Food Toxins Research Articles

Conducting Molecularly Imprinted Polymer (MIP) Chemical Sensors for Toxic N-Nitrosamines Selective Determination in Heat Processed Food of Animal Origin

High-temperature processing of meat food generates in it toxic heteroaromatic amines and nitrosamines (NAs)1,2 causing chronic diseases including hormonal dysfunctions and cancer.3 Therefore, we devised and fabricated chemical sensors for selective determination of the N-nitroso-L-proline (Pro-NO)4 and N-nitroso-thiazolidine-4-carboxylic acid (NT4A) toxins in this food. We applied films of conducting polymers molecularly imprinted (MIPs) with either the Pro-NO or NT4A template as recognition units of these chemosensors. For selection of most appropriate functional monomers, we used DFT at the B3LYP/3-21G(*) level modeling to find phenolic derivative of bis(bithiophene) and a thiosalenCo(III) complex (CS)5,6 suitable to form pre-polymerization complexes with Pro-NO and NT4A, respectively, at the 1:2 template/monomer stoichiometry. The calculated Gibbs free energy gain due to formation of these complexes with a series of NAs was proportional to the UV-vis spectroscopy determined stability constants of these complexes. These constants were relatively high equalling 3.14×109 M-2 and 2.72×1010 M-2for Pro-NO and NT4A, respectively. Then, we electrochemically polymerized these complexes under potentiodynamic conditions. This electropolymerization resulted in depositing thin MIP films on different electrode substrates. After subsequent extraction of the Pro-NO and NT4A templates from MIPs, we used the resulting MIPs with empty imprinted cavities for determination of Pro-NO and NT4A analytes in grilled pork neck samples. We confirmed the presence and then absence of NA templates in the MIP films before and after extraction, respectively, with FTIR spectroscopy and XPS, and then characterized morphology of the films with AFM. For analytical signal transduction, we used piezoelectric microgravimetry (PM) at the Au-QCR/MIP electrodes of EQCM as well as DPV and EIS at the Pt/MIP electrodes. Limits of detection of the chemosensors were 36.9 nM Pro-NO (with EIS) and 46 nM NT4A (with DPV). The chemosensors selectively responded to the target toxins in the presence of interfering compounds of similar composition and structure. Successful selective determination of Pro-NO and NT4A in the food extract samples indicates that the chemosensors are promising as tools for determination of these toxins in food of animal origin.

Cyanobacterial Toxins in Freshwater and Food: Important Sources of Exposure to Humans.

A recent ecological study demonstrated a significant association between an increased risk of nonalcoholic liver disease mortality and freshwater cyanobacterial blooms. Moreover, previous epidemiology studies highlighted a relationship between cyanotoxins in drinking water with liver cancer and damage and colorectal cancer. These associations identified cyanobacterial blooms as a global public health and environmental problem, affecting freshwater bodies that are important sources for drinking water, agriculture, and aquafarms. Furthermore, as a result of climate change, it is expected that our freshwater environments will become more favorable for producing harmful blooms that produce various cyanotoxins. Food is an important source of cyanotoxin exposure to humans, but it has been less addressed. This paper synthesizes information from the studies that have investigated cyanotoxins in freshwater and food on a global scale. We also review and summarize the health effects and exposure routes of cyanotoxins and candidates for cyanotoxin treatment methods that can be applied to food.

Nanosensors for food quality and safety assessment

Food toxins are produced as defense tools by microorganisms that use nutrients for their growth. Microorganisms thus spoil food, taste and can infect humans, sometimes leading to death. Food adulteration and brand protection are also major issues in the food industry. Here we review the use of nanomaterials for sensing food quality. Nanosensors can detect pathogenic bacteria, food-contaminating toxins, adulterant, vitamins, dyes, fertilizers, pesticides, taste and smell. Food freshness can be monitored using time–temperature and oxygen indicators. Product authenticity and brand protection can be assessed using invisible nanobarcodes. Overall, nanosensors with unique properties are improving food security.

