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Title: 

ORA Lab Manual Vol. IV Section 9 - Seafood Chemistry (IV-09) Page 1 of 25

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Sections in This Document

1. Introduction: Chemotherapeutics in Seafood............................................................................2

2. Triphenylmethane Dyes............................................................................................................3

2.1. Background....................................................................................................................3

2.2. Exercise .........................................................................................................................4

2.3. Questions.......................................................................................................................4

2.4. References.....................................................................................................................4

3. Nitrofurans/Chloramphenicol ....................................................................................................5

3.1. Background....................................................................................................................5

3.2. Exercise .........................................................................................................................5

3.3. Questions.......................................................................................................................5

3.4. References.....................................................................................................................5

4. MultiResidue Method................................................................................................................6

4.1. Background....................................................................................................................6

4.2. Exercise .........................................................................................................................6

4.3. Questions.......................................................................................................................6

4.4. References.....................................................................................................................7

5. Other Residue Methods............................................................................................................7

5.1. Background....................................................................................................................7

5.2. References.....................................................................................................................8

6. Answer Key...............................................................................................................................9

6.1. Triphenylmethane Dyes .................................................................................................9

6.2. Nitrofuran/Chloramphenicol ...........................................................................................9

6.3. Multi- residue methods.................................................................................................10

7. Chemical Indices of Decomposition........................................................................................10

7.1. Introduction ..................................................................................................................10

7.2. Histamine .....................................................................................................................12

7.2.1. Background....................................................................................................12

7.2.2. Exercise .........................................................................................................13

7.2.3. Questions.......................................................................................................14

7.2.4. References.....................................................................................................14

7.3. Indole ...........................................................................................................................15

7.3.1. Background....................................................................................................15
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7.3.2. Exercise .........................................................................................................15

7.3.3. Questions.......................................................................................................15

7.3.4. References.....................................................................................................15

7.4. Answer Key: Histamine................................................................................................16

7.5. Answer Key: Indole ......................................................................................................17

8. Marine Toxins .........................................................................................................................17

8.1. Background..................................................................................................................17

8.2. Saxitoxins and Tetrodotoxins.......................................................................................18

8.3. Ciguatoxins and Brevetoxins........................................................................................18

8.4. Diarrhetic Shellfish Toxins (DSTs) and Azaspiracids (AZAs).......................................19

8.5. Domoic Acid.................................................................................................................20

8.6. Exercise .......................................................................................................................22

8.7. Questions.....................................................................................................................22

8.8. References...................................................................................................................22

8.9. Answer Key: Marine Toxins .........................................................................................23

Document History ...................................................................................................................24

Change History.......................................................................................................................24

Attachments............................................................................................................................25

1. Introduction: Chemotherapeutics in Seafood

Over the past decade, there has been a significant increase in the commercial 

production and consumption of aquaculture products. As this industry grows, so does 

the use of approved and non-approved chemicals. Fish or seafood raised in a controlled 

environment can be better protected from catching wild parasites and fed to promote 

growth. On the other hand, fish/seafood raised in a high-density setting can cause quick 

exchange of disease, resulting in the need to pro-actively or actively treat with drugs. 

The use of non-approved chemical compounds on aquaculture products, or the misuse 

of approved chemicals, may have an impact on the safety of consumers. Some 

therapeutic agents are toxic and have been prohibited for use in aquaculture due to 

concerns for human health. Antibiotic residues can also find their way into the 

environment (by pond run-off, surviving sewage treatment, etc.) contributing to the 

development of antibiotic-resistant strains of bacteria; or by the chronic ingestion of 

antibiotics in our diet giving rise to antibiotic-resistant strains of bacteria. 

The FDA routinely monitors domestic and imported aquaculture and seafood products 

for drug residues under Compliance Program Guidance Manual 7304.018 

“Chemotherapeutics in Seafood.” The drugs (and species) are on surveillance status 
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and may change from year to year. The analytical methods are developed primarily by 

researchers in the Office of Regulatory Affairs (ORA) labs and may or may not be 

published in a scientific journal or the AOAC Official Methods of Analysis. Some 

methods are only circulated by the Office of Regulatory Science (ORS) or published in 

FDA’s Laboratory Information Bulletin (LIB). However, the compliance program will 

serve as guidance on which method is correct to use. Mass spectrometric methods 

described in this section provide either quantitative determination with confirmation of 

residue identity or provide qualitative screening for multiple classes of therapeutic 

residues above an established threshold concentration. The chemistry and instrumental 

analysis involved with these methods are some of the most difficult the analyst will find 

in the field laboratory because of the challenge of finding residue levels of analyte within 

a complex matrix. These methods are considered intermediate and advanced training. 

2. Triphenylmethane Dyes

2.1. Background

The triphenylmethane dyes malachite green (MG), crystal violet (CV), and brilliant 

green (BG) have antiseptic and antifungal properties with known uses in human 

and veterinary medicine. In aquaculture, dyes are primarily used as a treatment 

for fungal and external parasite infections in fish, and to protect incubating eggs 

from fungus. Though toxicity and mutagenicity of these compounds have led to 

worldwide restrictions and discontinuation of therapeutic dye treatments for 

aquaculture, the long history, efficacy and ready availability of these inexpensive 

dyes for infection control suggest that regulatory monitoring must continue (1-3). 

The cationic dyes and the leuco base form of the dyes are susceptible to 

oxidation/reduction and demethylation reactions in the presence of air and light. 

Following fish treatment by bath exposure, dyes are metabolized in fish to their 

reduced leuco forms, which have a several-month residence time in muscle 

compared to the cationic dye residue. Therefore, methods to determine prohibited 

residues of the triphenylmethane dyes must include detection of the leuco 

metabolites of MG and CV in addition to the dye forms, since the concentration of 

leucomalachite green (LMG) and leucocrystal violet (LCV) residues found will be 

significantly higher than the concentration of MG and CV. At low concentrations of 

residues, it is common to observe only LMG or LCV without any MG or CV 

present. Analytical methods have been developed for these compounds to either 

determine the total residue present (dye + leuco) following an oxidation reaction to 

convert the leuco form to the dye, or to measure the dye and leuco components 

individually. As shown in the compliance program, determinative methods for 

these compounds are required to quantify 0.5 ng/g as the sum of the contribution 

by the dye and the leuco metabolite (4).
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2.2. Exercise

Review the references. The trainer will provide a sample that has been either 

fortified or incurred with an unknown amount of a triphenylmethane dye and/or its 

leuco metabolite. Determine the quantity and confirm the identity of each residue 

present, using the AOAC Official Method of Analysis 2012.25 (1-3). Ensure all 

controls are extracted according to laboratory procedure. Determine the percent 

recovery of the residues in the laboratory positive control. Report all findings on a 

training analytical worksheet.

2.3. Questions

1. What is the Target Testing Level (TTL) for leucomalachite green?

2. Are there any special handling conditions for the triphenylmethane dyes and 

metabolites? If so, what are they?

3. What type of calibrants are used in AOAC Official Method of Analysis 

2012.25? 

