**3. Rapid immunoreagent monitoring in food safety**

Advanced techniques in liquid chromatography using different detectors (UV-Vis-PDA, FLD, MS and LC-MS/MS) have been introduced for the analysis of chemicals in different matrices (food, microbial/plant metabolites, and water). Chromatographic techniques provide the most reliable data due to their precision and accuracy of analysis; therefore, they have also been recommended for use in evaluating alternative rapid techniques. Analytical methods should be appropriate and efficient for each matrix array, i.e., each modification introduced must be in accordance with validation criteria and the specific requests of regulatory organization.

Incomplete extraction and matrix effects of crude extract in the cleaning step can lead to a subestimation of real concentrations in analysis; thus, a minimum preparation is advantageous. The multi-toxin methods for HPLC and sequential mass spectrometry (LC-MS/MS) provides a high selectivity, lower limits of quantification and detection, the possibility of generating structural information of the analyte with minimal sample treatment, and the reduction of errors associated with pre-and post-column derivatization. Analyses by LC-MS/MS has gained much interest in analytics [14, 15].

Although there have been other advances in analytics, HPLC coupled with fluorescence and ultraviolet detectors remain the main detection method in Brazil [16, 17, 18]. In addition, the unavoidable occurrence of mycotoxins has obliged several countries to adopt regulatory guidelines, and maximum tolerated levels vary widely among countries [19].

Current regulations are increasingly based on international organizations, such as the FAO/WHO Joint Expert Committee on Food Additives of the United Nations (JECFA), and the European Commission. Strict guidelines on mycotoxins have been imposed by importing countries, demanding a rigorous and continuous monitoring of the food chain. The prevailing guidelines for mycotoxins require different protocols of extraction and analysis, and foods for infants and young children with more restrictive limits increases the number of analyses [20]. Such diversity in extraction procedures results in costly work.

Safe raw materials should be tracked by reliable analytical methods, and rapid methods are useful tools, especially in food-producing countries. Immunoassays based on ic-ELISA with highly specific monoclonal antibodies (MAb) against ochratoxin (OTA), fumonisin (FB), aflatoxin (AF), deoxynivalenol (DON), zearalenone (ZEA) and microcystin (MC) have been developed, previously tested for cross-reactivity with each analogue group, and correlated with HPLC as the primary method (Table 1, 2 and 3). A careful evaluation of ic-ELISA was conducted in the analysis of natural toxins in the food chain targeted to field/storage stage, beginning with the monitoring of fumonisins in corn [21]. The successful rapid technique motivated to use of this analysis for OTA in coffee and wine [22, 23], aflatoxin [24, 25], DON [26, 27, 28] and ZEA [29].

fumonisin monitoring in 300 freshly harvested corn (2003 and 2004 crops) samples collected at two points of the production chain (reception and pre-drying) in Northern Paraná State. Based on the highest mean fumonisin levels being detected in the pre-drying samples (3.12 μg g-1) and the average consumption of corn-based products, the maximum probable daily intake (PDIM) of FB1 estimated in the Brazilian population (0.95 μg kg-1 body weight day-1) was below

Such monitoring allowed the identification of fumonisin levels in different regions of the state, enabling it to gain a prominent position in corn exportation. Currently, the State of Paraná is responsible for 14.3 million tons/year, corresponding to 17.9 % of the national

Advanced techniques in liquid chromatography using different detectors (UV-Vis-PDA, FLD, MS and LC-MS/MS) have been introduced for the analysis of chemicals in different matrices (food, microbial/plant metabolites, and water). Chromatographic techniques provide the most reliable data due to their precision and accuracy of analysis; therefore, they have also been recommended for use in evaluating alternative rapid techniques. Analytical methods should be appropriate and efficient for each matrix array, i.e., each modification introduced must be in accordance with validation criteria and the specific requests of regulatory organization.

