Service for the identification and quantification of algal toxins in aquatic and tissue matrices

Microcystin contamination in fish tissue is a recent detection in Italian lakes and its monitoring requires fast and precise analysis techniques, easy to apply and capable of providing results in real time. Instrumental chemical analysis is mostly used to confirm and identify the different variants of microcystins.

Given the majority percentage of toxins directly combined with the cells in the tissues (largely invisible to instrumental methods) compared to the free ones, and the need to use different standards for the detection of the single compounds, the simultaneous use of the ELISA analysis guarantees a rapid quantitative measurement of the total amount of MCs present, useful from the point of view of risk assessment. Studies performed on Italian lakes have shown that ELISA test results showed MC concentrations 3 to 8 times higher than the ones calculated from LC-MS/MS analyses¹⁾.

Chlorophyll is a molecule consisting of a porphyrin ring whose four central nitrogen atoms are coordinated with a central Mg²⁺ ion and a long phytol hydrocarbon side chain. It has the function of electron acceptor during the excitation caused by photons and therefore allows the start of the photochemical synthesis which allows plants and algae to have an autotrophic metabolism.

By determining the chlorophyll a in a sample, it is possible to evaluate the overall quantity of algae that constitute the phytoplankton in suspension. After having concentrated the phytoplankton cells on a filter by filtration of at least 250 mL of sample, the chlorophyll a is extracted in aqueous solution of acetone at 90% and its determination is carried out by spectrophotometry, making use of the capacity of this pigment to absorb in the lengths waveforms of the visible spectrum corresponding to red. The absorption peak of chlorophyll a, in fact, falls between 0.5 and 2.0 µm: the absorbance of the supernatant is measured at the wavelengths of 750, 663, 645, 630 nm using a blank consisting of the solution of 90% acetone. Reading the extract at 750 nm provides a measure of the turbidity of the extract which should not exceed 0.01 absorbance units. Otherwise, the clarification procedure must be repeated. Finally, to calculate the results, it is necessary to insert the absorbances (A) as indicated in Strickland & Parsons (1997) ²⁾:

where:

Ve = volume (mL) of the extract
Vf = volume (mL) of the filtrate

The samples are placed to settle in special plexiglass wells with a transparent bottom and a capacity of 25 mL for a period of 24 ~ 48 hours, then examined with an invertoscope. The algal counting techniques used are that by strip and that by field. The stripe technique recommends counting perpendicular strips through the center of the chamber. The width of the stripes is constant for each set of samples. The following formula allows the calculation of the number of cells per milliliter:

where C is the number of counted organisms; At is the total area of the chamber base in mm²; L is the length of a strip in mm; W is the width of a strip in mm; S is the number of strips counted and V is the sample volume in the chamber in mL. The field count technique involves randomly examining non-overlapping fields to count 100 units of the dominant species. The calculation is performed using the formula:

where Af is the area of a field in mm² and F is the number of fields counted.

The water samples are extracted as follows: aliquots of fresh phytoplankton (10 ~ 130 mg) obtained by centrifugation of water samples are suspended in 1 mL of sterile bidistilled water. The solution is stirred, sonicated for 5 minutes. at 30 ~ 40 °C in an ultrasonic bath (with vortexing for 20 seconds halfway through), then centrifuged for 10 min at 11,000 rpm (Beckman L5–55 Ultracentrifuge) to remove residue. The supernatant is collected, the pellet is suspended again and the whole process is repeated twice. The two supernatants are pooled and analyzed for intracellular microcystins³⁾.

Approximately 5 g of tissue is homogenized for 15 seconds with 10 mL of 100% methanol (MeOH) (HPLC purity) and centrifuged at 5000 g for 5 minutes. The extraction is repeated on the pellet and the collected supernatants are then filtered through paper filters, the filtrates pooled and made up to a volume of 25 mL with 100% MeOH; 5 mL of the volume obtained is diluted with 5 mL of distilled water and loaded onto a preconditioned WatersTM Oasis HLB 6 cc Vac Cartridge with 1 mL of 100% MeOH followed by 1 mL of distilled water. The sample is eluted with 1 mL of 5% MeOH/H2O and then collected with 1 mL of MeOH. The 100% MeOH fraction is dried and then dissolved in water (2 mL). This suspension is stored at -30°C for subsequent analysis of microcystins (MC)⁴⁾.

