of fish tissues may be a practical way of avoiding loss of HCN in the mixture. We will implement this suggestion.
· The recommendation of using ethylene diamine may be implemented, after it is evaluated by the BFAR/IMA laboratories.
· We generally try to bring the fish to the nearest laboratory or field office alive, and freeze it there. We will amend the SOP Manual with the current procedure.
· We doubt that cyanide is degraded after the sample is frozen. We have never had a problem in detecting cyanide, after digesting frozen samples in our CDT distillation apparatus.
· We will add the title in the SOP from “Procedure” to “Tissue Sample Procedure”
· We will modify the manual to elaborate on the parameters required for the testing to be undertaken (e.g. pH range, temperature etc.). The ISE measures the concentration of cyanide ion in a 10 g/L solution of sodium hydroxide at pH 12-13, since the cyanide ion is present at high pH values.
· It is possible to create replicate samples for testing from the same individual fish. Preliminary experiments conducted by IMA found almost identical results with replicate samples. Only one sample is usually tested, because of the time involved in digesting and refluxing replicate tissue samples.
· With small aquarium fishes (<10 g) the whole fish is digested and tested. It is impractical to be blending and testing larger fishes in the same way as small fish. Hence, the heart, liver, kidneys and anterior intestine are removed, and 10 grams of tissue weighed, blended and tested. The latter procedure is commonly used with groupers and snappers sampled from the food fish trade. The tissues tested are recorded on sample sheets that accompany the test results. We will add a description of these methods (already being done) to the SOP Manual.
· The test results for internal organs tend to yield higher cyanide concentrations, than tests with whole fish, since blood-born cyanide accumulates in these internal organs.
· Further CDT testing and analyses of the results are needed to quantify relative concentrations in different organs. Considering the size of the tropical/aquarium fish it can be assumed that it will very difficult to test the different organs of these small-sized specimen, unless otherwise a new device of test will be adopted such as isotope labeling or by examination of tissue sections using histology to determine the extent of damage of the biological system.
· As to the disposal of laboratory wastes, acidic wastes after the distillation are pre-treated with sodium hydroxide solution before disposal. Cyanide solutions e.g. standards, etc. are placed in amber-colored jars and sealed. Dichromate solution is not used at the laboratory for cleaning glassware. Instead, the glassware receives thorough cleaning using tap water and detergent. The final rinsing is done using distilled water. All glassware are washed, after each time they are used. Samples fixed with formalin (solution of formaldehyde) are rejected for cyanide analysis.
· Magnesium chloride is employed in the analysis as a catalyst and not because of the suspected presence of mercury in the sample.
· The reflux rate of 60 drops per minute that the group is suggesting is actually the same as what the SOP indicates, i.e. 1 bubble per second assuring that there will be no backing up of the samples during distillation.
· On the comment that “connect” shorting plugs (Sec 2.7, page 7), rather than “disconnect” (SOP page 13), as the case; the meter should have its shorting plugs closed or disconnected first during the self-test. After that, it is the time to connect the probes to the meter for electrode identification and running the calibration. This will be clarified in the amended SOP Manual.
· Most of the concerns brought up by the group are already being done. Those that were not discussed in the SOP Manual will be spelled out more clearly, when we modify the SOP.
2.6 Results and Data Equations
The equation used in the SOP is not far different from the equation the group recommends but the presentation of the latter is clearer, thus this will be included upon the revision of the manual.
2.7 Laboratory Conduct/Safety /Facilities
The three points cited by the group are all available, including first aid kit and common medicines e.g. paracetamol, aspirin and the like.
2.8 Literature Cited
The IMA keeps abreast of the scientific literature concerning cyanide toxicity to fish and cyanide testing methods. Some of this is cited in this response. The SOP will be modified to cite pertinent literature pertaining to the ASTM procedure etc.
2.9 Laboratory Accreditation/Chemists Registration Information
The chemists of CDT labs are all registered or licensed by the Philippine’s Professional Regulation Commission. Their names and professional qualifications will be listed in the amended SOP Manual.
2.10 Suggested Introductory Paragraphs
Relevant verbiage suggested by the MAC will be included in the revised SOP Manual. The first paragraph which discussed how cyanide complexes with heavy metals, is not considered relevant to the SOP Manual for testing fish (except possibly where fish are sampled near effluent sources for mine tailings). The second paragraph suggested is quite relevant to the CDT testing procedure.
3.0 Others
Compilation of the data as to the concentration of different species of fish is still on progress. IMA already has a database for that.
The IMA Sampling Officers (we call them MIS Officers) were given orientation as to what species should be taken from the source. They also have guidelines on how many fish samples have to be taken, depending on whether they are aquarium or food fishes. Samples are collected on boats, fish buying stations, airports, piers and other transshipment points.
-Besides the receipt issued for the samples taken from the facility/owner, another information sheet is filled out. This latter form can be used to record the time of the samples were taken at the holding facility or fish cages. Information is also obtained concerning approximately when and where the fish were caught (in the case of food fish).
-As to the selection of what species to pick up, the MIS Officers are familiar with the species commonly captured for the aquarium or the food fish trades. For example, most species tested in Puerto Princesa are groupers or snappers, obtained from local fishermen/collectors from the nearby municipalities, species mostly coming from NE coast of the Island of Palawan (Coron & Calamian Islands) are groupers. Where possible, aquarium fishes are sampled at random from collectors and from exporters’ holding facilities. Some resistance has been encountered with exporters, who refuse to supply higher priced species such as angelfish for CDT analyses. Despite these difficulties, the samples are generally representative of what is present in shipments within the country or destined for export.
-Further research is needed with the CDT database to determine whether certain species are being targeted with cyanide, or whether smaller fish species exhibit greater sensitivity to cyanide. These questions are best answered from a data set based on random sampling.
-It does not appear that certain tropical fish species are more likely to be cyanide collected. The data indicate that almost all the species exported by the aquarium trade are collected with cyanide. While the frequency of fishes found to contain cyanide has declined over the past three years, no species-specific patterns are apparent from visual inspection of the data. From a sampling perspective, it does not make sense to target certain species for CDT testing without a prior knowledge of which ones are contaminated with cyanide.
-Ice packs in styrofoam coolers are used to transport fish specimens to the CDT laboratories.
