The Fresh Fish Files: How to Perform TVB-N Analysis

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The food detectives are back in the office trying to figure out how Cornlumbo solved the last case using his stomach. As Cornlumbo explains how his internal digestion method led to the solution, Shallot Holmes says that internal digestion would be a terrible idea for the latest case. The detectives are intrigued and ask Holmes for an explanation. “We have been contacted by a new seafood company, “Reely Fresh.” They buy, store, and distribute fresh fish. They have invested in large refrigeration units and vehicles to ensure their produce is as fresh as possible when it reaches its customers. We have been asked if we can help them adhere to EU regulations to ensure the launch of their company goes smoothly,” explains Shallot Holmes. 

What is TVB-N?

“Surely, eating the fish is an excellent way to determine freshness,” says Cornlumbo. “Not if the fish contains high levels of TVB-N, which can lead to the presence of harmful bacteria like Salmonella, Listeria, and Vibrio. Such pathogens can cause severe food poisoning symptoms,” says Holmes. “What’s TVB-N?” asks Cornlumbo. “Total Volatile Basic Nitrogen; it’s a measure to determine the quality and freshness of fish and seafood. To meet EU regulations, the company will have to ensure the levels of nitrogenous compounds, such as ammonia (NH3), trimethylamine (TMA), and dimethylamine (DMA), are not too high,” says Eggcule Poirot. “Exactly! We need to inform the client of the applicable EU regulations and tell them what they must do to comply,” says Holmes. Immediately, the detectives jump into action. Nancy Beef and Miss Mapple begin researching the applicable EU regulations while Eggcule and Cornlumbo determine how best to perform the TVB-N analysis. 

What EU regulations apply to fishery products?

Nancy and Miss Mapple discover that new rules are being implemented for imported fishery products as part of a push to digitize and improve the certification process. EU importers must now use the IT tool ‘CATCH’ to submit their certificates to import fish into the EU market. They also make a list of the applicable EU laws that will have to be considered by Reely Fresh.

  • (EC) No 178/2002 - A general food law regulation that establishes the general principles and requirements of food law in the EU and includes traceability requirements.
  • (EC) No 852/2004 - Provides rules for the hygiene of foodstuffs and requires the implementation of HACCP (Hazard Analysis and Critical Control Points) principles to maintain cleanliness, ensure proper temperature control, and ensure staff hygiene.
  • (EC) No 853/2004 - Specific rules for food of animal origin, setting out detailed requirements for the handling, storing, and transporting of fishery products, including conditions for freezing and temperature control.
  • (EC) No 854/2004 - Rules for the organization of official controls on products of animal origin intended for human consumption, outlining inspection and control procedures to ensure compliance with hygiene and safety standards.
  • (EU) No 1169/2011 - The scope of this regulation is to provide rules on labeling of food information, including species, production method, origin, allergen information, and nutritional content. 
  • (EC) No 2073/2005 - Specifies microbiological criteria for certain microorganisms in foodstuffs. Sets contaminant limits and outlines sampling and testing methods to ensure food safety.
  • (EC) 2017/625 - Provides an official framework for controls permitted to ensure the application of food and feed law, covering all aspects of the food chain. 
  • (EC) No 95/149 - Sets limits for TVB-N in various fishery products to ensure freshness and quality and provides accepted methods of determination.
  • (EC) No 2074/2005 - This regulation implements measures for certain products under (EC) No 853/2004. It includes specific requirements for bivalve mollusks, fishery products, and aquaculture products, as well as health standards and labeling requirements.

How can you perform TVB-N analysis?

Eggcule Poirot and Cornlumbo outline a step-by-step guide for the client of an accurate method for TVB-N determination that complies with (EC) No 95/149 and No 2074/2005.

1. Preparation of Perchloric Acid Solution

  • Fill 90 mL of perchloric acid (6%) into a beaker.
  • Perchloric acid extracts nitrogenous compounds (such as ammonia, dimethylamine, and trimethylamine) from the fish sample. The acidic environment helps to release these compounds by breaking down protein structures.

2. Weighing the Fish Sample

  • Weigh 10 grams of fish sample and add it to the beaker (Using an indirect weighing technique).
  • Accurate measurement of the fish sample is crucial for reproducibility and to ensure the consistency of the results. Indirect weighing helps in handling samples that might be sticky or messy, ensuring precision. It involves weighing the empty beaker (the tare weight), adding the sample, and subtracting the tare weight. It is ideal for sticky samples like fish or samples that are volatile or hazardous. 

3. Homogenization

  • Homogenize the sample mixture.
  • Homogenization ensures that the fish sample is evenly mixed with the perchloric acid, allowing for efficient extraction of the nitrogenous compounds. It creates a uniform solution where all the components are finely dispersed.

4. Filtration

  • Filtrate the homogenized mixture.
  • Filtration removes solid particles from the homogenized mixture, leaving a clear filtrate that contains the dissolved nitrogenous compounds. This is necessary for the subsequent steps to avoid clogging the instruments and to ensure accurate measurement.

5. Transfer Filtrate

  • Transfer 50 mL of the filtrate to the sample tube.
  • A specific filtrate volume is required for the steam distillation process. Consistency in volume ensures that the analysis is standardized and comparable across different samples.

6. Steam Distillation

  • Insert the sample tube and start steam distillation.
  • Steam distillation separates volatile compounds (such as ammonia) from the sample solution. The heat and steam cause these compounds to evaporate and be carried over into a receiving solution.

7. Alkalization

  • Add a few drops of phenolphthalein to the sample tube.
  • Phenolphthalein is a pH indicator that turns pink in basic conditions (pH > 10). This step ensures the solution is sufficiently alkaline for the volatile nitrogen compounds to be distilled. Alkalization helps to convert ammonium ions to ammonia gas, which can be distilled.

8. Titration

  • Automatic titration with 0.01 M HCI solution.
  • The distilled ammonia is collected in a solution and titrated with a known concentration of hydrochloric acid (HCl). The endpoint of the titration, often indicated by a change in color (due to a pH indicator), determines the amount of ammonia (and other basic nitrogen compounds) present in the sample. This quantifies the TVB-N content.

“Using the correct equipment will ensure the client complies with the applicable regulations, and some instruments can even automate some of these steps to speed up the process. Modern distillation equipment features automatic titration directly started after distillation as well as automatic aspiration of the sample and receiving solution,” says Eggcule. 

“Great work, detectives; I’ll pass the information to the client. They’ll surely have Really Reely Fresh Fish now!” says Shallot Holmes.