Short inventory of EU legislation on plant toxins in food

Plant toxins, secondary metabolites that are not essential for the survival of the organism itself but are toxic to human health, are produced by many plants. Plant toxins can be present as inherent metabolites in daily foods such as potatoes, herbs and spices or in herbal preparations. Plant toxins can be present as contaminants in foods as a result of unintentionally co-harvested weeds, transfer from feed to products of animal origin, or as residue after application of plant toxins as natural pesticides. Incidents with plant toxins are reported in Europe, mainly as a result of mixing-up of plant species used in herbal remedies or similarities between edible crops and certain weeds. EU legislation on plant toxins in food, if existing at all, is scattered over different legal acts, such as acts on contaminants, flavourings and residues. Limits for plant toxins are, furthermore, mentioned in product specifications of approved novel foods and thus related to one specific plant product. It was concluded that there is a need for a more coherent legislation on plant toxins in food in the EU to ensure consumer health.

Analysis of Food Toxin Trends in the Federation of Bosnia and Herzegovina Over Five Years Period.

Introduction:Alimentary toxoinfections represent a significant public health problem. Globalization of the market and food production, significant impoverishment of a large part of the population, and traditional approach with food preparation and consumption, cause a significant increase in the rates of population infections around the world. The epidemiological surveillance of the illness occurrence plays a significant role in monitoring and controlling the population’s burden of diseases caused by unhygienically prepared and stored food.Aim:The aim of the article is to determine the rates and trends of food related diseases in the Federation of Bosnia and Herzegovina.Results:The results of the analysis have shown that the rate of illness in the Federation of Bosnia and Herzegovina is declining but is still significantly higher than in developed western countries. Particularly great burden is on the population of the Zenica-Doboj Canton (ZDC), which can be due to the traditional relation to the preparation and storage of food, as well as to the relatively poorer economic situation in ZDC.Conclusion:We can conclude that the strengthening of the monitoring system, laboratory capacities, the availability of monitoring guides will enable responsible FBiH/Bosnia and Herzegovina institutions to better control and implement safer food practice.

Eating when ill is risky: immune defense impairs food detoxification in the caterpillar Manduca sexta.

Mounting an immune response consumes resources, which should lead to increased feeding. However, activating the immune system reduces feeding (i.e. illness-induced anorexia) in both vertebrates and invertebrates, suggesting that it may be beneficial. We suggest that illness-induced anorexia may be an adaptive response to conflicts between immune defense and food detoxification. We found that activating an immune response in the caterpillar Manduca sexta increased its susceptibility to the toxin permethrin. Conversely, a sublethal dose of permethrin reduced resistance to the bacterium Serratia marcescens, demonstrating a negative interaction between detoxification and immune defense. Immune system activation and toxin challenge each depleted the amount of glutathione in the hemolymph. Increasing glutathione concentration in the hemolymph increased survival for both toxin- and immune+toxin-challenged groups. The results of this rescue experiment suggest that decreased glutathione availability, such as occurs during an immune response, impairs detoxification. We also found that the expression of some detoxification genes were not upregulated during a combined immune-toxin challenge, although they were when animals received a toxin challenge alone. These results suggest that immune defense reduces food detoxification capacity. Illness-induced anorexia may protect animals by decreasing exposure to food toxins when detoxification is impaired.

Preparation of Multi-target Fusarium Toxins (Zearalenone, Deoxynivalenol, T-2, and HT-2 Toxins) Immunoaffinity Column Using Polystyrene-Divinylbenzene as Matrix