4. If an analyst needs to test samples of shrimp, salmon, and eel on the same 

day, must six extracted calibrants be prepared in shrimp, salmon, and eel (18 

calibrants total)?

5. What steps can be taken to minimize the detection of crystal violet residues in 

negative control samples?

2.4. References

1. Andersen, W.C., et. al. “Triphenylmethane Dye Residue Analysis in Raw and 

Processed Aquaculture Products by AOAC Official Method of Analysis 

2012.25 (CARTS Projects IR01183 and IR01444)” Laboratory Information

Bulletin #4646, U.S. Food and Drug Administration 34 (2018) 1-23.

2. Andersen, W.C., et al. “Dye Residue Analysis in Raw and Processed 

Aquaculture Products: Matrix Extension of AOAC International Official Method 

2012.25” J. AOAC Int., 101 (2018) 1927-1939. 

3. Schneider, M.J., et al. “Determination of Triphenylmethane Dyes and Their 

Metabolites in Salmon, Catfish, and Shrimp by LC-MS/MS Using AOAC First 

Action Method 2012.25: Collaborative Study” J. AOAC Int., 98 (2015) 658-670.

4. Compliance Program 7304.018, Chemotherapeutics in Seafood Compliance 

Program (Issue Date06/23/2020).
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3. Nitrofurans/Chloramphenicol

3.1. Background

Nitrofurans (nitrofurantoin, furazolidone, nitrofurazone, and furaltadone) and 

chloramphenicol (CAP) are antimicrobial drugs that are widely used to treat 

bacteria in food-producing animals. CAP is used as an agent against both Grampositive and Gram-negative bacteria and nitrofurans are used for treatment of 

bacterial and protozoan infections (1-2). Currently, the U.S. Food and Drug 

Administration requires 12 individual subsamples to be analyzed for each sample 

collected for nitrofuran and CAP analysis (3). A two-subsample composite may 

be prepared provided that the lab has a documented study that demonstrates 

adequate sensitivity. While previous methods for nitrofurans and CAP had labor 

and time intensive liquid-liquid extraction procedures followed by analyses, the 

microwave-assisted derivatization using automated solid phase extraction 

simultaneously extracts all the analytes together.

3.2. Exercise

Review the references. The trainer will provide a sample that has been either 

fortified or incurred with an unknown amount of nitrofurans and/or CAP. Determine 

the quantity and confirm the identity of each residue present using the Laboratory 

Information Bulletin #4597 (2). Ensure all controls are extracted according to 

laboratory procedure. Determine the concentration of the residues in the 

laboratory positive control. Report all percent recoveries for matrix spikes and 

unknown findings.

3.3. Questions

1. What is the Target Testing Level (TTL) for the nitrofuran class of antibiotics? 

What is the TTL for chloramphenicol?

2. How often should 100 mM 2-nitrobenzaldehyde (2-NBA) be prepared?

3. What LCMS ionization techniques are used in LIB 4597 and for which 

compounds?

4. When utilizing the automated solid phase extraction system, samples are 

loaded on to SPE cartridges, washed, dried, and subsequently eluted with 

what solvent?

3.4. References

1. Veach, B.T., et al. “Quantitation of Chloramphenicol and Nitrofuran Metabolites 

in Aquaculture Products using Microwave-Assisted Derivatization, Automated 

SPE, and LC-MS/MS” J. AOAC Int., 98 (2015) 588-594.

2. Veach, B.T., et al. “Quantitation of Chloramphenicol and Nitrofuran Metabolites 

in Aquaculture Products using Microwave-Assisted Derivatization, Automated 
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Solid-Phase Extraction and LC-MS/MS” Laboratory Information Bulletin #4597, 

U.S. Food and Drug Administration 31 (2015) 1-27.

3. Compliance Program 7304.018, Chemotherapeutics in Seafood Compliance 

Program (Issue Date06/23/2022) 

4. MultiResidue Method

4.1. Background

Sulfonamide, quinolone, fluoroquinolone, amphenicol, and tetracycline antibiotics 

are effective for the treatment of bacterial infections in aquaculture products, and

these drugs have been monitored in single class regulatory analysis methods for 

years. Though these historical methods are quantitative; they are also labor 

intensive with respect to man hours and materials since most regulatory samples 

are found to be negative. Multi-residue screening methods have been developed 

to combine the analysis of individual classes of antibiotics with other therapeutic 

agents (e.g., hormones, antiparasitic or antifungal drugs) for time and cost savings 

(2-4). In a screening analysis, if a drug residue is identified with a response above 

a given threshold response, an additional analysis is performed to quantify the 

amount of the drug residue present.

4.2. Exercise

Review all references. The trainer will provide a sample that has been either 

fortified or incurred with an unknown amount of an analyte of interest. By utilizing 

a screening method, determine the presence of the unknown compound, using 

the method described in Compliance Program 7304.018 (1). Ensure all controls 

are extracted according to laboratory procedure. Additionally, perform a 

quantitative method on the unknown compound and determine the amount found. 

Report all findings on a training analytical worksheet.

4.3. Questions

1. What is the Target Testing Level (TTL) for the sulfonamide class of antibiotics? 

What is the TTL for ciprofloxacin?

2. During sample extraction, how many times do you transfer the upper organic 

layer?

3. Give an example of a situation (analyte/matrix) where the screening method 

does not provide suitable results for regulatory analysis. 

4. Explain the main differences between a screening method and a quantitative 

method?
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4.4. References

1. Compliance Program 7304.018, Chemotherapeutics in Seafood Compliance 

Program (Issue Date 06/23/2020) 

2. Clark, S. B., et al. “Analysis of sulfonamides, trimethoprim, fluoroquinolones, 

quinolones, triphenylmethane dyes (and their leuco metabolites) and 

methyltestosterone in fish and shrimp using liquid chromatography mass 

spectrometry” Laboratory Information Bulletin #4562, U.S. Food and Drug 

Administration 30 (2014) 1-23.

3. Storey, J.M., et al. “Analysis of sulfonamides, trimethoprim, fluoroquinolones, 

quinolones, triphenylmethane dyes and methyltestosterone in fish and shrimp 

using liquid chromatography–mass spectrometry” J. Chromatogr. B. 972

(2014) 38–47.

4. Nickel, T. J., et al. “Method transfer and Optimization of LIB 4562 (MultiResidue LC-MS/MS Screening Method for Veterinary Drugs in Aquaculture 

Tissues) from the Agilent 6490 LC-MS/MS to a SCIEX 5500 QTRAP LCMS/MS” Laboratory Information Bulletin #4614, U.S. Food and Drug 

Administration 32 (2016) 1-30.

5. Bunch, E. A., et al. “Homogeneous sample preparation of raw shrimp using dry 

ice” J. AOAC Int., 78 (1995) 883-887.

5. Other Residue Methods

5.1. Background

The Chemotherapeutics in Seafood Compliance Program Guidance Manual 

7304.018 also describes methodology and target testing levels for the analysis of 

additional therapeutic agents (1). Avermectin drugs such as ivermectin, 

emamectin, abamectin, and doramectin are macrocyclic lactone compounds that 

are used therapeutically to control parasites in veterinary medicine (2-4). 