Incomplete extraction and matrix effects of crude extract in the cleaning step can lead to a subestimation of real concentrations in analysis; thus, a minimum preparation is advantageous. The multi-toxin methods for HPLC and sequential mass spectrometry (LC-MS/MS) provides a high selectivity, lower limits of quantification and detection, the possibility of generating structural information of the analyte with minimal sample treatment, and the reduction of errors associated with pre-and post-column derivatization. Analyses by LC-MS/MS has gained

Although there have been other advances in analytics, HPLC coupled with fluorescence and ultraviolet detectors remain the main detection method in Brazil [16, 17, 18]. In addition, the unavoidable occurrence of mycotoxins has obliged several countries to adopt regulatory

Current regulations are increasingly based on international organizations, such as the FAO/WHO Joint Expert Committee on Food Additives of the United Nations (JECFA), and the European Commission. Strict guidelines on mycotoxins have been imposed by importing countries, demanding a rigorous and continuous monitoring of the food chain. The prevailing guidelines for mycotoxins require different protocols of extraction and analysis, and foods for infants and young children with more restrictive limits increases the number of analyses [20].

Safe raw materials should be tracked by reliable analytical methods, and rapid methods are useful tools, especially in food-producing countries. Immunoassays based on ic-ELISA with

guidelines, and maximum tolerated levels vary widely among countries [19].

Such diversity in extraction procedures results in costly work.

the tolerable daily intake (2.0 μg kg-1 body weight day-1).

**3. Rapid immunoreagent monitoring in food safety**

corn production [3].

142 Food Production and Industry

much interest in analytics [14, 15].


AFB1: Aflatoxin B1; AFB2: Aflatoxin B2; AFG1: Aflatoxin G1; AFG2: Aflatoxin G2; AFM1: Aflatoxin M1; DON: Deoxynivalenol; NIV: Nivalenol; ZEA: Zearalenone; OTA: Ochratoxin A; OTC: Ochratoxin C; OTB: Ochratoxin B; FB1: Fumonisin B1; FB2: Fumonisin B2; FB3: Fumonisin B3; MCLR: Microcystin-LR; MCRR: Microcystin-RR; MCYR: Microcystin-YR; and MCLA: Microcystin-LA.

\* Percentage of relative cross-reactivity was calculated as the amount of toxin required for 50 % binding inhibition/amount of other toxins requiring 50 % binding inhibition × 100.

\*\* Others: 3-acetyl DON, 3,4-diacetyl NIV, tetraacetyl NIV, Toxin T-2, Toxin T-2 acetyl, and diacetoxyscirpenol.

[30] Kawamura et al., 1988; [31] Kawamura, 2005; [32] Kawamura et al., 1989; [33] Iijima et al., 1996; [34] Kawamura e Emoto, 2006; [35] Nagata et al., 1995; [36] Tabuchi et al., 2015.

**Table 1.** Cross-reactivity of monoclonal antibodies (anti-mycotoxins & microcystins) applied in a monitoring study in Brazil.

Table 1 shows the cross-reactivity of MAb (anti-mycotoxins & microcystins). It confirmed the high specificity of selected hybridomas, which were adequate for application in rapid surveys. Cross-reaction in immunoassays would be expected due to the biosynthesis of natural toxins in a sequential cluster of closely related structural substances. Nevertheless, the crossreactivity within analogues can be advantageous in screening surveys of natural toxins compared with strongly specific individual analogue detection by HPLC.

Table 2 shows how ic-ELISA became established as reliable rapid technique to analyse mycotoxins and microcystins. Such local set-ups can allow safe supervision in one of the major food producing regions in Brazil, which was made possible due to joint research involving cell culture technologies, adaptation and proliferation of MAb producing hybridomas, and the development of immunoassays *in loco*. The standardized immunoassay was obtained through enhancing its sensitivity and adjusting to local conditions for the reagents, dilutions in ic-ELISA steps (upgrading crude extract preparation, antigen-protein conjugates for microplate coating, and dilutions of both the first and second antibody), and the analogue group for detection. The safety of the food, derived products, and water for analysis were amplified by awareness. These assays should be conducted for local consumption safety, the balance of agribusiness and exportation demand and importation independence.



ic-ELISA: Indirect competitive enzyme linked immunosorbent assay.