The aim of this study was to establish a purifying immunoaffinity column (IAC) for four kinds of Fusarium toxins (ZEN, DON, T-2, and HT-2 toxins) simultaneously, by using polystyrene-divinylbenzene (PS-DVB) as a column matrix. Four different types of PS-DVB resins were selected to prepare the IACs. Among them, two types of resins, GALAK and Agela, were chosen for IAC preparation. The coupling ratios of the monoclonal antibodies and GALAK or Agela resins were 98.37 and 97.64%, the recoveries of the four Fusarium toxins on the GALAK or Agela IACs were 73.02∼91.97 or 66.16∼112.80%, and the RSDs of recoveries were 2.17∼16.53 or 3.42∼12.25%. They can be applied for the purification of Fusarium toxins from the actual food samples. The GALAK resin was a type of matrix with hydrophilic treatment. The recoveries of four Fusarium toxins on Agela IAC were either too low or too high. The adsorption capacities of the GALAK IAC for the four Fusarium toxins were better than those of the Agela IAC. Hence, the research developed a new method for the preparation of multi-target IAC using PS-DVB resin as matrix of column for determination of Fusarium toxins in food. Then, the GALAK IAC was used for flour samples to detect the feasibility of the application in the actual sample.

A proteomics assay to detect eight CBRN-relevant toxins in food.

A proteomics assay was set up to analyze food substrates for eight toxins of the CBRN (chemical, biological, radiological and nuclear) threat, namely ricin, Clostridium perfringens epsilon toxin (ETX), Staphylococcus aureus enterotoxins (SEA, SEB and SED), shigatoxins from Shigella dysenteriae and entero-hemorragic Escherichia coli strains (STX1 and STX2) and Campylobacter jejuni cytolethal distending toxin (CDT). The assay developed was based on an antibody-free sample preparation followed by bottom-up LC-MS/MS analysis operated in targeted mode. Highly specific detection and absolute quantification were obtained using isotopically labeled proteins (PSAQ standards) spiked into the food matrix. The sensitivity of the assay for the eight toxins was lower than the oral LD50 which would likely be used in a criminal contamination of food supply. This assay should be useful in monitoring biological threats. In the public-health domain, it opens the way for multiplex investigation of food-borne toxins using targeted LC-MS/MS.

Survey of T-2/HT-2 toxins in unprocessed cereals, food and feed coming from Croatia and Bosnia & Herzegovina.

The aim of this study was to investigate into the level of T-2/HT-2 toxins in different unprocessed cereals (n=201), as well as in marketed cereal-based products (n=58), feed components (n=191) and feedstuffs (n=91) coming from Croatia and Bosnia & Herzegovina. The number of positive samples of unprocessed cereals for food production (>LOD) ranged from 30.4% in barley to 68.8% in oat whereas for feed components ranged from 26.9% in wheat to 86.1% in oat. The maximal values found in unprocessed oat and oat-based feed components were 304.2μg/kg and 521.0μg/kg, respectively. As for final products, the highest T-2/HT-2 concentrations were determined in oat flakes (89.4μg/kg) and calf feed (129.3μg/kg). Despite of the increased T-2/HT-2 concentrations found in some of the samples, the obtained values were unanimously lower than the indicative levels given as recommendations above which further investigations should be necessary performed.

Conceptual study of Dooshivisha (Cumulative toxicity) w.r.t. Ancient classics and Modern science

Ayurveda describes the vast turnover of beauty aid products. More and more new synthetic substances are introduced every day. Many of these are not adequately tested for safety, in particular for routes other than oral or parenteral. Increased incidence of skin conditions like eczema and contact dermatitis after using newer cosmetics suggest that this may be due to local toxic effects or toxic effects manifested on this phenomenon as Dooshivisha. They are relevant to the current way of life considering the prevalence of pollution, occupational hazards and extent of exposure to toxins in food and other articles used in daily life. Present generation is much more likely to consume junk foods-cold drinks, to have unhealthy travelling habits, working late nights, working with computers in A.C rooms. This is referred to by terms like Hetusatatya, Vegavarodha, Diwaswapa, Viparit Chesta, Virudhannasevana which in turn hamper the normal physiology of human body. Same thing is mentioned in Ayurvedic texts also under the topic of Pradnyaparadh which is the basic cause for all diseases. This Pradnyaparadha is responsible to disturb the daily routine life and person has to face with the complaints of ill-health.

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris).

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee's taste system.

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (<em>Bombus terrestris</em>)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior. The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated. In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested. This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee's taste system.

Multiplexed Biosensors for Mycotoxins.