Isoeugenol is used as a fish sedative in aquaculture operations (5). The stilbene 

drugs diethylstilbestrol, dienestrol, and hexestrol are non-steroidal synthetic 

estrogens that have been previously used in veterinary medicine for growth 

promotion; they are now banned for use in food producing animals as the 

stilbenes are known endocrine disruptors (6). Tetracycline antibiotics have also 

been used in aquaculture operations. In addition to these specific drug classes, 

wide scope screening methods have been developed to simultaneously identify 

hundreds of veterinary drug residues in seafood products (7-9). Training on 

avermectin, isoeugenol, stilbene, and wide scope screening methods should be 

completed as needed.
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5.2. References

1. Compliance Program 7304.018, Chemotherapeutics in Seafood Compliance 

Program (Issue Date 06/23/2020) 

2. Casey, C.R., et al. “Liquid Chromatography-Tandem Mass Spectrometry 

Method for the Confirmation and Quantitative Analysis of Avermectin Residues 

in Salmon” Laboratory Information Bulletin #4496, U.S. Food and Drug 

Administration 27 (2011) 1-12.

3. Casey, C.R., et al. “Liquid Chromatography-Tandem Mass Spectrometry 

Method for the Determination and Confirmation Analysis of Emamectin 

Residues in Salmon Tissue” Laboratory Information Bulletin #4567, U.S. Food 

and Drug Administration 30 (2014) 1-15.

4. Casey, C.R., et al. “A Rapid Liquid Chromatography-Fluorescence Detection 

(UPLC/FLD) for the Quantitative Analysis of Avermectin Residues in Salmon 

and Trout (CARTS No. IR01392)” Laboratory Information Bulletin #4636, U.S. 

Food and Drug Administration 34 (2018) 1-13.

5. Ye, L. “Development and validation of a LC/MS/MS method for the 

determination of isoeugenol in finfish with rapid extraction and simple off-line 

derivatization” Laboratory Information Bulletin #4619, U.S. Food and Drug 

Administration 32 (2016) 1-19.

6. Lohne, J.J., et al. “Analysis of Stilbene Residues in Aquacultured Finfish using 

LC-MS/MS” Laboratory Information Bulletin #4535, U.S. Food and Drug 

Administration 29 (2013) 1-16.

7. Storey, J.M., et al. “Screening for veterinary drug residues in fish, shrimp and 

eel using LC-HRMS. Part 1. Optimization of a cleanup and extraction 

procedure” Laboratory Information Bulletin #4615, U.S. Food and Drug 

Administration 32 (2016) 1-16.

8. Turnipseed, S.B., et al. “Screening for veterinary drug residues in fish, shrimp 

and eel using LC-HRMS. Part 2: Optimization of MS acquisition and validation 

of method” Laboratory Information Bulletin #4616, U.S. Food and Drug 

Administration 32 (2016) 1-31.

9. Turnipseed, S.B., et al. "Wide-scope screening method for multiclass 

veterinary drug residues in fish, shrimp, and eel using liquid chromatography–

quadrupole high-resolution mass spectrometry" J. Agric. Food Chem. 65

(2017) 7252-7267.
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6. Answer Key

6.1. Triphenylmethane Dyes

1. CP 7304.018: The TTL for the sum of leucomalachite green and malachite 

green is 0.5 ng/g (0.5 ppb). If only LMG is present, the TTL for LMG is 0.5 

ng/g. If both LMG and MG are present, the method must be able to quantify 

LMG below 0.5 ng/g. 

2. AOAC 2012.25: Samples, standards, and solutions need to be protected from 

light exposure by storing standard solutions, samples, and extracts on a dark 

cabinet, in amber colored glass or covered with foil when not in use. 

Triphenylmethane dyes and metabolites have mutagenic and teratogenic 

properties; proper personal protective equipment is worn while preparing 

standards and performing the extraction steps to minimize one’s exposure to 

these compounds.

3. AOAC 2012.25: The calibrants are internal standard corrected, matrix-matched 

extracted calibrants. Despite internal standard correction, differences in 

method accuracy may occur for some analytes if the calibration curve is 

prepared from extracted samples of a different type of negative control fish 

than the fish that is being tested. 

4. AOAC 2012.25: Not necessarily. In addition to the full quantitative procedure, 

AOAC 2012.25 has been also validated as a screening method. Samples can 

be analyzed, and the response compared to one matrix-matched extracted 

calibrant at the 0.5 ng/g level; further quantitative analysis with the full matrix 

matched extracted calibration curve is only required for suspected positive 

samples.

5. AOAC 2012.25: Take care to clean bench areas after standard preparation. 

Avoid using black permanent markers which may contain crystal violet. Use an 

injection needle wash protocol between sample injections. Inject water blanks 

between sample injections.

6.2. Nitrofuran/Chloramphenicol

1. CP 7304.018: 0.5 ng/g (ppb) is the TTL for the nitrofuran antibiotic residues. 

0.15 ng/g (ppb) is the TTL for chloramphenicol.

2. LIB 4597: 100 mM 2-NBA should be prepared fresh daily.

3. AOAC 2015:98: Positive atmospheric pressure chemical ionization (APCI) was 

used for the nitrofuran metabolites and negative electrospray ionization (ESI) 

was used for CAP analysis to achieve quantitation.

4. LIB 4597: The SPE cartridges are eluted with ethyl acetate.
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6.3. Multi- residue methods

1. CP 7304.018: 10.0 ng/g (ppb) is the TTL for sulfonamide antibiotic residues. 

5.0 ng/g (ppb) is the TTL for the combined contribution of ciprofloxacin and 

enrofloxacin. Ciprofloxacin is the metabolite of enrofloxacin treatment, so these 

residues are often found together.

2. LIB 4562/4614: 2 times

3. LIB 4562/J. Chromatogr. B paper: Although the 4562 method provides 

accurate results for the tilapia matrix, this method does not perform well for 

residues of malachite green, crystal violet, or brilliant green in matrices such as 

salmon, trout, eel, or shrimp. 

4. LIB 4562: The 4562-screening method utilizes a single point calibrant to 

establish a threshold response for each drug included in the method. The 

retention time and mass spectrometric product ion transition ratios can be 

used to identify the compounds of interest. These methods are also referred to 

as qualitative methods, or when threshold response data is used, as semiquantitative methods. A quantitative method utilizes a calibration curve over a 

concentration range to calculate the concentration of the unknown sample. 

Note: Screening methods DO NOT accurately determine the concentration of a 

drug residue in a sample.

7. Chemical Indices of Decomposition 

7.1. Introduction

While sensory examinations are the primary method used by the agency to 

determine decomposition in seafood products, chemical indices provide a 

significant support mechanism to sensory findings in some products.