Table 1 shows the cross-reactivity of MAb (anti-mycotoxins & microcystins). It confirmed the high specificity of selected hybridomas, which were adequate for application in rapid surveys. Cross-reaction in immunoassays would be expected due to the biosynthesis of natural toxins in a sequential cluster of closely related structural substances. Nevertheless, the crossreactivity within analogues can be advantageous in screening surveys of natural toxins

Table 2 shows how ic-ELISA became established as reliable rapid technique to analyse mycotoxins and microcystins. Such local set-ups can allow safe supervision in one of the major food producing regions in Brazil, which was made possible due to joint research involving cell culture technologies, adaptation and proliferation of MAb producing hybridomas, and the development of immunoassays *in loco*. The standardized immunoassay was obtained through enhancing its sensitivity and adjusting to local conditions for the reagents, dilutions in ic-ELISA steps (upgrading crude extract preparation, antigen-protein conjugates for microplate coating, and dilutions of both the first and second antibody), and the analogue group for detection. The safety of the food, derived products, and water for analysis were amplified by awareness. These assays should be conducted for local consumption safety, the balance of

**Cell line products Mycotoxin/**

**LOD / LOQ (µg kg-1)**

**Second Ab: IgGenzyme**

2 μg mL-1 1200 μg mL-1 1:2000 177.1 / - 0.93 Wheat grain[26];

2 μg mL-1 19.2 μg mL-1 1:1000 113.5 / 445.3 Wheat grain[28]

2 μg mL-1 10.9 μg mL-1 1:2000 159.3 / 370 - Biscuita

2.5 μg mL-1 10.3 μg mL-1 1:2000 33.7 / 87 - Wheat graina

2.5 μg mL-1 10.3 μg mL-1 1:2000 9.7 / 23.7 - Biscuita

0.77 μg mL-1 1:50 1:5000 93 / - 0.94

0.25 μg mL-1 0.094 μg mL-1 1:2000 2.0 / 4.6 -

2 μg mL-1 10.9 μg mL-1 1:2000 302.8 / 589.3 -

**ELISA/ HPLC (r)**

**Cereal &**

wheat flour[27]

Corn graina,[21]

compared with strongly specific individual analogue detection by HPLC.

agribusiness and exportation demand and importation independence.

**Cyanotoxin Coating First MAb**

DON-HG-OVA

DON-HS-OVA

DON-HS-OVA

ZEN-OVA

ZEN-OVA

FB1- OVA

AFB1-BSA

DON-HS-OVA

9 ZEN.2 ZEA ZEN-OVA 10.3 μg mL-1 1:2000 51.7 / 93.2 0.91

**Hybridoma Toxins Reagents: ic-ELISA steps**

**Item**

144 Food Production and Industry

1

2

3

4

5

DON.3 DON

ZEN.2 ZEA

6 FB 1-2 FB1

7 AF.2 AF

8 DON.3 DON

DON: Deoxynivalenol; ZEA: Zearalenone; FB1: Fumonisin B1; AF: Aflatoxin; OTA: Ochratoxin A; MCLR: Microcystin-LR; DON-HG-OVA: Deoxynivalenol-hemiglutarate-ovalbumin; DON-HS-OVA: Deoxynivalenol-hemisuccinateovalbumin; ZEN-OVA: Zearalenone-ovalbumin; AFB1-BSA: Aflatoxin B1- Bovine Serum Albumin; OTA-BSA: ochratoxin A- Bovine Serum Albumin; and MCLR-BSA: Microcystin-LR - Bovine Serum Albumin.

[26]Santos et al. 2011; [27] Santos et al. 2013; [28] Souza et al. 2014; [21] Ono et al. 2001; [24] Rossi et al. 2013a; [25] Rossi et al. 2013b; [22] Fujii et al, 2006; [37] Kamogae et al. 2006; a Data not published.

**Table 2.** Development of ic-ELISA: standardized immunoassay for mycotoxins and microcystins analysis.

The optimized ic-ELISA showed a correlation coefficient of >0.9 with HPLC (Table 2). The result obtained with anti-OTA MAb produced by hybridoma OTA.1 was adequate to analyse wine using 1:10,000 anti-OTA MAb and 1:30,000 OTA-BSA. However, the matrix interference in the OTA analysis in wine by ic-ELISA should be considered. In analysing 60 wine samples, only one was OTA positive by HPLC (0.12 ± 0.01 ng mL-1), whereas 11 false-positives were observed by ic-ELISA (range from 0.32 ± 0.02 to 0.47 ± 0.14 ng mL-1). False-positive data in red wine may be attributed to the interference of anthocyanins and other pigments on OTAbinding to the antibody [38, 39]. The influence of matrix interference in OTA detection by ic-ELISA could be explained using a principal component analysis through the relationship of higher *trans*-resveratrol and OTA levels in the positive samples (Figure 2). In contrast, the addition of condensed tannins can inhibit the binding activity of antibodies in ELISA [40].

