3.3.1 Immunoassays
Specific antibodies, either monoclonal or polyclonal have been produced against a large variety of biotoxins, and a high number of assays based on different formats have been reported, showing high sensitivities for quantitative or semiquantitative
measurements. In the case of marine biotoxins, substantial efforts to develop immunoassays have been undertaken to detect most common groups of toxins in shellfish, DSP, PSP, ASP, NSP, and CFP toxins. Some of them are excellent candidates for the development of a range of functional immunochemical-based detection assays for this group of toxins, as, for example, the assay reported by Steward et al. based on monoclonal antibodies against okadaic acid and dinophysistixins-1,-2 [203]. One of the main problems associated to immunochem-ical approaches to detect marine biotoxins is the lack of the adequate cross-reactivity to detect all the analogs of each group. For some groups, this creates an uncertainty gap that prevents the antibody approach as a useful tool to detect marine biotoxins on a routine basis. For those chemical groups with a reduced number of analogs, as is the case of Domoic acid, immunochemical methods could be suitable approaches. Despite these limitations, immunoassays seem to be a promising tool for routine analysis of shellfish toxins, due to the high sample throughput and relative low cost. Moreover, they require neither sophisticated and expensive equipment nor skilled personnel.
3.3.2 Bioassays
The more classical approach to assess the presence of marine biotoxins in seafood is the in vivo mouse bioassay. It is based on the administration of suspicious extracted shellfish samples to mice, the evaluation of the lethal dose and the toxicity calcula-tion according to reference dose response curves, established with reference mate-rial. It provides an indication about the overall toxicity of the sample, as it is not able to differentiate among individual toxins. This is a laborious and time-consuming procedure; the accuracy is poor, it is nonspecific and generally not acceptably robust. Moreover, the mouse bioassay suffers from ethical implications and it is in conflict with the EU Directive 86/609 on the Protection of Laboratory Animals. Despite the drawbacks, this bioassay is still the method of reference for almost all types of marine toxins, and is the official method for PSP toxins.
The investigation of the presence of marine biotoxins in water, phytoplankton, and food has been achieved by several in vitro assays. However, alternatives to the animal bioassay for marine toxins have not been sufficiently evaluated in interlaboratory studies needed to demonstrate their scientific validity. In addition, these methods continue to be time consuming and expensive for intensive monitor-ing programs, and present some difficulties for their automation.
3.3.3 Instrumental Techniques
A high number of works have been reported the development of MS and LC-MS/MS for the determination of biotoxins in aquatic environments with limits of detection in the range of ng-pg/L concentrations; however, much less work have been performed to detect emerging groups of marine biotoxins such as
(CFP, Palytoxin, Spirolide). Emerging groups of marine biotoxins comprises those groups less studied and without a specific regulation. The main advantages of instrumental approaches based on LC-MS are high selectivity, specificity, and sensitivity. For example, a method based on LC-QqQ-MS operating in selected ion monitoring (SIM) and MRM mode under positive ionization to detect palytoxin has been developed [31]., and it was decisive for the confirmation of these toxins for first time along the Italian coast. Other examples of works carried out during the last year for the analysis of emerging biotoxins are [75, 82,204–206]. In this work, main advantages of instrumental applications have been the confirmation of target groups of toxins. For example, Gonza´lez et al. [82], the presence of free okadaic acid (OA) and dinophysistoxin-2 (DTX-2) as well as esters of these toxins. The results also revealed the presence of minor amounts of 13-desmethyl spirolide C (SPX-1) in the analyzed samples, although this toxin has never been reported before in Spain. The combination of different MS modes of operation, just as enhanced MS (EMS) and MS3experiments, allowed to confirm the first occurrence of spirolides in Spanish shellfish.
However, the major limitations of instrumental analysis for marine biotoxins are, first sometimes the lack of standards, second the required time of analysis, are expensive techniques to be applied in routine analysis but the main limitation is the lack of information about the possible presence of other nontarget marine biotoxins.
4 Occurrence of Selected Groups in Food Samples
In this section, different studies reporting the concentration levels of selected compounds in food samples. A summary is presented in Table2.