Histamine development in fish with high free histidine levels and indole in canned, 

cooked, and fresh/frozen shrimp are two important decomposition indicators 

supported by the agency. These indices may be used to support sensory findings

in these products. Furthermore, since histamine can develop in the absence of 

detectable odors of decomposition and since the shrimp industry has employed 

treatments that mask odors of decomposition, chemical indices meeting the 

established criteria may be used to support regulatory action in the absence of 

sensory evidence in some cases. However, since decomposition pathways may 

develop which do not yield these metabolites, chemical analyses should not 

substitute or eliminate the need for sensory examination. Product guidance should 

be referred to for more information on the criteria used to support actions based 

on chemical indicators of decomposition. The original and check analyses should 

be used when supporting regulatory action based on chemical indices.
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In addition to histamine and indole, there are several other indices that may have 

utility in determining the decomposed state of seafood. Two of the more promising 

ones are the diamines, putrescine and cadaverine. There may be other 

decomposition metabolites that may also be supportive if problems arise using the 

established methods. However, the laboratories should consult with the Office of 

Regulatory Science (ORS) and the Center for Food Safety and Nutrition (CFSAN) 

before employing any of these methods during their regulatory activities.

The formation of chemical indicators of decomposition is associated with seafood 

that was not held at a proper temperature for storage after being caught or from 

mishandling during subsequent storage or processing. Most time-temperature 

abuse of histamine-susceptible fish occurs soon after catch. At that point the 

enzyme produced by bacteria is present and can produce more histamine at any 

point in the supply chain when fish is thawed, until fish products are cooked. In 

restaurant situations, storage of “good” product at improper temperatures can 

result in histamine (scombrotoxin) formation. Other chemical markers of 

decomposition have been found in spoiled fish, but their relationship to 

scombrotoxin fish poisoning has not been determined. Histamine can form in both 

high and low temperature storage conditions, and even before the associated 

odors of decomposition are apparent. Histamine-forming bacteria seem to be 

more sensitive to freezing than spoilage-producing bacteria. According to the 

FDA’s Compliance Policy Guide 540.525, significant decomposition and histamine 

formation can be avoided by following good handling practices. This includes icing 

or rapid immersion of the fresh catch in chilled water (at –1° C) followed by 

continuous frozen storage. Leaving fresh catch lying about on deck of a fishing 

vessel for an extended period of time or interruption of frozen storage are 

common occurrences in the histories of histamine-contaminated products. The 

canning of fish provides additional opportunity for problems associated with poor 

handling. Frozen fish are received at the cannery and thawed prior to processing, 

at which point temperature abuse (letting the product get too warm or 

inadvertently allowing it to thaw) has another chance to occur. Additionally, 

temperature abuse can occur during transportation or retail display if cooling 

equipment is not held at the correct temperature.

The seafood industry has implemented programs to establish Hazard Analysis 

Critical Control Point (HACCP) plans to help producers prevent cases of 

contamination and foodborne illness. HACCP plans delineate the most likely 

locations and scenarios for something to go “wrong” in a process that would result 

in the food product becoming unfit for consumption. The HACCP theory can 

simply be summarized as: if it is known where the problems are most likely to 

occur, then a prevention and monitoring plan can be put in place to effectively 

control them. It is a proactive approach that places the burden on industry, not a 

reactive approach to be countered by the government and tax dollars.
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7.2. Histamine

7.2.1. Background 

Scombrotoxin fish poisoning is otherwise known as histamine or scombroid 

poisoning. The name “scombroid poisoning” was coined because histamine 

(“scombrotoxin”) was first associated with fish species of the family Scombridae. 

However, it is now known that histamine can be produced in any fish species 

containing high levels of free L-histidine, which are typically dark meat fish and

include some non-scombroid fish (e.g., from the families Coryphaenidae and 

Pomatomidae). Histamine-producing fish include tuna, mahi-mahi, escolar, bonito, 

bluefish, sardine, pilchard, swordfish, marlin, and mackerel, to name a few. 

Histamine survives processing, and thus can be present in fresh, canned, or

cooked product. The formation of histamine is typically associated with 

decomposed product. However, decomposed product (determined 

organoleptically) does not always produce histamine, and the presence of 

histamine does not always occur in decomposed product – thus sensory analysis 

cannot ensure the presence or absence of histamine. Histamine can reliably be 

quantitated by chemical analysis down to 1-3 ppm (an acceptable level often 

found in fresh fish) (CPG 540.525). However, LOQs vary based on instrument and 

analytical method.

The aforementioned fish are inherently high in levels of free L-histidine, from 

which histamine is formed in the muscle after death. The amino acid L-histidine is 

decarboxylated by histidine decarboxylase (HDC), an enzyme produced by certain 

bacteria common in fish. Since histamine-producing bacteria are commonly found 

in the viscera and gills of the fish, a fillet from the anterior section is more likely to 

be contaminated as the intestine and gills decompose. Formation of histamine is 

dependent upon the growth of these bacteria, which is a function of time and 

temperature. However, it is critical to understand that once the bacteria produce 

HDC, more histamine can be produced even when the bacteria are no longer 

viable. This is true until the enzyme is destroyed by cooking or denaturation (e.g., 

during a methanolic extraction). Excess L-histidine may also be produced by 

proteolysis during the advanced stages of spoilage, which can further contribute to 

the formation of histamine. In this case, however, sensory analysis would likely 

detect decomposition. Interestingly, histamine can also be found in cheeses (such 

as Swiss cheese) that rely on the action of bacteria to form the product. The 

distribution of histamine within an individual fish fillet is heterogeneous. One 

portion of the fish may cause poisoning, while another may cause little to no 

reaction. It follows then that cans of processed product can have inconsistent 

histamine levels even within the same case lot (FDA, 1998). 

Scombrotoxin fish poisoning manifests as an allergic reaction. Onset of the 

reaction can be immediate to within one hour. Symptoms may include 
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tingling/burning mouth and lips, rash, headache, or nausea and vomiting. The 

symptoms may last for several hours, and recovery is generally rapid. 

Antihistamine drugs are an effective treatment, however sensitive individuals may 

need further medical treatment. When multiple cases occur in an outbreak, attack 

rate can also be a useful clue in the differential diagnosis of scombrotoxin fish 

poisoning versus seafood allergy: Multiple cases support scombrotoxin fish 

poisoning while seafood allergies will impact a low percentage of those consuming 

the suspect fish. Patient histories are important also. The suspect food should be 

collected and analyzed as soon as possible to confirm the presence of histamine. 

A good indicator of undesirable fish is a sharp, metallic or peppery taste. Also, fish 

with an “off-smell” should be avoided (FDA, 1998).

Scombrotoxin fish poisoning knows no geographic boundaries. The network for 

harvesting, processing, and distributing fisheries products is worldwide. Finished 

seafood products are sold fresh, frozen, or processed to homes, restaurants or 

various institutions. That adds up to a lot of opportunities for decomposition to 

occur. The FDA monitors fresh, frozen, and canned seafood for decomposition 

through organoleptic analysis. Products that might form histamine can be 

subjected to further chemical testing. Aside from the results of organoleptic 

analysis, product is also considered decomposed if it contains at least 50 ppm of 

histamine. However, regulatory action is considered on a case-by-case basis 

(CPG 540.525).

