Acid Occurrence And Effect In Shelfish Mussels

Published Date: 02 Nov 2017

INTRODUCTION

Okadaic acid (OA) and the structurally related Dinophysistoxins (DTX-1, DTX-2 and DTX-3) are the principal toxic compounds causing diarrhetic shellfish poisoning (DSP) in humans ( Carmody et al.,1996). The DSP toxins (DST) are lipophilic polyether molecules produced by dinoflagellates of the Dinophysis sp. And Prorocentrum sp. genera and accumulate in the digestive system of bivalve molluscs such as oysters, mussels, scallops, and clams when feeding on these plankton species (Murata et al., 1982). DST occurs in bivalves in all parts of the world and pose a serious threat to both public health and a sustainable aquaculture .This toxin is lipophilic and heat stable, and can be found in various species of shellfish, mainly in filter feeding bivalve molluscs such as oysters, mussels, scallops, and clams.

OA- toxins cause Diarrhoeic Shellfish Poisoning (DSP), which is characterized by symptoms such as diarrhoea, nausea, vomiting and abdominal pain. These symptoms may occur in humans shortly after consumption of contaminated bivalve molluscs such as mussels,scallops, oysters or clams. Inhibition of serine/threonine phosphor protein phosphatases is assumed to constitute the mode of action of OA- toxins (EFSA, 2008).

Different methods of detecting harmful algal toxins have been developed in different laboratories. An ELISA (Enzyme Linked Immune Sorbent Assay) is one of the methods for the detection of Okadaic acid in shellfish and is commercially available as a test kit (Gas et al. 2009). Enzyme-linked immune sorbent assay (ELISA), is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The test combines the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily assayed enzyme that possesses a high turnover number. ELISAs can provide a useful measurement of antigen or antibody concentration.

The objective of this exercise was to quantify the amount of Okadaic acid present in homogenized muscle tissue which contains an unknown concentration of the toxin by applying the antigen-antibody reaction

MATERIALS AND METHODS

Materials and Reagents

The materials and reagent used during the lab test were: Spectrophotometer,Pipette tips Micropipettes, Eppendorf tubes ,Abraxis Okadaic Acid test kit, Parafilm, Gloves and Paper tissue

Methodologies and Principles

Principles

The test is a direct competitive ELISA based on the recognition of Okadaic Acid by specific antibodies. Okadaic Acid, when present in a sample and an okadaic acid-enzyme-conjugate compete for the binding sites of rabbit anti-okadaic acid antibodies in solution. The okadaic acid antibodies are then bound by a second antibody (goat anti-rabbit) immobilized on the plate. After a washing step and addition of the substrate solution, a color signal is produced. The intensity of the blue color is inversely proportional to the concentration of Okadaic Acid present in the sample. The color reaction is stopped after a specified time and the color is evaluated using an ELISA reader. The concentrations of the samples are determined by interpolation using the standard curve constructed with each run.

Methodologies

A homogenized mussel tissue positive for toxic-OA has been initially prepared as a sample for the test. Conjointly, standards of 0, .2, 0.5, 1.0, 2.0 and 5.0ppbs of okadaic acid were also prepared prior to the test. The different standards are helpful in generating the absorption standard curve later.

A 100µL of each of the standards was then added into separate wells with a micropipette and new pipette tip for each sample so as to avoid contamination or mixing between different samples. The wells were then marked/labeled for easier identification.

Then after, 50µL of enzyme conjugate was added to each well followed by 50µL of anti-OA antibody solution to each of the wells again. The strip was then covered with a parafilm layer, manually gently mixed and incubated for an hour. The principle is that, the specific anti-OA antibody will bind with the antigen (toxin) in the well and sticks to the well, as this may take time, an hour incubation was prescribed and done too. The role of the enzyme conjugate is that, it will bind (form conjugation) with the antigen-bound specific antibodies and remain in the well together so that in the final step a substance is added that the enzyme can convert to some detectable signal, a colour change in a chemical substrate.