4.1 Industrial Origin Compounds
4.1.1 PFCs
A relatively low number of studies have been published on the analysis of PFCs in food samples. A recent study looking at wild fish from Northern Germany revealed levels for PFOS ranging from 8.2 to 225 ng/g fresh weight (fw) [215], which were notably lower than values reported for fish from Japan (3–7,900 ng/g fw) [83] and fish from Spain (0.1–50 ng/g fw) [73] but higher than levels found in freshwater fish from Sweden (0.5–23 ng/g fw). The differences observed in the above studies can be probably attributed to variations in the fat content of the samples analyzed. Mussels and oysters have also shown the presence of PFOS in ranges 0.11–0.59 ng/g ww, along with PFHS (0.06–0.51 ng/g ww), PFBS (0.009–0.030 ng/g ww), PFOSA (0.04–2.96 ng/g ww), PFDoDA (0.2 ng/g ww), PFDA (0.13 and 0.12 ng/g) [70].
Table 2 Occurrence of emerging contaminants in the food chain
Analytes Matrix Levels Reference
PBDEs
PBDEs Chicken fat ∑PBDEs: 1.76–39.43 ng/g [37]
BDE #47, #85, #99, #100,
27 PBDEs congeners Fish and Crabs ∑PBDEs: 135–518 ng/g [90]
BDE #28, #47, #66, #71,
Table 2 (continued)
Edible Fish 0.23-23.04 ng/g fw [73]
PFOS, PFHS, PFBS, PFOSA, PFDoDA, PFUnDA, PFDA, PFNA, PFOA, PFHpA, PFHxA
Mussels, oysters PFOS: 114–586 pg/g (w/w), PFHS: 63–512 pg/g
Farm, pet animals PFOS: 67 ng/g (Chicken), 54 ng/g (pigs), 34 ng/g (cattle)
[72]
(continued)
Table 2 (continued)
Analytes Matrix Levels Reference
4 PFSs, PFOSA, 10 PFCAs Freshwater fish PFOS: 0.5–23 ng/g fresh wt., PFHsS:
0.02–0.8 ng/g, PFOSA:
<0.3–3.3 ng/g
[214]
PFOS, PFOA Wild fish PFOS: 8.2–225 ng/g fw [215]
PFOS, PFOSA, C7-C14
Florfenicol Liver, meat 186.2–1423.5mg/kg [55]
Amphenicols: CAP,
Putative Palytoxin Plankton 1,350 ng (plankton pellet), 1,950 ng (butanol
HBCD hexabromocyclododecane, HCB hexachlorobenzene, p,p0-DDT Dieldrin, 1,1,1-trichloro-2,2-bis-(4-chlorophenyl)-ethane, p,p0-DDE 1,10-dichloro-2,20-bis(4-chlorophenyl) ethylene, p,p0-DDD 1,10-dichloro-2,20-bis-(4-chlorophenyl)ethane, PFOS perfluorooctane sulfonate, PFHS perfluorohexane sulfonate, PFBS perfluorobutane sulfonate
Chicken, pig, and cattle samples have revealed PFOS mean values of 67 ng/g, 54 ng/g, and 34 ng/g, respectively [72] .
4.1.2 PBDEs, PCBs, PBBs
On the other hand, there is a plethora of studies monitoring the occurrence of PBDEs, PCBs, and PBBs in food samples, with the majority of results reporting the occurrence of these compounds in fish, shellfish, and crustacean samples. Levels detected are in the range 3.5–604 ng/g lipid weight (lw) [208], 14–60 ng/g lw [217], 43–192 ng/g lw [44] for the sum of 12 and 10 PBDEs in biota, 7 PBDEs in fish, 20 PBDEs in lower-trophic-level coastal marine species, respectively. An Elisa screen-ing on Hawaiian euryhaline fish and crabs showed ranges of 135–518 ng/g lw [90].
A study on HBCD revealed levels between 90 and 4,863 ng/g ww in fish [218].