Methods used for histamine analysis include AOAC fluorometric method 977.13 

for original and check analysis, and the ELISA-based Veratox Quantitative 

Histamine Test for rapid screening purposes.

Caution: When preparing fish flesh composites for testing, those prepared from 

unprocessed fish products should be considered thermally labile due to the 

potential presence of active histidine decarboxylase.

7.2.2. Exercise

The student should familiarize him/herself with the analytical guidance for 

histamine analysis: AOAC fluorometric method 977.13; Veratox for Histamine test 

kit; CPGM 7303.842 and 7303.844; the ORS-wide SOP for histamine analysis; 

and local laboratory’s SOP for histamine analysis, as appropriate. The student will 

analyze either canned or fresh/frozen tuna. Both may be analyzed if time and 

resources permit. The trainer spikes duplicate samples at 50 ppm histamine 

[spiking done after the trainee has measured out an aliquot of the sample 

composite]. Alternatively, previously prepared canned tuna packs of known 

histamine concentration can be used. Analyze duplicate samples using the AOAC 

method and following any additional analytical directives in the CPGM and SOP.
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7.2.3. Questions

1. How many sub-samples are needed for histamine analysis when no odors of 

decomposition are present? When is it optional to do histamine analysis on 

product found by organoleptic analysis to be decomposed?

2. Why does histamine analysis need to be performed immediately following 

organoleptic analysis? If that is not possible, how should the sample be 

handled?

3. For fresh/frozen fish, why is the anterior portion preferred for histamine 

analysis?

4. What is the purpose of the ion-exchange column? What chemistry is 

involved?

5. What is the purpose of the OPT reagent? What chemistry is involved?

6. Why is it important to “read” derivatized samples in a timely fashion?

7. Why does the slit width of the xenon lamp need to be less than 6 nm?

7.2.4. References

1. AOAC official methods of analysis (current ed.). Method 977.13 Histamine in 

Seafood, Fluorometric Method. Gaithersburg, MD: AOAC International.

2. U.S. Food & Drug Administration. Compliance program guidance manual. 

Compliance Programs 7303.842 Domestic fish & fishery products, and 

7303.844 Imported seafood products. Washington DC: U.S. Government 

Printing Office. Available via Internet at: http://www.cfsan.fda.gov/~comm/cptoc.html.

3. Staruszkiewicz, W., Jr., Waldron, E. and. Bond, J. (1977). Fluorometric 

determination of histamine in tuna: development of method. Journal of 

Association of Official Analytical Chemists, 60, 1125-1130.

4. ORS-wide SOP for histamine analysis, and local laboratory SOP for histamine 

analysis (as appropriate).

5. U.S. Food & Drug Administration. Guidance for the Industry: Fish and Fishery 

Products Hazards and Controls Guidance. Fourth edition, March 2020.

6. Hungerford, J.M., 2021. Histamine and scombrotoxins. Toxicon, 201, pp.115-

126.

7. Lupo A, Mozola M. Validation study of a rapid ELISA for detection of histamine 

in tuna. Journal of AOAC International. 2011 May 25;94(3):886-99.

8. Veratox for Histamine kit insert
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7.3. Indole

7.3.1. Background

The presence of indole may serve as a chemical indicator for the evaluation of 

incipient spoilage of crustaceans and other seafood products. Indole analysis can 

be used to enhance and reinforce sensory data. Indole is formed in shrimp, 

crabmeat, and other seafood products by bacterial decomposition of proteins in 

seafood products.

7.3.2. Exercise

The student should familiarize him/herself with the analytical guidance for indole 

analysis: AOAC LC-fluorometric method 981.07; CPGM 7303.842 and 7303.844; 

and the local laboratory’s SOP for indole analysis. The student will analyze either 

canned or fresh/frozen shrimp. Multiple sample types may be analyzed if time and 

resources permit. The trainer spikes duplicate samples at 10 ug indole /100 g 

tissue (= 0.1 ug/g = 0.1 ppm) [spiking done after the trainee has measured out an 

aliquot of the sample composite].

Alternatively, previously prepared canned shrimp packs of known indole 

concentration can be used. Analyze duplicate samples using the AOAC method 

and following any additional analytical directives in the CPGM and SOP.

7.3.3. Questions

1. What is the purpose of spiking samples with 2-methylindole?

2. What is the logic behind making standard solutions A, B and C first, instead of 

making calibrations solutions directly?

3. How does the detector type influence the extent of the extraction chemistry?

4. If a matrix is too “dirty” to allow baseline separation of the indole peak, what 

other options might the analyst have? Hint: see LIB#4016.

7.3.4. References

1. AOAC official methods of analysis (current ed.) Method 981.07 Indole in 

seafood, liquid chromatographic fluorometric method. Gaithersburg, MD: 

AOAC International.

2. U.S. Food and Drug Administration. Compliance program guidance manual. 

Compliance Programs 7303.842 Domestic fish and fishery products, and 

7303.844 Imported seafood products. Washington DC: U.S. Government 

Printing Office. Available via Internet at: 

http://www.fda.gov/ICECI/ComplianceManuals/default.htm
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3. Berg, R. and Carley, C. Modification of HPLC method for indole in shrimp. FDA 

Laboratory Information Bulletin, No. 4016.

4. Local laboratory’s SOP for Indole Analysis.

7.4. Answer Key: Histamine

1. How many subsamples are needed for histamine analysis when no odors of 

decomposition are present? When is it optional to do histamine analysis on 

product found by organoleptic analysis to be decomposed? Six sub-samples 

are needed. Analysis is optional when a confirmation-qualified organoleptic 

analyst performs a check exam, or if additional sample is sent to another 

servicing laboratory for check exam. The remaining sub-sample should be 

analyzed if histamine is detected at greater than or equal to 35 ppm in any of

the initial sub samples. Alternatively, if a product was processed with chemical 

treatment (e.g., chlorine dip, CO, salt, smoke, etc.) that could mask odors of 

decomposition, all sub- samples should be analyzed for histamine.

2. Why does histamine analysis need to be performed immediately following 

organoleptic analysis? If that is not possible, how should the sample be 

handled? Because the enzymes that cause histamine formation can be still 

active. If can’t analyze immediately, the sample should be frozen.

3. For fresh/frozen fish, why is the anterior portion preferred for histamine 

analysis? This is the portion of the fish near the gut. It is the gut bacteria that 

provide the enzyme to form histamine. So, if histamine is present in the fish, it 

is more likely to be at the anterior end.

4. What is the purpose of the ion-exchange column? What chemistry is involved? 

It functions as a clean-up mechanism: the anion-exchange column traps the 

amino acid contaminants but allows histamine to pass through.