The undesired matrix effect and be minimized by diluting the crude extract prior to ic-ELISA; a 1:100 dilution of coffee extract minimized the matrix effect on OTA detection, regardless of the maturity stage [22]. Additionally, a dilution factor of 1:80 minimized the matrix effect when anti-DON MAb produced by Hybridoma DON.3 was used in ic-ELISA for wheat grain.

**Figure 2.** Principal Component Analysis in the evaluation of wine samples, Paraná State: OTA (ochratoxin A), CAO (antioxidant capacity), *trans*-resveratrol and chromatic (hgraus, L\*, C\*). a) graphical representation in two dimensions (Principal Component 1- CP1 and Principal Component 2- CP2) and b) cluster analysis of parameters.

Table 3 shows the monitoring of natural toxins (mycotoxins & microcystins) by ic-ELISA developed for different food specimens, as well as in the freshwater since the 1990s. Corn, coffee, wheat, grain-derived products, wine, broiler and laying hen feeds, and fresh water in agricultural regions were analysed (Table 3). The table also shows some maximum limits established by Brazilian guidelines, the European Commission, and the World Health Organization.



ic-ELISA: Indirect competitive enzyme linked immunosorbent assay.

\* These analysis were carried out using a commercial Kit (Beacon Microcystin Plate Kit, USA).

PR: Paraná State; RS: Rio Grande do Sul State.

Table 3 shows the monitoring of natural toxins (mycotoxins & microcystins) by ic-ELISA developed for different food specimens, as well as in the freshwater since the 1990s. Corn, coffee, wheat, grain-derived products, wine, broiler and laying hen feeds, and fresh water in agricultural regions were analysed (Table 3). The table also shows some maximum limits established by Brazilian guidelines, the European Commission, and the World Health

**Figure 2.** Principal Component Analysis in the evaluation of wine samples, Paraná State: OTA (ochratoxin A), CAO (antioxidant capacity), *trans*-resveratrol and chromatic (hgraus, L\*, C\*). a) graphical representation in two dimensions

(Principal Component 1- CP1 and Principal Component 2- CP2) and b) cluster analysis of parameters.

**SAMPLING / MONITORING Ic-ELISA**

**Feed, Wine[41, 42] Mycotoxina (n) (µg kg-1)**

2006 – 2008

Flour North/ PR 2009 21 / 23 2455.9

2010 - 2011

DON

North/PR 2009 36 / 50 2379.4

North /PR 159 / 160 848.9

2006 - 2008 7 / 23 1578.6

2013 29 / 56 742.4

**Toxins +/total Mean**

15 / 15 2918.1

84 / 84 1879.3

**Locality Crop Year**

North-West, North-East, South-West /RS

North, Central, South-West /PR

Market)

4 Grain North, Central-South 2010 - 2011 ZEA 11 / 125 161.4

Organization.

146 Food Production and Industry

**Cereal & products,**

2 Grain Central-South /PR

<sup>3</sup> Biscuits North /PR (Retail

1 Wheat Grain

**Item**

[41] The Brazilian Health Surveillance Agency (ANVISA, 2011) established the maximum levels of DON in flour and biscuits (1,750.0 μg kg-1), ZEA for biscuits (200.0 μg kg-1), AF for corn (20.0 μg kg-1), and OTA for wine (2.0 μg kg-1). The deadlines were established in RDC 07/2011 and will be extended to January 1, 2017 for DON in wheat and corn (3,000.0 μg kg-1), ZEA in wheat and corn (400.0 μg kg-1), and FB1 + FB2 in corn (5,000.0 μg kg-1) [42].

[43] The World Health Organization (WHO, 1998) established maximum levels of 1 μg of MCLR L-1 for drinking water and a Tolerable Daily Intake of 0.04 μg of MCLR kg-1 body weight.

c Mycotoxin / Cyanotoxin: DON, Deoxynivalenol; ZEA, Zearalenone; FB1, Fumonisin B1; AF, Aflatoxin; OTA, Ochratoxin A; and MCLR, Microcystin-LR.

[44] European Commission (2003). The maximum limit allowed by the European Commission is 0.02 mg aflatoxin B1 kg-1.