-For the water samples, keeping it at 4°C is not always possible. Sampling is done sometimes in far-flung areas like islands, where there is no electricity or ice available. But, when there is ice or a refrigerator available, the water samples are subjected to lower temperature. We will try transporting water samples from field sites with “Blue-Ice” packs and/or by adding sodium hydroxide to the water samples, following the U.S.EPA methods discussed in the MAC Report .
2.5 Sample Preparation·
Keeping the solution alkaline during the crushing/blending of fish tissues may be a practical way of avoiding loss of HCN in the mixture. We will implement this suggestion.
· The recommendation of using ethylene diamine may be implemented, after it is evaluated by the BFAR/IMA laboratories.
· We generally try to bring the fish to the nearest laboratory or field office alive, and freeze it there. We will amend the SOP Manual with the current procedure.
· We doubt that cyanide is degraded after the sample is frozen. We have never had a problem in detecting cyanide, after digesting frozen samples in our CDT distillation apparatus.
· We will add the title in the SOP from “Procedure” to “Tissue Sample Procedure”
· We will modify the manual to elaborate on the parameters required for the testing to be undertaken (e.g. pH range, temperature etc.). The ISE measures the concentration of cyanide ion in a 10 g/L solution of sodium hydroxide at pH 12-13, since the cyanide ion is present at high pH values.
· It is possible to create replicate samples for testing from the same individual fish. Preliminary experiments conducted by IMA found almost identical results with replicate samples. Only one sample is usually tested, because of the time involved in digesting and refluxing replicate tissue samples.
· With small aquarium fishes (<10 g) the whole fish is digested and tested. It is impractical to be blending and testing larger fishes in the same way as small fish. Hence, the heart, liver, kidneys and anterior intestine are removed, and 10 grams of tissue weighed, blended and tested. The latter procedure is commonly used with groupers and snappers sampled from the food fish trade. The tissues tested are recorded on sample sheets that accompany the test results. We will add a description of these methods (already being done) to the SOP Manual.
· The test results for internal organs tend to yield higher cyanide concentrations, than tests with whole fish, since blood-born cyanide accumulates in these internal organs.
· Further CDT testing and analyses of the results are needed to quantify relative concentrations in different organs. Considering the size of the tropical/aquarium fish it can be assumed that it will very difficult to test the different organs of these small-sized specimen, unless otherwise a new device of test will be adopted such as isotope labeling or by examination of tissue sections using histology to determine the extent of damage of the biological system.
· As to the disposal of laboratory wastes, acidic wastes after the distillation are pre-treated with sodium hydroxide solution before disposal. Cyanide solutions e.g. standards, etc. are placed in amber-colored jars and sealed. Dichromate solution is not used at the laboratory for cleaning glassware. Instead, the glassware receives thorough cleaning using tap water and detergent. The final rinsing is done using distilled water. All glassware are washed, after each time they are used. Samples fixed with formalin (solution of formaldehyde) are rejected for cyanide analysis.
· Magnesium chloride is employed in the analysis as a catalyst and not because of the suspected presence of mercury in the sample.
· The reflux rate of 60 drops per minute that the group is suggesting is actually the same as what the SOP indicates, i.e. 1 bubble per second assuring that there will be no backing up of the samples during distillation.
· On the comment that “connect” shorting plugs (Sec 2.7, page 7), rather than “disconnect” (SOP page 13), as the case; the meter should have its shorting plugs closed or disconnected first during the self-test. After that, it is the time to connect the probes to the meter for electrode identification and running the calibration. This will be clarified in the amended SOP Manual.
· Most of the concerns brought up by the group are already being done. Those that were not discussed in the SOP Manual will be spelled out more clearly, when we modify the SOP.
2.6 Results and Data Equations
The equation used in the SOP is not far different from the equation the group recommends but the presentation of the latter is clearer, thus this will be included upon the revision of the manual.
2.7 Laboratory Conduct/Safety /Facilities
The three points cited by the group are all available, including first aid kit and common medicines e.g. paracetamol, aspirin and the like.
2.8 Literature Cited
The IMA keeps abreast of the scientific literature concerning cyanide toxicity to fish and cyanide testing methods. Some of this is cited in this response. The SOP will be modified to cite pertinent literature pertaining to the ASTM procedure etc.
2.9 Laboratory Accreditation/Chemists Registration Information
The chemists of CDT labs are all registered or licensed by the Philippine’s Professional Regulation Commission. Their names and professional qualifications will be listed in the amended SOP Manual.
2.10 Suggested Introductory Paragraphs
Relevant verbiage suggested by the MAC will be included in the revised SOP Manual. The first paragraph which discussed how cyanide complexes with heavy metals, is not considered relevant to the SOP Manual for testing fish (except possibly where fish are sampled near effluent sources for mine tailings). The second paragraph suggested is quite relevant to the CDT testing procedure.
3.0 Others
Compilation of the data as to the concentration of different species of fish is still on progress. IMA already has a database for that.
4.0 Independent Laboratory Evaluation of the CDT Methods
An unidentified laboratory conducted experiments intended to duplicate the BFAR/IMA testing procedures. Unfortunately, they changed the apparatus, and conducted experiments that did not completely mimic the SOP described in the CDT Manual (Manipula et al 1995). Apparently, a midi-distillation apparatus was used in addition to the full sized distillation flask and reflux condenser described in the CDT Manual. The manufacturer of the apparatus was not identified. A midi distillation apparatus for cyanide determination is sold by Wheaton Science in the U.S.A.
The consultant evaluated a cyanide ion selective electrode (Orion Catalogue #9406BN). The first ISE electrode obtained by the consultant gave erratic results. Both the consultant and the CDT laboratories used the same ISE electrode. The ISE brand and model number will be specified in the revised CDT SOP manual.
Since, the main purpose of using the ISE is the determination of the presence of cyanide in fish samples, it is not of great concern that U.S. EPA recommends the use of the pyridine-barbituric acid colorimetric procedure for the detection of cyanide in wastewater effluents (APHA et al. 1998). It is recognized that different cyanide testing procedures may be better suited for different applications. The ISE method works well with testing fish samples for the presence of cyanide over the 0.05-10 mg/L range as recommended by Orion.