5. What is the purpose of the OPT (o-phthaldialdehyde) reagent? What chemistry 

is involved? OPT converts histamine to a fluorophore making it visible for 

quantitative analysis. OPT is phthalate ortho-substituted with aldehyde. The 

two aldehyde groups reach out like arms to form a new ring with the amine 

function of an amino acid (in the presence of an organic acid), giving form to a 

fluorescent conjugated system which is water soluble. [Note that OPT is also 

abbreviated as OPA by chemists and may be written as o- phthalaldehyde.]

6. Why is it important to “read” derivatized samples in a timely fashion? Because 

the OPT-histamine complex breaks down over time from UV light energy which 

would give a false low reading.

7. Why does the slit width of the xenon lamp need to be less than 6 nm? 

Because a larger slit allows too much energy through, which degrades the 

OPT-histamine complex.
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7.5. Answer Key: Indole

1. What is the purpose of spiking samples with 2-methylindole? This compound is 

an internal standard with similar extraction efficiencies and absorbance 

characteristics to indole yet is able to be chromatographed separately. An 

internal standard is used to help determine extraction recovery of the analyte, 

while providing a correction for extraction losses from the method.

2. What is the logic behind making standard solutions A, B and C first, instead of 

making calibrations solutions directly? Because the solutions are so dilute, it 

would be difficult to accurately weigh out indole standard in such a small 

amount. By starting with a large amount in a large volume, any error is 

minimized by subsequent dilutions (assuming no dilution errors!).

3. How does the detector type influence the extent of the extraction chemistry? 

Because the fluorescence detector is relatively specific to the compound of 

interest, it eliminates the need to do extensive clean-up of the sample. If using 

UV detection, minimal clean-up could leave behind matrix components that 

could interfere with detection of the analyte.

4. If a matrix is too “dirty” to allow baseline separation of the indole peak, what 

other options might analyst have? Hint: see LIB#4016. Use a SPE-column to 

help clean-up matrix interferences.

8. Marine Toxins

8.1. Background 

Most human intoxications by marine toxins in the US are due to the accumulation 

and metabolism of planktonic algae by filter-feeding marine organisms especially 

molluscan shellfish (e.g., mussels, clams, cockles, oysters). Among these 

microalgae, certain dinoflagellates produce most marine toxins except for the rare 

amino acid domoic acid and tetrodotoxin, which are produced by certain species 

in a specific genus of diatoms and bacteria, respectively.

The toxic microorganisms responsible for these seafood intoxications often form 

as algal blooms, referred to generally as harmful algal blooms (HABs), that occur 

when regional environmental conditions exist that are favorable to rapid 

proliferation of their populations. These conditions may include water temperature, 

sunlight, sea currents, and nutrients. Control factors, such as predation, also play 

a role in whether a bloom develops. Historically these HABs have been termed 

“red tides”, due to discoloration of the water caused by the presence of large 

numbers of these organisms. However, that term is a misnomer: HABs can occur 

without any noticeable change in the appearance of the water, and the presence 

of a red tide does not mean that the organisms are necessarily toxic. 
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Marine toxins are extremely diverse in their structures and mechanisms of action. 

The most toxic are neurotoxins exerting their physiologic effect by specific binding 

to voltage gated sodium channels (VGSCs) which are integral membrane proteins 

expressed in neurons, glia, and muscle cells. 

8.2. Saxitoxins and Tetrodotoxins

Channel blockers such as saxitoxins (STXs) and tetrodotoxins (TTXs) bind to site 

1 of the VGSCs and occlude the extracellular pore of the channel. This results in a 

polarization of net charge of the cell culminating in the toxins’ respective 

pathologies. Among the toxins presenting the greatest risk to human health, they 

are small water-soluble neurotoxins. These guanidinium-based toxins block nerve 

function to cause respiratory paralysis. Saxitoxins are produced by certain species 

of dinoflagellates (e.g., Alexandrium spp., Pyrodinium bahamense, Gymnodinium 

catenatum) but studies have shown that the toxin profiles observed in 

dinoflagellates reflect both the dinoflagellate species of their origin and their 

subsequent metabolism in shellfish. Consequently, the very diverse toxin profiles 

observed in shellfish vary by shellfish species. Of all the marine toxins, the 

saxitoxins have the greatest impact on shellfish safety due to their global 

distribution, frequent occurrence, and well documented fatalities. Thus, the STXs 

are extensively monitored in US waters and around the world. TTXs, well known 

for their occurrence in pufferfish and occasionally fatal Fugu poisoning incidents in 

Japan, have only in recent years been reported in molluscan shellfish (initially in 

Europe). Tetrodotoxins have similar toxicity to saxitoxin, and yet they have an 

endosymbiotic bacterial origin. Their occurrence in molluscan shellfish is 

considered an emerging issue for consideration which may be correlated with 

climate change.

8.3. Ciguatoxins and Brevetoxins

Channel activators such as the brevetoxins (BTXs) and ciguatoxins (CTXs), bind 

to site 5 of the channel, leaving it in an open state, destroying the ion gradient, 

and depolarizing the cell. 

Ciguatera fish poisoning is caused by the CTXs and is another seafood 

intoxication associated with certain species of toxic dinoflagellates. The 

commodity impacted is fish rather than shellfish, especially top predators like 

grouper, barracuda, and amberjack. Second only to Scombrotoxin fish poisoning, 

ciguatera is among the leading causes of illness (approximately 50,000/year) 

associated with consumption of fish. Ciguatera may present with gastroenteritis, 

cardiovascular issues including bradycardia (slow heart rate) and hypotension. 

There is also accompanying fatigue, insomnia, and malaise. The most 

diagnostically relevant symptoms are neurologic; temperature reversal (cold 

allodynia), paresthesia (tingling or numbness) most often in the extremities, dental 

pain, or a sensation of loosening teeth. Fatal intoxications are uncommon but do 
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occur especially in the case of ciguatoxins originating in the Pacific Ocean. The 

potency of ciguatoxins is illustrated by the FDA advisory level of 0.01 ug Pacific 

ciguatoxin (CTX1B) equivalents per kg. Currently the only assay capable of 

detecting these low levels for ciguatoxins is a cell bioassay (N2A-MTT) using a 

mouse neuroblastoma cell line (N2A) and a cell viability indicator (MTT) 

developed at the Applied Technology Center (ATC) formerly the Seafood 

Products Research Center at the Pacific Northwest Laboratory. Collaborations 

between FDA, Japan, and Australia have been critical in addressing the 

ciguatoxins threat. Regardless of origin, investigations of ciguatera outbreaks 

using LC-MS/MS methods require gifted purified toxins from researchers as no 

analytical standards are commercially available. Cell bioassay of ciguatoxins can 

use commercially available standards such as CTX3C to determine CTX-like 

activity. Currently two laboratories in FDA (NFFL and GCSL/CFSAN) are 

equipped to perform the assay.