Significant progress has been made in the development of biosensors that can be used to detect low-MW toxins produced by fungi (mycotoxins). The number of formats that have been investigated is impressive and is an indication of the importance attached to finding easy-to-use, accurate, and rapid methods for detecting these toxins in commodities and foods. This review explores the details of multiplexed biosensors based on many formats, including multiplexed immunoassays, suspension arrays, membrane-based devices (flow-through and immunochromatographic), and planar microarrays. Each assay format has its own strengths and areas that need improvement. Certain formats, such as multiplexed immunochromatographic devices, are well developed and relatively easy to use, and in some cases, commercial products are being sold. Others, such as the suspension arrays and microarrays, are laboratory-based assays that, although more complicated, are also more amenable to a larger scale of multiplexing. The diversity of such efforts and the multitude of formats under investigation suggest that multiple solutions will be found to satisfy the need for multiplexed toxin detection.

Bioaccumulation of heavy metals in marine fishes (Siganus sutor, Lethrinus harak, and Rastrelliger kanagurta) from Dar es Salaam Tanzania

Toxic metals that bioaccumulate and magnify along food chains are a concern to human health worldwide. This study determined heavy metal concentrations in three commercial fish species; viz: whitespotted rabbitfish (Siganus sutor), thumbprint emperor (Lethrinus harak), and the Indian Mackerel (Rastrelliger kanagurta) from Kunduchi fish market in Dar es Salaam, Tanzania, assessed potential risks to human health, and the suitability of their fins as a non-destructive monitoring organ. Metal concentrations in fish muscle differed significantly among fish species as a result of their diverse ecological needs, metabolism, feeding habits, and habitats. The results also indicated that fish fin may not be a very good non-destructive monitoring organ for most metals, but can be used selectively for some metals and fish species. The levels of metal intake (aluminum (Al), cadmium (Cd), copper (Cu), iron (Fe), lead (Pb) and zinc (Zn)) in fish muscles were below the FAO/WHO maximum levels for contaminants and toxins in foods for human consumption except for arsenic (As) which were higher than the recommended levels in Lethrinus harakand Rastrelliger kanagurta. It is recommended to reduce the amount of L. harakand R. kanagurta in daily meals of especially fishers and coastal communities who may eat fish up to three meals daily. Recommendation for further studies is provided.

Rapid Detection of Escherichia coli O157 and Shiga Toxins by Lateral Flow Immunoassays.

Shiga toxin-producing Escherichia coli O157:H7 (STEC) cause food-borne illness that may be fatal. STEC strains enumerate two types of potent Shiga toxins (Stx1 and Stx2) that are responsible for causing diseases. It is important to detect the E. coli O157 and Shiga toxins in food to prevent outbreak of diseases. We describe the development of two multi-analyte antibody-based lateral flow immunoassays (LFIA); one for the detection of Stx1 and Stx2 and one for the detection of E. coli O157 that may be used simultaneously to detect pathogenic E. coli O157:H7. The LFIA strips were developed by conjugating nano colloidal gold particles with monoclonal antibodies against Stx1 and Stx2 and anti-lipid A antibodies to capture Shiga toxins and O157 antigen, respectively. Our results indicate that the LFIA for Stx is highly specific and detected Stx1 and Stx2 within three hours of induction of STEC with ciprofloxacin at 37 °C. The limit of detection for E. coli O157 LFIA was found to be 105 CFU/mL in ground beef spiked with the pathogen. The LFIAs are rapid, accurate and easy to use and do not require sophisticated equipment or trained personnel. Following the assay, colored bands on the membrane develop for end-point detection. The LFIAs may be used for screening STEC in food and the environment.