**Table 3.** Monitoring of natural toxins (mycotoxins & microcystins) by the rapid ic-ELISA method.

The application of ic-ELISA to monitoring freshly harvested corn from Paraná State (1991 to 2004 crops) indicated the widespread occurrence of fumonisins but a low occurrence of aflatoxins. In a recent study conducted in Paraná State, 74 corn samples were contaminated with an average of 1,840 μg of fumonisin kg-1, 36 of poultry feeds with 239 μg of fumonisin kg-1, and 9 corn factory residues with 23,676 μg of fumonisin kg-1, whereas the aflatoxin and trichothecene levels were approximately at the LOD values [45]. Ic-ELISAs, using monoclonal mAb produced by hybridoma cells (AF.2, ZEN.2 and DON.3), were developed and optimized for AFs, ZEA and DON detection (Table 2). In corn samples from an experimental farm in central-southern Paraná State, 12 samples were found to be positive for AF (mean of 8.1 μg kg-1), 36 samples for ZEA (mean of 522.3 μg kg-1) and 6 samples for DON (mean of 2142.3 μg kg-1) (Table 3).

An emphasis was placed on DON evaluations by ic-ELISA in wheat from 2006 to 2011 (Table 3). Paraná and Rio Grande do Sul States in southern Brazil produce 90 % of the national wheat [1]. This country depends on the importation of 5 to 6 million ton per year to provide for an annual domestic consumption of approx. 11 million ton, mainly used in bakery (55 %), pasta (17 %) and biscuit (13 %) processing [2, 46, 47]. Brazil is the world's second-largest biscuit producer, but the current low exportation (54,083 tons) results in nearly all production earmarked for domestic consumption, despite its ranking [48, 49]. In the wheat samples from experimental farms of north and central-southern of Parana State analysed by ic-ELISA, DON was detected in almost all of samples (243 positive samples of 244) and ZEA was detected in 10 of 125 samples (Table 3). In, wheat-based biscuits acquired from a local retail market in Londrina, Paraná State (56 samples) DON was detected in 29 samples (mean of 742.4 μg kg-1) and ZEA in 17 samples (mean of 56.1 μg kg-1) (Table 3). A study [50] analysed 23 cracker biscuit samples produced in Southern Brazil and group A trichothecene was non-detectable, but 18 samples were contaminated with DON (378 – 5295 μg kg-1), with 22 % of the samples at level over the Brazilian guideline limit (1,750 μg kg-1). When zearalenone was analysed in cornbased products (51 samples of popcorn and 50 corn grits) and cracked wheat (*n* = 109) commercialized in 18 counties of the Paraná state, ZEA was non-detected in cracked wheat samples, but one cracked corn sample contaminated 64 μg of ZEA kg-1 [50]. Fusariotoxin monitoring in wheat should be conducted in both domestic production and in imported wheat, which represents 50 % of the category.

Due to the possible carry-over of mycotoxins to tissues, the degree of exposure of broiler chicken and laying hens to fumonisins and aflatoxins through naturally contaminated feeds has been assessed (Table 3). Occurrence of fumonisins and aflatoxins were evaluated in four feed types intended for broilers (n=158), collected from a poultry breeding farm in Northern Paraná State [24]. Fumonisins were detected in 94.9 % of the feed samples at mean levels ranging from 0.52 μg g-1 (finisher) to 0.68 μg g-1 (pre-starter and grower), and aflatoxins were detected in 72.1 % of the feed samples at mean levels ranging from 2.22 ng g-1 (pre-starter) to 6.41 ng g-1(grower). The maximum estimated daily intake of FB1 for broilers (0.057 mg/kg body weight/day) was below the Lowest Observed Adverse Effect Level (2 mg kg-1 body weight day-1). Most of the aflatoxin positive samples (97 %) showed levels below the maximum limit allowed by the European Commission (0.02 mg aflatoxin B1 kg-1). To estimate the degree of exposure of laying hens to mycotoxins, a total of 95 mash feed samples were collected from January to December 2010 from the Experimental Farm at the University, Northern Paraná State, Brazil. Aflatoxins and fumonisins were detected in 69.7 % and 89.5 % of the feed (n=95) intended for laying hens at mean levels of 9.61 ng g-1 and 1.28 μg g-1, respectively. The estimated daily intake of FB1 for laying hens (0.038 mg kg-1 body weight day-1) was below the Lowest Observed Adverse Effect Level (2 mg kg-1 body weight day-1). Aflatoxin levels were below the maximum allowed limit by the European Commission in the majority of the positive samples (85.1 %), which indicated that some of the feed samples could have a negative effect on animal health and performance, but the risk would be very low.