The BTXs cause Neurotoxic Shellfish Poisoning (NSP) and are unusual in that 

they have been found only in the Gulf of Mexico, the southern Atlantic seaboard of 

the US, and New Zealand. The BTXs are far less toxic than ciguatoxins and there 

are no documented fatalities. In addition to the usual gastrointestinal symptoms, 

NSP does share two noteworthy symptoms with CFP, temperature reversal and 

paresthesia which likely reflect their shared receptor, site 5 on the VGSC. Long 

monitored by using a mouse bioassay, the BTXs are also the subject of studies of 

validation efforts applying enzyme linked immunosorbent assay (ELISA) and LCMS/MS confirmation. Although BTXs can also be detected using the N2A-MTT 

assay as with CFP, applying this assay to the BTXs is plagued by matrix effects 

due to their lower potency versus the CTXs.

8.4. Diarrhetic Shellfish Toxins (DSTs) and Azaspiracids (AZAs)

Like the brevetoxins and ciguatoxins, the DSTs and AZAs are polyether 

compounds and are produced by certain dinoflagellates. DSTs and AZAs which 

cause diarrhetic shellfish poisoning (DSP) and Azaspiracid Shellfish Poisoning 

(AZP) have much lower toxicity and are not neurotoxic. Further, the DSTs and 

AZAs can be found in temperate waters in contrast to the tropical waters where 

BTXs and CTXs occur. DSTs have a much wider global distribution than AZAs. 

Like the brevetoxins, the DSTs and AZAs are not associated with human fatalities. 

DSTs have a global distribution. In contrast, while AZAs have been detected 

globally, their largest commercial impact is in harvest waters off the coast of 

Ireland. The symptoms of both DST and AZA intoxications are primarily 

gastrointestinal and include vomiting, diarrhea, body aches, fever and chills. The 

onset of symptoms occurs from 30 minutes to 14 hours of eating contaminated 

shellfish. Although both cause primarily diarrhea, the mechanisms of action are 

different. Detection methods for both the DSTs and AZAs use predominantly LCMS/MS. The DSTs can be screened using an enzymatic assay based on protein 
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phosphatase subunit 2A (PP2A). The DSTs impacted Europe and Asia long 

before they were reported in the US. The DSTs caused a recreational harvestrelated outbreak in the US Pacific Northwest while US illnesses due to AZAs have 

thus far only been due to imported mussels harvested off the coast of Ireland.

8.5. Domoic Acid

Domoic acid is a small hydrophilic and neurotoxic amino acid which can cause 

brain damage manifesting as loss of short-term memory. It was first discovered as 

a shellfish toxin in Canada in 1987 and first impacted the US in 1993. Many cases 

of permanent memory impairment resulted from the Canadian outbreak, and 

several fatalities occurred among the elderly and the immune-compromised. Other 

neuropathologic symptoms (confusion, memory loss, disorientation, seizure, and 

coma) were also manifest during the outbreak. The symptomatic memory loss 

observed in the illness led to the intoxication to be called amnesic shellfish 

poisoning (ASP). Accompanying the previously described neurologic symptoms, 

ASP is characterized by gastrointestinal disorders such as vomiting, diarrhea, and 

abdominal cramping. There have been no documented outbreaks of ASP in the 

US, and it is noteworthy that the first occurrence of domoic acid contamination in 

the US was signaled by its impact not on humans but on wildlife (pelicans and 

marine mammals) in California waters. Defying the narrow classification implied 

by ASP as a shellfish toxin, the vector for these intoxications in the wildlife 

intoxications was anchovies. Like shellfish, anchovies are filter feeders and 

bioaccumulate the toxins. Since the Canadian and US events, the occurrence of 

domoic acid in both molluscan and crustacean shellfish has caused numerous 

harvest closures and is closely monitored.

Commercial harvest of molluscan shellfish in the US is monitored by shellfishproducing states under the National Shellfish Sanitation Program (NSSP), which 

is the federal/state cooperative program recognized by the FDA and Interstate 

Shellfish Sanitation Conference (ISSC) for the sanitary control of shellfish 

produced and sold for human consumption. Although shellfish producing states 

have programs to post harvest warnings, most shellfish intoxications that occur in 

the US are due to recreational harvesters who ignore posted warnings and 

closures. 

All humans are susceptible to intoxications by marine toxins. They are heat stable 

non-protein molecules, not inactivated by usual food preparation practices, i.e., 

cooking, freezing, pickling, etc. Ingestion of contaminated shellfish results in a 

wide variety of symptoms, depending upon the toxin(s) present, their 

concentrations in the shellfish, and the amount of contaminated shellfish 

consumed. Diagnosis of shellfish poisoning is based initially on observed 

symptomatology and recent dietary history, followed by analysis of unconsumed 

consumer portions if available.
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At the time of writing, there are no active surveillance assignments to collect and 

analyze seafood for marine toxins. While import and domestic compliance 

programs (7303.844 and 7303.842, respectively) have provisions for ASP and 

PSP sample collection and analysis, only local field and center work is being done 

on other toxins. In both compliance programs, two methods are mentioned: the 

mouse bioassay (MBA) for PSP and HPLC for ASP. Indeed the “gold standard” 

method for the detection of marine toxins has traditionally been the MBA. 

However, in the past few decades, many technologies have been exploited, and 

validated methods have been developed using them. Some of these methods 

have been adopted for regulatory use, such as AOAC 2011.02 (RBA) and AOAC 

2011.27 (Post-column HPLC), both of which have been tailored and adopted for 

specific use in some cases by the NSSP. 

Currently, for the analysis of regulatory samples, ORS suggests the use of the 

RBA in the analysis for PSTs (NSSP recognizes this method for mussels and 

limited use for clams and scallops). For domoic acid the HPLC method described 

in Quilliam et al.1995, is approved by the NSSP and for use in ORA laboratories. 

Prior to the implementation of an alternative, validated method, a suitable 

laboratory verification should be performed, consistent with the servicing 

laboratory’s Quality Management plan.

Procedures for performing the RBA are given in AOAC 2011.27 and useful 

information is also found in the AOAC interlaboratory study. In addition, 

experience in ORA laboratories has shown that it is advantageous to have the 

following considerations in mind. First, ensure that the ORA laboratory is cleared 

for use of radioactive materials, and that proper radiation safety use and disposal 

protocols are adhered to. The amount of tritium used for the assay is within 

Nuclear Regulatory Commission Exempt status levels. However, if the performing 

laboratory already operates under an NRC License, the license should be 

amended to include tritium. Prior to purchasing tritium labeled saxitoxin, contact 

the ORA RSO and follow guidelines in the ORA Radiation Safety Manual. Next, 

rat brain synaptosome aliquots should be prepared and tested in advance of 

sample arrival. During preparation of the synaptosomes, all reagents should be 

kept on ice, and the completed aliquots should be stored at -80°C. Furthermore, 

protein concentration determination of the synaptosome preparation is best done 

by the bicinchoninic acid (BCA) protein assay. Bradford-based methods are not 

advisable due to interference, presumably by cell membrane lipid components, 

leading to an overestimation of protein concentration. Finally, to ensure well-towell uniformity during the assay, it is advised to combine the following 

components into multiwell tubes: sample extract (or standard) dilution, 3H-STX 

working stock, and synaptosome prep. The tube contents should be mixed well 

by multichannel micro pipettor aspiration prior to, and throughout plating.
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Safety Levels in FDA regulated commodities 