4.1 Calibration of Electrode
The consultant was finally able to calibrate the electrode and found a linear relationship (Table 1) with semi-log plots (Figures 1 and 2) similar to those routinely obtained by the BFAR/IMA CDT laboratories. The consultant noted that each time the electrode is calibrated the analyst should use calibration solutions that cover the entire range of the electrode, and verify that the electrode is responding with a linear calibration over the entire working range. The SOP CDT Manual states that the BFAR/IMA laboratories calibrate their equipment daily using standard cyanide solutions with cyanide ion concentrations of 0.03, 0.1, 1.0 and 10 mg/L (Manipula et al. 1995). It would have been helpful if the consultant had used the same concentrations that mimic the SOP Manual rather than the concentrations used (0.092, 0.230, 0.460, 0.920, 0.920, 4.60, 9.20 mg/L) specified in Table 1 of the report (Holthus 1999).
4.2 Sample Preparation and Quality Control
The consultant correctly states “The distillation is the biggest source of errors in the analysis. Particular causes for the errors lie in the vigor of the distillation, the rate of flow of the vapors through the absorption tube, and the inherent matrix interference’s in the samples that is present and released during distillation of the sample. Because of these well known points of difficulty quality controls have been devised and are present in most current analytical protocols for cyanide analysis.”
Two quality control (QA/QC) methods discussed by the consultant include the use of: a) a laboratory control sample (LCS) and b) the matrix spike procedure. These methods are applied by the consultant supposedly to evaluate the CDT SOP.
-The implication being made is that IMA is not aware of these QA/QC methods. This is not the case. It is true that they are not discussed in the SOP Manual. The QA/QC methods have been applied in the CDT laboratories.
-Perhaps of more concern is that in the four experiments described the consultant has not correctly applied the test procedures to obtain reliable results. Tables 2, 3, 4, and 5 indicate percent recoveries of cyanide ion ranging from 31-77%. If the ASTM (1996) procedure is correctly conducted one should expect recoveries of CN- in excess of 90%.
The published procedure states “Equivalent apparatus is acceptable provided cyanide recoveries of 100% +4% or -4% are documented (ASTM 1996). Hence, the low percent cyanide ion recoveries reported brings into serious question the validity of the experimental results (Holthus 1999).
The low percent recoveries might be related to the use of the midi distillation apparatus (Tables 2 and 4). However, the fact that low recoveries also occurred with the full sized 500 ml distillation apparatus (Tables 3 and 5) suggests that the apparatus used was not the problem.
Another possible explanation for the discrepancy is presented in the MAC report. The consultant states (top of page 14) “The distillation was set to run as fast as possible without actual flooding of the apparatus, and the vacuum set to achieve the maximum flow-through of the vapor, without loss of liquid from the absorption tube.” This may be interpreted to mean that the consultant accelerated the distillation either by overheating the distillation flask and/or by allowing an air flow in excess of the 1 bubble per second (60 bubbles per minute) recommended (ASTM 1996, APHA et al. 1998).
4.3 Matrix Spike Experiments
Fresh halibut tissue was weighed and homogenized in a blender with 300 ml of water. During blending a cyanide spike solution was added. The contents of the blender was then transferred to a volumetric flask, where additional water was added to bring the volume to 500 ml. A 50 ml portion was transferred to the midi-distillation flask, distilled and tested as described in the SOP Manual. The conclusion from results of this experiment (Table 2) stating “that the simple presence of fish tissue is not a significant
matrix interference to the cyanide analysis” is probably a valid conclusion.
The consultant notes (page 15, second paragraph) that “A significant effect was noted for the trials with spiked homogenates where storage was attempted. The Yellow Tang #2 and Klein Butterfly homogenates were preserved to pH 12 with sodium hydroxide, when placed in the refrigerator. .... After five days storage the samples were distilled and analyzed as listed in Table 3, resulting in only traces of cyanide, whereas prompt analysis gave recoveries of 45% and 31% respectively. The implications are that fish samples must be analyzed immediately to maximize the possibility of cyanide recovery from the sample”.
In Table 3, the samples were distilled using the 500 ml distillation flask and the ISE method used by the IMA/BFAR CDT laboratories. Another five fish were similarly prepared and digested in a 50 ml midi distillation apparatus and tested for cyanide ion using the colorimetric method (Table 4). Both experiments found only traces of cyanide below the detection limit (BLD) present when the spiked homogenates were held for five days after addition of sodium hydroxide in a refrigerator prior to testing.
The sample preparation procedure described above (Tables 3 and 4) differs from the methods described in the CDT SOP Manual where samples which are not immediately tested are frozen. In the tests (Tables 3 and 4), it is possible that the cyanide spiked tissue reacted with the rhodanese enzyme to convert the cyanide ion to thiocyanate (SCN-). The SCN ion is not detectable by the cyanide ion ISE electrode. Despite the addition of sodium hydroxide at pH 12 to the sample, the rhodanese enzyme may still have been active at the 4 degree centigrade temperature in the refrigerator (the sample was not frozen).
While it is true that the fish tissue is an aggressive matrix to the HCN present under the experimental condition, this is not the method utilized by the CDT laboratories. Freezing the sample would serve to arrest the action of the rhodanese enzyme. Since, the consultant’s methods differ from the CDT SOP, the conclusion that “fish samples must be analyzed immediately to maximize recovery” appears to be invalid.
An unidentified laboratory conducted experiments intended to duplicate the BFAR/IMA testing procedures. Unfortunately, they changed the apparatus, and conducted experiments that did not completely mimic the SOP described in the CDT Manual (Manipula et al 1995). Apparently, a midi-distillation apparatus was used in addition to the full sized distillation flask and reflux condenser described in the CDT Manual. The manufacturer of the apparatus was not identified. A midi distillation apparatus for cyanide determination is sold by Wheaton Science in the U.S.A.
The consultant evaluated a cyanide ion selective electrode (Orion Catalogue #9406BN). The first ISE electrode obtained by the consultant gave erratic results. Both the consultant and the CDT laboratories used the same ISE electrode. The ISE brand and model number will be specified in the revised CDT SOP manual.
Since, the main purpose of using the ISE is the determination of the presence of cyanide in fish samples, it is not of great concern that U.S. EPA recommends the use of the pyridine-barbituric acid colorimetric procedure for the detection of cyanide in wastewater effluents (APHA et al. 1998). It is recognized that different cyanide testing procedures may be better suited for different applications. The ISE method works well with testing fish samples for the presence of cyanide over the 0.05-10 mg/L range as recommended by Orion.