The application of ic-ELISA to monitoring freshly harvested corn from Paraná State (1991 to 2004 crops) indicated the widespread occurrence of fumonisins but a low occurrence of aflatoxins. In a recent study conducted in Paraná State, 74 corn samples were contaminated with an average of 1,840 μg of fumonisin kg-1, 36 of poultry feeds with 239 μg of fumonisin kg-1, and 9 corn factory residues with 23,676 μg of fumonisin kg-1, whereas the aflatoxin and trichothecene levels were approximately at the LOD values [45]. Ic-ELISAs, using monoclonal mAb produced by hybridoma cells (AF.2, ZEN.2 and DON.3), were developed and optimized for AFs, ZEA and DON detection (Table 2). In corn samples from an experimental farm in central-southern Paraná State, 12 samples were found to be positive for AF (mean of 8.1 μg kg-1), 36 samples for ZEA (mean of 522.3 μg kg-1) and 6 samples for DON (mean of 2142.3 μg

An emphasis was placed on DON evaluations by ic-ELISA in wheat from 2006 to 2011 (Table 3). Paraná and Rio Grande do Sul States in southern Brazil produce 90 % of the national wheat [1]. This country depends on the importation of 5 to 6 million ton per year to provide for an annual domestic consumption of approx. 11 million ton, mainly used in bakery (55 %), pasta (17 %) and biscuit (13 %) processing [2, 46, 47]. Brazil is the world's second-largest biscuit producer, but the current low exportation (54,083 tons) results in nearly all production earmarked for domestic consumption, despite its ranking [48, 49]. In the wheat samples from experimental farms of north and central-southern of Parana State analysed by ic-ELISA, DON was detected in almost all of samples (243 positive samples of 244) and ZEA was detected in 10 of 125 samples (Table 3). In, wheat-based biscuits acquired from a local retail market in Londrina, Paraná State (56 samples) DON was detected in 29 samples (mean of 742.4 μg kg-1) and ZEA in 17 samples (mean of 56.1 μg kg-1) (Table 3). A study [50] analysed 23 cracker biscuit samples produced in Southern Brazil and group A trichothecene was non-detectable, but 18 samples were contaminated with DON (378 – 5295 μg kg-1), with 22 % of the samples at level over the Brazilian guideline limit (1,750 μg kg-1). When zearalenone was analysed in cornbased products (51 samples of popcorn and 50 corn grits) and cracked wheat (*n* = 109) commercialized in 18 counties of the Paraná state, ZEA was non-detected in cracked wheat samples, but one cracked corn sample contaminated 64 μg of ZEA kg-1 [50]. Fusariotoxin monitoring in wheat should be conducted in both domestic production and in imported wheat,

Due to the possible carry-over of mycotoxins to tissues, the degree of exposure of broiler chicken and laying hens to fumonisins and aflatoxins through naturally contaminated feeds has been assessed (Table 3). Occurrence of fumonisins and aflatoxins were evaluated in four feed types intended for broilers (n=158), collected from a poultry breeding farm in Northern Paraná State [24]. Fumonisins were detected in 94.9 % of the feed samples at mean levels ranging from 0.52 μg g-1 (finisher) to 0.68 μg g-1 (pre-starter and grower), and aflatoxins were detected in 72.1 % of the feed samples at mean levels ranging from 2.22 ng g-1 (pre-starter) to 6.41 ng g-1(grower). The maximum estimated daily intake of FB1 for broilers (0.057 mg/kg body weight/day) was below the Lowest Observed Adverse Effect Level (2 mg kg-1 body weight day-1). Most of the aflatoxin positive samples (97 %) showed levels below the maximum limit allowed by the European Commission (0.02 mg aflatoxin B1 kg-1). To estimate the degree of

kg-1) (Table 3).

148 Food Production and Industry

which represents 50 % of the category.

Intensive agricultural activity has become an increasing concern due to the eutrophication of aquatic environments. Microcystins (MCs) were monitored in Itaipu Lake and Tibagi River in the north and west of Paraná State, respectively (1999 to 2000 and 2014). The reduction of microcystin levels in Itaipu Lake was likely a consequence of ecological programs encouraging the recovery of riparian forests, in addition to a change in planting management (Table 3). Such a reliable MAb-based rapid immunoassay has been a good choice for tracking mycotoxins and cyanotoxins and determining actions to be performed in a control strategy.