However, the levels found for PCBs in bivalves, crabs, and fish samples were significantly higher (14–7,340 ng/g) than PBDE levels [44]. Recent monitoring of dairy products has shown PBDE concentrations in the range of 0.31 ng/g and 0.02–0.96 ng/g for 17 PBDEs in milk and 12 PBDEs in butter, respectively.
Also in the case of butter, PCB levels were higher than the ones for PBDEs with values ranging from 0.14 to 2.52 ng/g [219].
4.1.3 Pesticides
As has been discussed before, recent developments are focused on large multiresidue methods of analysis. Some examples of recent applications of multiresidue methods in cooked and processed food are those reported recently by Kitacawa et al. [210], and Lee et al. [211], Kitakawa et al., presented a methods for the analysis of 222 pesticides in different processed foods. On the other hand, Lee et al. presented a LC-MS/MS method for the quantitative determination of 44 pesticide residues with hydrolyzable functional group in five types of vegetables. In another example [212]. a simple multiresidue method has been developed for the routine determination of 236 pesticides and degradation products in meat-based baby food. This approach combines a modified QuEChERS sample preparation method using a triple partitioning extraction step with water/ACN/hexane and a system composed of GC with programmable temperature vaporization injector hyphenated to an IT-MS.
Another import field of development is the investigation of pesticides transformation products in food. Some examples are the investigation of the acaricide amitraz and its transformation products, dimethylaniline (DMA), 2,4-dimethylformamidine (DMF), and N-2,4-dimethylphenyl-N-methylformamidine (DMPF) in pears [220] Antioxidant pesticides as well as their metabolites used in postharvest treatment have been investigated in pears and apples with concentrations in the ranges 0.002–0.672 ng/g (ethoxyquin), 0.94–11.86 ng/g (imazalil), 0.024–0.902 ng/g (diphenylamine), 0.012–2.59 ng/g (thiabendazole).
The study reveals for the first time the presence of some EQ metabolites in fruits, with levels exceeding several times those of the parent compounds (Pico´ 2010).
About new emerging pesticides such as chiral pesticides most of the works are focused on environmental occurrence [221,222].
4.1.4 Nanomaterials
As we mentioned previously, the research on the field of nanomaterials is at a primitive stage and literature mainly focuses on the benefits of using such particles for environmental load reduction, waste treatment, and source pollution control, as well as the toxicological and health issues accompanying the use of such materials.
As a consequence, there are still few methods developed for food matrices and even lesser monitoring schemes applied. Currently, no data have been noticed reporting the occurrence on nanomaterial residues in food and just one work has been published till now reporting the occurrence of nanoparticle (fullerenes) in real environmental samples [6].
4.2 Pharmaceutical Residues: Antibiotics and Coccidiostats
Unfortunately, in the case of antibiotics and coccidiostats, considering the some-times conflicting interests between the pharmaceutical industry, the regulatory agencies and the scientific community, not many results can be found in the literature regarding survey studies for those substances. A small number of publications are therefore available, mostly in an abstract form, which usually do not emphasize values detected. An application of an enzyme immunoassay for monitoring of milk samples revealed 41.3% of samples (151 total samples) containing antimicrobial residues in Brazil, with one sample exceeding the corresponding MRL (200 mg/kg) for streptomycin-dihydrostreptomycin. Also, 4 samples were above the zero tolerance level for chloramphenicol [223]. In a similar study using a semiquantitative ELISA to monitor 60 ultra-heat-treatment milk samples from Turkey, the authors detected high incidence rate of chloramphenicol (28 samples) and tetracycline (40 samples) [228]. Another study on raw cow’s milk with an LC-UV method revealed low concentrations of tetracycline residues and 51% of the samples containing oxytetracycline [224]. Dairy products, eggs and meat, poultry, and meat tissue samples from Kuwait were screened by the Charm II test and confirmed by LC-MS/MS for tetracycline residues. The study showed 5%
of poultry and 18% of milk samples were above the permitted limits [225]. Finally, honey samples from the Italian market showed a total of 6.3% of all samples containing the antibacterial drugs analyzed with sulphonamides being the most occurring, followed by tetracyclines, streptomycin, tylosin, and chloramphenicol [226].