4.1 Calibration of Electrode
The consultant was finally able to calibrate the electrode and found a linear relationship (Table 1) with semi-log plots (Figures 1 and 2) similar to those routinely obtained by the BFAR/IMA CDT laboratories. The consultant noted that each time the electrode is calibrated the analyst should use calibration solutions that cover the entire range of the electrode, and verify that the electrode is responding with a linear calibration over the entire working range. The SOP CDT Manual states that the BFAR/IMA laboratories calibrate their equipment daily using standard cyanide solutions with cyanide ion concentrations of 0.03, 0.1, 1.0 and 10 mg/L (Manipula et al. 1995). It would have been helpful if the consultant had used the same concentrations that mimic the SOP Manual rather than the concentrations used (0.092, 0.230, 0.460, 0.920, 0.920, 4.60, 9.20 mg/L) specified in Table 1 of the report (Holthus 1999).
4.2 Sample Preparation and Quality Control
The consultant correctly states “The distillation is the biggest source of errors in the analysis. Particular causes for the errors lie in the vigor of the distillation, the rate of flow of the vapors through the absorption tube, and the inherent matrix interference’s in the samples that is present and released during distillation of the sample. Because of these well known points of difficulty quality controls have been devised and are present in most current analytical protocols for cyanide analysis.”
Two quality control (QA/QC) methods discussed by the consultant include the use of: a) a laboratory control sample (LCS) and b) the matrix spike procedure. These methods are applied by the consultant supposedly to evaluate the CDT SOP.
-The implication being made is that IMA is not aware of these QA/QC methods. This is not the case. It is true that they are not discussed in the SOP Manual. The QA/QC methods have been applied in the CDT laboratories.
-Perhaps of more concern is that in the four experiments described the consultant has not correctly applied the test procedures to obtain reliable results. Tables 2, 3, 4, and 5 indicate percent recoveries of cyanide ion ranging from 31-77%. If the ASTM (1996) procedure is correctly conducted one should expect recoveries of CN- in excess of 90%.
The published procedure states “Equivalent apparatus is acceptable provided cyanide recoveries of 100% +4% or -4% are documented (ASTM 1996). Hence, the low percent cyanide ion recoveries reported brings into serious question the validity of the experimental results (Holthus 1999).
The low percent recoveries might be related to the use of the midi distillation apparatus (Tables 2 and 4). However, the fact that low recoveries also occurred with the full sized 500 ml distillation apparatus (Tables 3 and 5) suggests that the apparatus used was not the problem.
Another possible explanation for the discrepancy is presented in the MAC report. The consultant states (top of page 14) “The distillation was set to run as fast as possible without actual flooding of the apparatus, and the vacuum set to achieve the maximum flow-through of the vapor, without loss of liquid from the absorption tube.” This may be interpreted to mean that the consultant accelerated the distillation either by overheating the distillation flask and/or by allowing an air flow in excess of the 1 bubble per second (60 bubbles per minute) recommended (ASTM 1996, APHA et al. 1998).
4.3 Matrix Spike Experiments
Fresh halibut tissue was weighed and homogenized in a blender with 300 ml of water. During blending a cyanide spike solution was added. The contents of the blender was then transferred to a volumetric flask, where additional water was added to bring the volume to 500 ml. A 50 ml portion was transferred to the midi-distillation flask, distilled and tested as described in the SOP Manual. The conclusion from results of this experiment (Table 2) stating “that the simple presence of fish tissue is not a significant
matrix interference to the cyanide analysis” is probably a valid conclusion.
The consultant notes (page 15, second paragraph) that “A significant effect was noted for the trials with spiked homogenates where storage was attempted. The Yellow Tang #2 and Klein Butterfly homogenates were preserved to pH 12 with sodium hydroxide, when placed in the refrigerator. .... After five days storage the samples were distilled and analyzed as listed in Table 3, resulting in only traces of cyanide, whereas prompt analysis gave recoveries of 45% and 31% respectively. The implications are that fish samples must be analyzed immediately to maximize the possibility of cyanide recovery from the sample”.
In Table 3, the samples were distilled using the 500 ml distillation flask and the ISE method used by the IMA/BFAR CDT laboratories. Another five fish were similarly prepared and digested in a 50 ml midi distillation apparatus and tested for cyanide ion using the colorimetric method (Table 4). Both experiments found only traces of cyanide below the detection limit (BLD) present when the spiked homogenates were held for five days after addition of sodium hydroxide in a refrigerator prior to testing.
The sample preparation procedure described above (Tables 3 and 4) differs from the methods described in the CDT SOP Manual where samples which are not immediately tested are frozen. In the tests (Tables 3 and 4), it is possible that the cyanide spiked tissue reacted with the rhodanese enzyme to convert the cyanide ion to thiocyanate (SCN-). The SCN ion is not detectable by the cyanide ion ISE electrode. Despite the addition of sodium hydroxide at pH 12 to the sample, the rhodanese enzyme may still have been active at the 4 degree centigrade temperature in the refrigerator (the sample was not frozen).
While it is true that the fish tissue is an aggressive matrix to the HCN present under the experimental condition, this is not the method utilized by the CDT laboratories. Freezing the sample would serve to arrest the action of the rhodanese enzyme. Since, the consultant’s methods differ from the CDT SOP, the conclusion that “fish samples must be analyzed immediately to maximize recovery” appears to be invalid.
Comments
The fish sample sizes (Tables 2-5) are relatively small (<7). Hence, the tests require further confirmation.
The consultant reported a loss of cyanide when two fish samples were refrigerated after being mixed with sodium hydroxide. The concentrations reported for the Yellow tang (0.0003 mg/g) and the Klein butterflyfish (0.0001 mg/g) were interpreted as being below detectable limits (BLD). If the concentrations stated are correct, they are not below the limit of detection of the ISE for cyanide ion.
-One part per million wet weight is equivalent to 1 mg/kg (or 1 micro- gram/g). Hence, a concentration of 0.0003 mg/g is the same as 0.3 mg/kg (ppm). Hence, the two measurements are not below the detectable limit using the ion selective electrode (ISE) for cyanide ion.
4.4 Cyanide Recovery from Exposed Fish
The final experiment describes how five live fish specimens were dosed with 1.0 mg/L in 1.00 ml portions with mixing for an unspecified period until the fish were immobilized. Then, the fish were rinsed in water and allowed to recover (5 minutes for a clownfish, longer for the three yellow tangs). After this, the fish were killed, blended, and then analyzed for the presence of cyanide using the 500 ml distillation flask and the ISE method. The test results (Table 5) indicate levels below detection limits (BLD) ranging from 0.0001-0.0005 mg/g.