An hour later, the parafilm was taken off and the liquid dumped out into a sink. The wells were taped upside down against a tissue to further remove the remaining liquid inside the wells. The wells were then filled with 250µL of washing buffer and dumped out two times. The washing is done so that unbound antibody will be removed (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")

Then after, 150µL of substrate/chromogen was added to each well, mixed gently manually and incubated for 25 minutes at room temperature in a dark place because access to light may result in errors during absorbance reading. Substrate/chromogen was added to produce a visible signal (chromoflurecent), which indicates the quantity of antigen in the sample. That is, chromogenic reporters and substrates produce some kind of observable colour change to indicate the presence of antigen.

Finally, 100µL of stop solution was added to each well, mixed gently and then the absorbance was measured at 450nm using a spectrophotometer apparatus. Reading was taken within 15 minutes after addition of stop solution.

RESULTS

The spectrophotometric absorbance results and the percentage absorbance values relative to the zero standard (100%) are calculated as follows:

% Absorbance of 0.2 concentration= (1.609/1.6368)x100= 98.302%

Table 1. Percentage Absorbance results of all the standard samples.

Mussel (Group1)

 

 

 

 

Concentrations

Log (concentrations)

Absorbance

% Absorbance

0

 

1.6368

100

0.2

-0.698970004

1.609

98.30156403

0.5

-0.301029996

1.2877

78.67179863

1

0

0.9847

60.16006843

2

0.301029996

0.8427

51.48460411

5

0.698970004

0.4593

28.06085044

Figure 1. The Absorbance standard curve.

The absorbance values read for the samples 1 and two were, 1.5441 and 1.4727 respectively. The Absorbance percentage of these samples was calculated as follow:

Sample 1(SAM1),has an absorbance of 1.5441 ,SAM2 =1.4727and A0=1.6368 so,

SAM 1= (1.5441/1.6368)x100= 94.34%

SAM 2= (1.4727/1.6368)x100= 89.97%

Therefore, using the regression equation, we can now calculate the OA concentration of the samples:

Y= -49.45x + 63.336 Y= -49.45x + 63.336

94.34= -49.45x + 63.336 89.97= -49.45x + 63.336

94.34-63.34=-49.45x 89.97-63.336=-49.45x

31.004=-49.45x 26.634=-49.45x

31.004/-49.45=x 26.634/-49.45=x

-0.626976744=x -0.538604651=x

X1= -0.6269 X2= -0.5386

Table 2. Percentage Absorbance and concentration results of sample 1 and 2.

For the sample duplicates

Concentrations

Log (concentrations)

Absorbance

% Absorbance

0

1.6368

100

0.236060464

-0.626976744

1.5441

94.34

0.289331254

-0.538604651

1.4727

89.97

0.262695859

The okadaic acid concentration in the sample is, therefore, 0.263ppb.

DISCUSION

Diarrhetic shellfish poisoning (DSP) is a gastrointestinal syndrome that occurs in humans following the consumption of bivalve molluscs contaminated with OA with the main symptoms of diarrhoea, nausea, vomiting and abdominal pain. It is recognized as a worldwide public health problem in relation to the shellfish industry. Strict regulations have been implemented in most European Union countries, where a limit of 160µg/kg for these toxins in shellfish has been established (Commission Regulation, 2004). In addition, Regulation (EC) No 853/20045 repealing the previous Directives, prescribes in chapter VI: "Health Standards for Live Bivalve Molluscs" that "food business operators must ensure that live bivalve molluscs placed on the market must not contain marine bio toxins in total quantities (measured in the whole body or any part edible separately) that exceed the following limits: for okadaic acid, dinophysis toxins and pecteno toxins, 160 μg of OA equivalents per kg"(EFSA,2008).In our experiment the result revealed a toxin concentration of 0.263ppb which is equal to 0.263µg/kg. These indicate that the shellfish mussel tissues contain less amounts of OA-toxins and still can be consumed.

In conclusion, consumption of OA-toxin contaminated of shellfish products in general and mussel products in particular have a zoonotic impact on human consumers. Competitive ELISA is and remains a specific, most effective and less costly and easily available diagnostic tool for OA-toxin identification in shellfish products.