Approximately 5 g of tissue is homogenized in 10 mL 100% MeOH for 15 min, then sonicated 5 min at 30 ~ 40 °C in an ultrasonic bath (at 25 kHz) at room temperature, to disrupt the cells. The homogenate is then centrifuged for 5 min at 5000 g and the supernatant decanted and filtered. The extraction is repeated on the pellet, the sample is centrifuged and the supernatant is filtered on the same filter previously used. The filter and funnel are washed three times with small volumes of MeOH; the two extracts and the washings are collected together, then dried with a Rotavapor at 40°C; the residue resuspended in 2 mL of distilled water is then stored at -30 °C until analysis.

Approximately 5 g of muscle tissue is homogenized for 15 min and then freeze-dried. The lyophilisate is pulverized and a 100 mg aliquot is placed in a polypropylene vial with 3 mL of 0.1 N trichloroacetic acid (TCA), dispersed by vortexing and extracted on a roller mixer for 1.5 h. After centrifugation for 10 minutes at 3500 g, the supernatant is transferred to filter tubes (PVDF filter, 5 µm) and further centrifuged for 10 minutes at 3500 g. The supernatant is then purified using cation exchange cartridges (Oasis-MCX, 6 cc, Waters) previously conditioned and activated with 5 mL of methanol followed by 5 mL of distilled water; after loading the sample, the cartridge is washed with 5 mL of 0.1 N hydrochloric acid; the toxin is eluted with 4 mL of 2% ammonium hydroxide solution in methanol/water (75/25, v/v). The eluted solution is then dried at 50 °C under a gentle stream of nitrogen and the residue dissolved in 5 mL of distilled water for ELISA examination.

The anti-ADDA assay (ABRAXIS, USA) is an indirect competitive ELISA (Enzyme Linked Immunosorbent Assay) for the independent detection of microcystin and nodularin congeners. It is based on the recognition of microcystins, nodularins and their congeners by specific antibodies. The toxin, if present in a sample, and an immobilized microcystin peptide analog on the plate compete for the binding sites of anti-microcystin/nodularin antibodies in solution. The plate is then washed and a second antibody-HRP (Horseradish Peroxidase) is added.

After a second washing step and addition of substrate, a blue color is generated in the solution. The intensity of the color is inversely proportional to the concentration of microcystins present in the sample. The colorimetric reaction is stopped after a specific time and the color intensity is measured using an ELISA spectrophotometric reader. Sample concentrations are determined by interpolation using the standard curve constructed with each reading. The test does not distinguish between Microcystin-LR and other microcystin variants, but detects their global presence. The Limit of Detection (LOD) for this test, based on MC-LR, is 0.10 ppb (μg/L). The coefficients of variation (CV%) for the standards are less than 10%; for samples below 15%.

Samples of water, cyanobacterial cell extracts, and tissue extracts are analyzed using the Abraxis ELISA for cylindrospermopsins (Abraxis Bioscience CA), following the manufacturer's instructions and using the suggested calibration concentrations. The Abraxis ELISA immunoassay claims a Limit of Detection (LOD) of 0.04 ng/mL, with a percent coefficient of variation (CV%) of less than 10% for the standard and less than 15% for the samples.

The currently available test (ABRAXIS, USA) is a direct competitive ELISA based on the recognition of BMAA by specific antibodies. BMAA, if present in a sample, and a BMAA-HRP analogue compete for the binding sites of rabbit anti-BMAA antibodies in solution. The BMAA antibodies are then bound by a second immobilized antibody on the wells of the microtitre plate. After a wash step and addition of substrate solution, a color signal is generated. The intensity of the blue color is inversely proportional to the concentration of BMAA present in the sample. The color reaction is stopped after a specified time and the color is evaluated using an ELISA microplate reader. Sample concentrations are determined by interpolation using the standard curve constructed with each test. The LOD for this test, based on MC-LR, is 4 μg/L. The coefficients of variation (CV%) for the standards are less than 10%; for samples below 15%.