Comments
· Again, the consultant reports BLD cyanide measurements for a Clownfish and three Yellow tangs ranging from <0.0001 to 0.0005 mg/g.
This is a range of 0.1-0.5 ppm (mg/kg). The measurements are not below the limit of detection of the ISE apparatus.
· If the consultant made a mistake, and meant to report data in the range 0.0001-0.0005 mg/kg some of the following points might explain the low cyanide recoveries.
-The concentration of cyanide solution (Table 5) used by the consultant (1-2 mg/L) to dose the fish is much lower than the concentration used by the aquarium fish collectors (1,500-2,000 mg/L) as previously discussed.
-The time of exposure of the live fish (Table 5) was not stated. It might be too quick as compared to the time interval from exposure up to the time the fish was removed from the vessel with cyanide solution.
-The manner of rinsing (Table 5) was not stated. Did the consultant use salt water or tap water? Was the rinsing by way of using running water (faucet) or by submerging the fishes in a container with tap water or seawater?
-It is also possible that the HCN present in the fish was lost during the blending process. Sodium hydroxide should be added during the blending process, not afterwards. This was suggested by one of the MAC panel of experts.
The Manila BFAR/IMA laboratory conducted similar experiments, wherein live fish were dosed with 5 mg/L cyanide for one minute and then tested. We did not have the problem with cyanide measurements below detectable levels encountered by the consultant. Cyanide levels in the range 0.1-1.4 mg/kg were measured. Hence, the problem encountered by the consultant is not a problem for the CDT laboratories in the Philippines.
The fish sample sizes (Tables 2-5) are relatively small (<7). Hence, the tests require further confirmation.
The consultant reported a loss of cyanide when two fish samples were refrigerated after being mixed with sodium hydroxide. The concentrations reported for the Yellow tang (0.0003 mg/g) and the Klein butterflyfish (0.0001 mg/g) were interpreted as being below detectable limits (BLD). If the concentrations stated are correct, they are not below the limit of detection of the ISE for cyanide ion.
-One part per million wet weight is equivalent to 1 mg/kg (or 1 micro- gram/g). Hence, a concentration of 0.0003 mg/g is the same as 0.3 mg/kg (ppm). Hence, the two measurements are not below the detectable limit using the ion selective electrode (ISE) for cyanide ion.
4.4 Cyanide Recovery from Exposed Fish
The final experiment describes how five live fish specimens were dosed with 1.0 mg/L in 1.00 ml portions with mixing for an unspecified period until the fish were immobilized. Then, the fish were rinsed in water and allowed to recover (5 minutes for a clownfish, longer for the three yellow tangs). After this, the fish were killed, blended, and then analyzed for the presence of cyanide using the 500 ml distillation flask and the ISE method. The test results (Table 5) indicate levels below detection limits (BLD) ranging from 0.0001-0.0005 mg/g.
Comments
· Again, the consultant reports BLD cyanide measurements for a Clownfish and three Yellow tangs ranging from <0.0001 to 0.0005 mg/g.
This is a range of 0.1-0.5 ppm (mg/kg). The measurements are not below the limit of detection of the ISE apparatus.
· If the consultant made a mistake, and meant to report data in the range 0.0001-0.0005 mg/kg some of the following points might explain the low cyanide recoveries.
-The concentration of cyanide solution (Table 5) used by the consultant (1-2 mg/L) to dose the fish is much lower than the concentration used by the aquarium fish collectors (1,500-2,000 mg/L) as previously discussed.
-The time of exposure of the live fish (Table 5) was not stated. It might be too quick as compared to the time interval from exposure up to the time the fish was removed from the vessel with cyanide solution.
-The manner of rinsing (Table 5) was not stated. Did the consultant use salt water or tap water? Was the rinsing by way of using running water (faucet) or by submerging the fishes in a container with tap water or seawater?
-It is also possible that the HCN present in the fish was lost during the blending process. Sodium hydroxide should be added during the blending process, not afterwards. This was suggested by one of the MAC panel of experts.
The Manila BFAR/IMA laboratory conducted similar experiments, wherein live fish were dosed with 5 mg/L cyanide for one minute and then tested. We did not have the problem with cyanide measurements below detectable levels encountered by the consultant. Cyanide levels in the range 0.1-1.4 mg/kg were measured. Hence, the problem encountered by the consultant is not a problem for the CDT laboratories in the Philippines.
The IMA believes the consultant made an incorrect conclusion based on misinterpretation of the results discussed in Table 5 (Holthus 1999). Certainly, more research is needed before one could conclude that “the SOP is insufficiently sensitive to determine if a fish has been subjected to cyanide treatment during the capture process.”
General Conclusions
The report by the independent consultant has serious flaws. It is doubtful that the conclusions reached would survive scientific review. The most serious problem appears to be the low recovery of the spiked samples (Tables 2-4). This may be related to how the distillation flasks were heated or how the air bubbles were added to the apparatus. This may also have contributed to the low recoveries noted in Table 5. The independent consultant raised more questions than were answered by using methods that differed from those described in the
BFAR/IMA CDT manual.
The IMA remains convinced that the ASTM (1996) procedure being utilized by the BFAR/IMA CDT laboratories yields valid scientific results. More work is needed to clarify and revise the SOP, if it is to be applied elsewhere. Certain sections such as the “Rules of Conduct in the Laboratory”, “Selection and Preparation of Specimen for Grouper Aggregation and Breeding Studies” should be removed from the CDT SOP.
The review of the CDT SOP (Holthus 1999) has been helpful. It will help the IMA-Philippines revise its manual (Manipula et al 1995). The efforts of the MAC to improve the SOP manual are appreciated. Significant progress has been made to reduce cyanide fishing in the Philippines. Hopefully, the CDT can be used to help put an end to cyanide fishing in other countries where the problem is still out of control.
References Cited
APHA, AWWA, WPCF 1991. Standard Methods for the Examination of Water and Wastewater, 19th Edition, American Public Health Association, Washington, DC.
APHA, AWWA, WPCF 1998. Method 4500-CN-E. Standard Methods for the Examination of Water and Wastewater, 20th Edition, American Public Health Association.
ASTM 1996. Annual Book Of Standards” Vol. 11.02, (D2036-91), American Society of Testing and Materials (ASTM), Philadelphia, PA.