(FDA, 2020, and FDA and EPA, 2020, Appendix 5)

PSP ≥ 80μg STX equivalents/100g

NSP ≥ 20 MU/100g or 0.8 mg brevetoxin-2 

equivalents/kg

AZP ≥ 0.16 mg AZA-1 equivalents/kg (0.16 ppm)

DSP ≥ 0.16 μg OA equivalents/kg

ASP ≥ 20 mg DA/kg

> 30 mg DA/kg in Dungeness crab viscera

CFP ≥ Caribbean: 0.1 μg C-CTX-1 equivalents/kg

≥ Pacific: 0.01 μg P-CTX-1 equivalents/kg

Laboratory analysis for marine toxins is restricted to specialized FDA field 

laboratories; training in this area is therefore up to the discretion of each servicing 

laboratory. Laboratories may contact the Applied Technology Branch of Pacific 

Northwest Laboratory or the Northeast Food and Feed Laboratory. Historically the 

Gulf Coast Seafood Laboratory branch of CFSAN has also provided assistance. 

These laboratories should be contacted for additional guidance and training 

suggestions.

8.6. Exercise

Review references, as well as the analytical portions of Compliance programs 

7303.842 and 7303.844. Instructor will assign trainee with a PST-spiked sample 

at three toxin levels: below action level, action level and above action level, as 

well as unspiked. Trainee should perform extractions, prepare dilutions (along 

with standard dilutions), run the RBA, and analyze data with Graph Pad Prism (or 

equivalent software)

8.7. Questions

1. What are the criteria for an acceptable Standard Dilution curve in the 

competitive Receptor Binding Assay?

2. What is the purpose of Tryptophan as a reagent in the Domoic acid HPLC 

analysis?

8.8. References

1. National Shellfish Sanitation Program (NSSP) | FDA

2. U.S. Food & Drug Administration SGR 129 Fish and Fishery Products Hazards 
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and Controls Guidance, June 2022 Edition

3. FDA and EPA Safety Levels in Regulations and Guidance Appendix 5 (2020) 

4. AOAC Official Methods of Analysis Method 2006.02 Domoic Acid Toxins in 

Shellfish Biosense ASP ELISA- A Competitive Direct Enzyme-Linked 

Immunosorbent Assay

5. Quilliam MA, Xie M, Hardstaff WR. Rapid extraction and cleanup for liquid 

chromatographic determination of domoic acid in unsalted seafood. Journal of 

AOAC International. 1995 Mar 1;78(2):543-54.

6. AOAC Official Method 2011.27. Paralytic Shellfish Toxins (PSTs) in Shellfish. 

Receptor Binding Assay

7. AOAC Official Method 9 59.08 Paralytic Shellfish Poison. Biological Method

8. AOAC Official Method 2011.02 Paralytic Shellfish Toxins in Mussels, Clams, 

Oysters, and Scallops. Post-Column Oxidation (PCOX) Method

9. AOAC Official Method 2005.06 Paralytic Shellfish Poisoning Toxins in 

Shellfish. Prechromatographic Oxidation and Liquid Chromatography with 

Fluorescence Detection

10.Determination of Paralytic Shellfish Toxins in Shellfish by Receptor Binding 

Assay: Collaborative Study. Dolah FM, Fire SE, Leighfield TA, Mikulski CM, 

Doucette JAOAC Int.2012; 95(3): 795-812 

11.Manger, R.L., Leja, L.S., Lee, S.Y., Hungerford, J.M., Hokama, Y., Dickey, 

R.W., Granade, H.R., Lewis, R., Yasumoto, T. and Wekell, M.M., 1995. 

Detection of sodium channel toxins: directed cytotoxicity assays of purified 

ciguatoxins, brevetoxins, saxitoxins, and seafood extracts. Journal of AOAC 

International, 78(2), pp.521-527.Abraham A, Jester EL, Granade HR, Plakas 

SM, Dickey RW. Caribbean ciguatoxin profile in raw and cooked fish 

implicated in ciguatera. Food chemistry. 2012 Mar 1;131(1):192-8.

12.Abraham A, Jester EL, Granade HR, Plakas SM, Dickey RW. Caribbean 

ciguatoxin profile in raw and cooked fish implicated in ciguatera. Food 

chemistry. 2012 Mar 1;131(1):192-8.

8.9. Answer Key: Marine Toxins

1. The standard curve should have a slope of -1 ± 0.2 and an EC50 of 2 ηM ± 

30%

2. Tryptophan is present in shellfish extracts and will elute close to domoic acid, 

potentially resulting in a false positive result. The inclusion of Tryptophan 

ensures that the method can adequately address this potential interference.
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Science

Document Number:

MAN-000062

Revision #: 03

Revised: 

07 Oct 2022

Title: 

ORA Lab Manual Vol. IV Section 9 - Seafood Chemistry (IV-09) Page 24 of 25

For the most current and official copy, check QMiS.

Document History

Revision

#

Status*

(D, I, R) Date Author Name and Title Approving Official Name and Title

1.3 R 01/17/2008 LMEB LMEB

1.4 R 02/02/2010 LMEB LMEB

1.5 R 02/13/2014 LMEB LMEB

02 R 06/15/2020 LMEB LMEB

03 R REFER TO 

QMIS 

CONNIE DRAKE & HEATHER 

HAWK, PROGRAM MANAGERS 

OFFLO

LMEB

* - D: Draft, I: Initial, R: Revision

Change History

Revision 

# Change

1.3

9.2, para 2, line 3 – changed “confirmation” to “confirmatory”

9.3.2. A. – updated website

9.3.2. C. – changed “OK” to “permissible”

9.5, 9.2.1 1. – revised answer

9.5, 9.3.2 5. – changed “spectra” to “spectrum”

1.4

9.2 D. 2. – updated web link 

9.3.1 D. 2. – updated 

9.3.2 A. – deleted last sentence 

9.3.2 D. – revised 

9.5 9.3.1 1. and 3. – chemical names corrected 

Footer – updated web link

1.5 Header – Division of Field Science changed to Office of Regulatory Science 

9.2 – Division of Field Science (DFS) changed to Office of Regulatory Science (ORS)

02

Areas shaded gray were revised:

• Reformatted document which led to change in numbering and bullets

• Chemotherapeutics: complete revision 

• Marine toxins: complete revision

• Chemical Indices of Decomposition: updated information related to time/temperature 

abuse, fish species, enzyme action and implications for analytical testing, outbreaks, 

Veratox kits, ORS-wide SOPs, literature references, and other highlighted information.

03 Questions for Nitrofuran/Chloramphenicol were revised. References and hyperlinks were 

updated. All edits within document are shaded gray.
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Science

Document Number:

MAN-000062

Revision #: 03

Revised:

07 Oct 2022

Title: 

ORA Lab Manual Vol. IV Section 9 - Seafood Chemistry (IV-09) Page 25 of 25

For the most current and official copy, check QMiS.

Attachments

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