Barber, C.V., and V.R. Pratt. 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.
Barber, C.V. and V.R. Pratt. 1998. Poison for profits: cyanide fishing in the Indo-Pacific. Environment 40(8):5-9, 28-34.
Brown, D.G., R.P. Lanno, M.R. van Den Heuval, and D.G. Dixon. 1995. HPLC determination of plasma thiocyanate concentrations in fish blood:application to laboratory pharmakinetic and field monitoring studies. Ecotoxicology and Environmental Safety 30:302-308.
Dempster, R.P., and M. S. Donaldson. 1974. Cyanide-tranquilizer or poison? Aquarium Digest International Tetra 2(4):21-22. Issue No.8.
Duodoroff, P., 1976. Toxicity to Fish of Cyanides and Related Compounds (A Review), EPA-600/3-76-038. National Technical Information Service.
Duodoroff, P. 1980. A Critical Review of Recent Literature on the Toxicity of Cyanides on Fish. American Petroleum Institute, Washington DC (ISBN- 0-89364-039-5) 71 pp.
Eisler, R. 1991. Cyanide Hazards to Fish, Wildlife, and Invertebrates: A Synoptic Review. U.S. Department of the Interior, Fish and Wildlife Service, Biological Report 85(1.23), Contaminant Hazard Reviews Report 23: 64 pp.
Johannes, R.E., and M. Riepen. 1995. Environmental, economic, and social implications of the live fish trade in Asia and the Western Pacific. Report submitted to the Nature Conservancy, 87 pp.
Jones, R.J. 1997. Effects of cyanide on corals. South Pacific Commission, Noumea Cedex, New Caledonia, Live Reef Fish Information Bulletin 3:3-8.
Jones, R. J., and O.Hoegh-Guldberg. 1999. Effects of cyanide on coral photosynthesis: implications for identifying the cause of coral bleaching and for assessing the environmental effects of cyanide fishing. Marine Ecology Progress Series 177:83-91.
Jones, R.J., T. Kildea, and O. Hoegh-Guldberg. In Press. PAM Chlorophyll Fluorometry: a new in situ technique for stress assessment in scleratine corals, used to examine the effects of cyanide from cyanide fishing. Marine Pollution Bulletin Vol.
Jones, R.J. and A.L. Steven. 1997. Effects of cyanide on corals in relation to cyanide fishing on reefs. Mar. Freshwater Res. 48:517-522.
Hall, K.C., and D.R. Bellwood. 1995. Histological effects of cyanide, stress, and starvation on the intestinal mucosa of Pomacentrus coelestis, marine aquarium fish species. J. Fish Biology 47:438-454.
Hanawa, M., L. Harris, M. Graham, A.P.Farrell, and L.I. Bendall-Young. 1998. Effects of cyanide exposure on Dascyllus aruanus, a tropical marine fish species: lethality, anaesthesia and physiological effects. Aquarium Sciences and Conservation 2:21-34.
Holthus, P. 1999. Peer Review of Cyanide Detection Testing Methods Employed in the Philippines. Draft Report by the Marine Aquarium Council to the World Bank Marine Market Transformation Initiative, 23 pp.
Ireland, P.J., and C.C. Robertson 1974. A review of evidence relating to the use and effects of sodium cyanide and other methods commercially employed in coralfish collecting. The British Marine Aquarists Association, Booklet No. 2, 21pp.
.
Leduc, G., R.C. Pierce, and I.R. McCracken. 1982. The Effects of Cyanides on Aquatic Organisms With Emphasis on Freshwater Fish. National Research Council of Canada, Associate Committee On Scientific Criteria For Environmental Quality, NRCC No. 19246:139 pp., Ottawa.
Leduc, G. 1984. Cyanides in water: toxicological significance. Pages 153-224, In: L.J. Weber (ed.), Aquatic Toxicology, Vol. 2, Raven Press, New York.
Manipula, B.E., E.R. Suplido, N.M. Astillero. 1995. Manual of Standard Operating Procedures For Cyanide Analysis. International Marinelife Alliance/Philippine Department of Agriculture-Bureau of Fisheries and Aquatic Resources, 26 pp.
Pet, J.S., and R.H. Djohani. Combating destructive fishing practices in Komodo National Park: ban the hookah compressor! Report to the Nature Conservancy. Web URL address http://www.spc.org.nc/coastfish/news/lrf/4/pet.htm
Pratt, V.R. 1996. The growing threat of cyanide fishing in the Asia Pacific region and emerging stategies to combat it. Coastal Management in Tropical Asia 6:9-11.
Robinson, 1985a. Collecting tropical marines-An interview with Earl Kennedy: the founding father of the Philippine fish trade speaks out. Part 1. Freshwater And Marine Aquarium 8(2):80-
86.
Robinson, 1985b. Collecting tropical marines-An interview with Earl Kennedy: the founding father of the Philippine fish trade speaks out. Part 2. Freshwater And Marine Aquarium 8(3):27-28, 30-31.
Raymond, P., G. Leduc, and J. A. Kornblatt. 1986. Investigation sur la toxicodynamique du cyanure et sur la biotransformation chez la truit arc-en-ciel (Salmo gaidneri). Can. J. Fish. Aquat. Sci. 43:2017-2024.
Rubec, P.J. 1986. The effects of sodium cyanide on coral reefs and marine fish in the Philippines. Pages 297-302, In: J.L. Maclean, L.B. Dizon, and L.V. Hosilos (eds.) The First Asian Fisheries Forum, Asian Fisheries Society, Manila, Philippines.
Rubec, P.J. 1987. The effects of sodium cyanide on coral reefs and marine fish 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. 1988. The need for conservation and management of Philippine coral reefs. Environmental Biology of Fishes 23(1-2):141-154.
Rubec, P.J., and V.R. Pratt. 1984. Scientific Data concerning the effects of cyanide on marine fish. Freshwater And Marine Aquarium 7(5);4-6, 78-80, 82-86, 90-91.
Rubec, P.J., and R. Soundararajan. 1991. Chronic toxic effects of cyanide on tropical marine fish. Pages 243-251, In: P. Chapman et al. (eds.) Proceedings of the Seventeenth Annual Toxicity Workshop: November 507, 1990, Vancouver, B.C. Vol. 1, Canadian Technical Report of Fisheries and Aquatic Sciences No. 1774 (Vol. 1).
Smith, L.S. 1982. Osmoregulation. Chapter 2, Pages 19-86, In: Introduction to Fish Physiology, TFH Publications, Inc., Neptune, New Jersey.
Williams, J.L. 1948. Cyanogen Compounds. Edward Arnold & Company.
General Conclusions
The report by the independent consultant has serious flaws. It is doubtful that the conclusions reached would survive scientific review. The most serious problem appears to be the low recovery of the spiked samples (Tables 2-4). This may be related to how the distillation flasks were heated or how the air bubbles were added to the apparatus. This may also have contributed to the low recoveries noted in Table 5. The independent consultant raised more questions than were answered by using methods that differed from those described in the
BFAR/IMA CDT manual.
The IMA remains convinced that the ASTM (1996) procedure being utilized by the BFAR/IMA CDT laboratories yields valid scientific results. More work is needed to clarify and revise the SOP, if it is to be applied elsewhere. Certain sections such as the “Rules of Conduct in the Laboratory”, “Selection and Preparation of Specimen for Grouper Aggregation and Breeding Studies” should be removed from the CDT SOP.
The review of the CDT SOP (Holthus 1999) has been helpful. It will help the IMA-Philippines revise its manual (Manipula et al 1995). The efforts of the MAC to improve the SOP manual are appreciated. Significant progress has been made to reduce cyanide fishing in the Philippines. Hopefully, the CDT can be used to help put an end to cyanide fishing in other countries where the problem is still out of control.
References Cited
APHA, AWWA, WPCF 1991. Standard Methods for the Examination of Water and Wastewater, 19th Edition, American Public Health Association, Washington, DC.
APHA, AWWA, WPCF 1998. Method 4500-CN-E. Standard Methods for the Examination of Water and Wastewater, 20th Edition, American Public Health Association.
ASTM 1996. Annual Book Of Standards” Vol. 11.02, (D2036-91), American Society of Testing and Materials (ASTM), Philadelphia, PA.
Barber, C.V., and V.R. Pratt. 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.
Barber, C.V. and V.R. Pratt. 1998. Poison for profits: cyanide fishing in the Indo-Pacific. Environment 40(8):5-9, 28-34.
Brown, D.G., R.P. Lanno, M.R. van Den Heuval, and D.G. Dixon. 1995. HPLC determination of plasma thiocyanate concentrations in fish blood:application to laboratory pharmakinetic and field monitoring studies. Ecotoxicology and Environmental Safety 30:302-308.
Dempster, R.P., and M. S. Donaldson. 1974. Cyanide-tranquilizer or poison? Aquarium Digest International Tetra 2(4):21-22. Issue No.8.
Duodoroff, P., 1976. Toxicity to Fish of Cyanides and Related Compounds (A Review), EPA-600/3-76-038. National Technical Information Service.
Duodoroff, P. 1980. A Critical Review of Recent Literature on the Toxicity of Cyanides on Fish. American Petroleum Institute, Washington DC (ISBN- 0-89364-039-5) 71 pp.
Eisler, R. 1991. Cyanide Hazards to Fish, Wildlife, and Invertebrates: A Synoptic Review. U.S. Department of the Interior, Fish and Wildlife Service, Biological Report 85(1.23), Contaminant Hazard Reviews Report 23: 64 pp.
Johannes, R.E., and M. Riepen. 1995. Environmental, economic, and social implications of the live fish trade in Asia and the Western Pacific. Report submitted to the Nature Conservancy, 87 pp.
Jones, R.J. 1997. Effects of cyanide on corals. South Pacific Commission, Noumea Cedex, New Caledonia, Live Reef Fish Information Bulletin 3:3-8.
Jones, R. J., and O.Hoegh-Guldberg. 1999. Effects of cyanide on coral photosynthesis: implications for identifying the cause of coral bleaching and for assessing the environmental effects of cyanide fishing. Marine Ecology Progress Series 177:83-91.
Jones, R.J., T. Kildea, and O. Hoegh-Guldberg. In Press. PAM Chlorophyll Fluorometry: a new in situ technique for stress assessment in scleratine corals, used to examine the effects of cyanide from cyanide fishing. Marine Pollution Bulletin Vol.
Jones, R.J. and A.L. Steven. 1997. Effects of cyanide on corals in relation to cyanide fishing on reefs. Mar. Freshwater Res. 48:517-522.
Hall, K.C., and D.R. Bellwood. 1995. Histological effects of cyanide, stress, and starvation on the intestinal mucosa of Pomacentrus coelestis, marine aquarium fish species. J. Fish Biology 47:438-454.
Hanawa, M., L. Harris, M. Graham, A.P.Farrell, and L.I. Bendall-Young. 1998. Effects of cyanide exposure on Dascyllus aruanus, a tropical marine fish species: lethality, anaesthesia and physiological effects. Aquarium Sciences and Conservation 2:21-34.
Holthus, P. 1999. Peer Review of Cyanide Detection Testing Methods Employed in the Philippines. Draft Report by the Marine Aquarium Council to the World Bank Marine Market Transformation Initiative, 23 pp.
Ireland, P.J., and C.C. Robertson 1974. A review of evidence relating to the use and effects of sodium cyanide and other methods commercially employed in coralfish collecting. The British Marine Aquarists Association, Booklet No. 2, 21pp.
.
Leduc, G., R.C. Pierce, and I.R. McCracken. 1982. The Effects of Cyanides on Aquatic Organisms With Emphasis on Freshwater Fish. National Research Council of Canada, Associate Committee On Scientific Criteria For Environmental Quality, NRCC No. 19246:139 pp., Ottawa.
Leduc, G. 1984. Cyanides in water: toxicological significance. Pages 153-224, In: L.J. Weber (ed.), Aquatic Toxicology, Vol. 2, Raven Press, New York.
Manipula, B.E., E.R. Suplido, N.M. Astillero. 1995. Manual of Standard Operating Procedures For Cyanide Analysis. International Marinelife Alliance/Philippine Department of Agriculture-Bureau of Fisheries and Aquatic Resources, 26 pp.
Pet, J.S., and R.H. Djohani. Combating destructive fishing practices in Komodo National Park: ban the hookah compressor! Report to the Nature Conservancy. Web URL address http://www.spc.org.nc/coastfish/news/lrf/4/pet.htm
Pratt, V.R. 1996. The growing threat of cyanide fishing in the Asia Pacific region and emerging stategies to combat it. Coastal Management in Tropical Asia 6:9-11.
Robinson, 1985a. Collecting tropical marines-An interview with Earl Kennedy: the founding father of the Philippine fish trade speaks out. Part 1. Freshwater And Marine Aquarium 8(2):80-
86.
Robinson, 1985b. Collecting tropical marines-An interview with Earl Kennedy: the founding father of the Philippine fish trade speaks out. Part 2. Freshwater And Marine Aquarium 8(3):27-28, 30-31.
Raymond, P., G. Leduc, and J. A. Kornblatt. 1986. Investigation sur la toxicodynamique du cyanure et sur la biotransformation chez la truit arc-en-ciel (Salmo gaidneri). Can. J. Fish. Aquat. Sci. 43:2017-2024.
Rubec, P.J. 1986. The effects of sodium cyanide on coral reefs and marine fish in the Philippines. Pages 297-302, In: J.L. Maclean, L.B. Dizon, and L.V. Hosilos (eds.) The First Asian Fisheries Forum, Asian Fisheries Society, Manila, Philippines.
Rubec, P.J. 1987. The effects of sodium cyanide on coral reefs and marine fish 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. 1988. The need for conservation and management of Philippine coral reefs. Environmental Biology of Fishes 23(1-2):141-154.
Rubec, P.J., and V.R. Pratt. 1984. Scientific Data concerning the effects of cyanide on marine fish. Freshwater And Marine Aquarium 7(5);4-6, 78-80, 82-86, 90-91.
Rubec, P.J., and R. Soundararajan. 1991. Chronic toxic effects of cyanide on tropical marine fish. Pages 243-251, In: P. Chapman et al. (eds.) Proceedings of the Seventeenth Annual Toxicity Workshop: November 507, 1990, Vancouver, B.C. Vol. 1, Canadian Technical Report of Fisheries and Aquatic Sciences No. 1774 (Vol. 1).
Smith, L.S. 1982. Osmoregulation. Chapter 2, Pages 19-86, In: Introduction to Fish Physiology, TFH Publications, Inc., Neptune, New Jersey.
Williams, J.L. 1948. Cyanogen Compounds. Edward Arnold & Company.
Thank you for your researched summary on the issue of cyanide tests Dr. Rubec. Dr. is certainly the authority on this issue.
I hope that David of MAC abandons yet another delay supported by some members of his board and institues cyanide testing forthwith.
May we hear from you David?
Thank you
Wayne Ryan
I hope that David of MAC abandons yet another delay supported by some members of his board and institues cyanide testing forthwith.
May we hear from you David?
Thank you
Wayne Ryan
I should note that the IMA used the recommendations made by the MAC review committee and revised the SOP to include QA/QC procedures. There presently are four SOP manuals that IMA prepared and turned over to BFAR when the IMA contract with BFAR teminated at the end of September 2001. The SOP manuals focus on a) Sampling, b) Sample preparation, c) CDT analyses, and d) use of the Thermo Orion ISE probes and use of the Thermo Orion pH/ISE meter. These are the CDT manuals presently being used by BFAR in their laboratories to measure cyanide concentrations in fish and water samples.
Peter Rubec
CDT SOP Manuals
Alban, J., B.E. Manipula, P.J. Rubec. 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, 24 pp.
Manipula, B., E.R. Suplido, N.M. Astillero. 2001. 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/International Marinelife Alliance, 19 pp.
Manipula, B.E., E.R. Suplico, N.M. Astillero. 2001. Standard Operating Procedures For Cyanide Testing Used By The Philippines Cyanide Detection Test (CDT) Network. Philippine Department of Agriculture-Bureau of Fisheries and Aquatic Resources/International Marinelife Alliance, 33 pp.
Manipula, B.E., P.J. Rubec, and M. Frant. 2001. 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/International Marinelife Alliance, 12 pp.
Peter Rubec
CDT SOP Manuals
Alban, J., B.E. Manipula, P.J. Rubec. 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, 24 pp.
Manipula, B., E.R. Suplido, N.M. Astillero. 2001. 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/International Marinelife Alliance, 19 pp.
Manipula, B.E., E.R. Suplico, N.M. Astillero. 2001. Standard Operating Procedures For Cyanide Testing Used By The Philippines Cyanide Detection Test (CDT) Network. Philippine Department of Agriculture-Bureau of Fisheries and Aquatic Resources/International Marinelife Alliance, 33 pp.
Manipula, B.E., P.J. Rubec, and M. Frant. 2001. 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/International Marinelife Alliance, 12 pp.
I was curious concerning what the MAC committed itself to with regard to CDT under the World Bank GEF MAMTI grant (Philippines and Indonesia Marine Aquarium Market Transformation Initiative). Page 52 of the MAMTI proposal describes the commitment that MAC Interim Exective Director David Mainenti is now obligated to carry out. I assume that whomever gets the MAC/Reefcheck grant for $100,000 will be assigned with the following tasks specified in the MAMTI proposal.
Project Component and Outputs
8.5
Cyanide Detection Test (CDT) Potential Evaluated, Sampling Protocol and Program Developed and Pilot Labs Established.
Targets
*CDT methods evaluated for appropriatness and effectiveness including possible import country CDT.
*Sampling protocol and program developed.
* 2 pilot CDT labs established (1 each in Philippines and Indonesia)
* A significant % of MAC Certified fish are sampled beginning in year 2
of MAMTI.
* Decrease in % of fish that test positive for cyanide.
Monitoring Mechanisms
* Report evaluating CDT methods and possible import level CDT.
* Document of sampling protocol.
* Report of pilot lab operations.
* Lab report on test results.
Key Assumptions
There are CDT methods that could be appropriate and effective at export and/import level.
Project Component and Outputs
8.5
Cyanide Detection Test (CDT) Potential Evaluated, Sampling Protocol and Program Developed and Pilot Labs Established.
Targets
*CDT methods evaluated for appropriatness and effectiveness including possible import country CDT.
*Sampling protocol and program developed.
* 2 pilot CDT labs established (1 each in Philippines and Indonesia)
* A significant % of MAC Certified fish are sampled beginning in year 2
of MAMTI.
* Decrease in % of fish that test positive for cyanide.
Monitoring Mechanisms
* Report evaluating CDT methods and possible import level CDT.
* Document of sampling protocol.
* Report of pilot lab operations.
* Lab report on test results.
Key Assumptions
There are CDT methods that could be appropriate and effective at export and/import level.
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