Development of a sensitive monoclonal-based enzyme-linked immunosorbent assay for monitoring T-2 toxin in food and feed

ABSTRACTThe consumption of food or feed contaminated with high levels of T-2 toxin may cause adverse health effects in humans and other animals. In this study, to monitor T-2 toxin rapidly in food and feed, a sensitive and specific monoclonal antibody (mAb) against T-2 toxin was generated and a simple and rapid indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) developed. T-2 toxin was first converted to T-2-hemisuccinate (T-2HS) and T-2-hemiglutarate (T-2HG), which were then conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to prepare an immunogen and coating antigen, respectively. After the inoculation of female Balb/c mice and cell fusions, one cell line, 4D8, with the IgG1 isotype was obtained. The 4D8 antibody exhibited the ability specifically to recognise T-2 toxin with IC50 1.46 µg l−1. Based on this 4D8 mAb, an optimised ic-ELISA protocol was developed using only methanol–water (7:3, v/v) in feed and cereal samples and ethyl acetate in muscle samples. The limits of detection of T-2 toxin in various sample matrices varied from 0.07 to 15.8 µg kg−1; the recoveries ranged from 50.3% to 113.6%; and the CVs were less than 19.0%. These results suggest that the prepared mAb and the developed ic-ELISA method will be a useful tool for detecting T-2 toxin in foods and feeds.

Star shaped zinc sulphide quantum dots self-assembled monolayers: Preparation and applications in food toxin detection

We report the results of studies relating to the fabrication of an electrochemical immunosensor based on self-assembled of star shaped thioglycolic acid capped zinc sulphide quantum dots (ZnS QDs) synthesized by hot injection method. Structural and morphological investigations of ZnS QDs have been accomplished via transmission electron microscopy (TEM), X-ray diffraction spectroscopy (XRD), UV–visible spectroscopy (UV–vis) and Photoluminescence spectroscopic (PL) techniques. Further, monoclonal aflatoxin-B1 antibodies (anti-AFB1) have been covalently immobilized onto the ZnS/APTES/ITO electrode surface for the detection of aflatoxin-B1, a potential food toxin using electrochemical impedance spectroscopy. The impedemetric response of the proposed immunosensor shows high sensitivity (176.58Ω/(ng/mL)/cm2), improved detection limit (0.02ng/mL) and wide detection range (0.25–2.0ng/mL). The ZnS QDs based platform provides an efficient platform for the fabrication of immunosensor for the detection of aflatoxin- B1 and these results have implications in the design of highly efficient biosensors for other potential toxins.

The levels of mercury, methylmercury and selenium and the selenium health benefit value in grey-eel catfish (Plotosus canius) and giant mudskipper (Periophthalmodon schlosseri) from the Strait of Malacca

The present study examined the concentrations of mercury (Hg), methylmercury (MeHg), and selenium (Se) in the multiple tissues of the Plotosus canius and Periophthalmodon schlosseri collected from the Strait of Malacca. The mean value in mg kg−1 of Hg (P. canius: 0.34 ± 0.19; P. schlosseri: 0.32 ± 0.18) and MeHg in muscle (P. canius: 0.14 ± 0.11; P. schlosseri: 0.17 ± 0.11) were below the Codex general standard for contaminants and toxins in food and feed (CODEX STAN 193-1995), the Malaysian Food Regulation 1985 and the Japan Food Sanitation Law. For P. canius, the liver contained the highest concentrations of Hg (0.48 ± 0.07 mg kg−1) and MeHg (0.21 ± 0.00 mg kg−1), whereas for P. schlosseri, the gill contained the highest concentrations of Hg (0.36 ± 0.06 mg kg−1) and MeHg (0.21 ± 0.05 mg kg−1). The highest concentration of 80Se (mg kg−1) was observed in the liver of P. canius (20.34 ± 5.68) and in the gastrointestinal tract (3.18 ± 0.42) of P. schlosseri. The selenium:mercury (Se:Hg) molar ratios were above 1 and the positive selenium health benefit value (HBVSe) suggesting the possible protective effects of Se against Hg toxicity. The estimate weekly intakes (EWIs) in μg kg−1 body weight (bw) week−1 of Hg (P. canius: 0.27; P. schlosseri: 0.15) and MeHg (P. canius: 0.11; P. schlosseri: 0.08) were found to be lower than the provisional tolerable weekly intake established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Based on the calculated EWIs, P. canius, and P. schlosseri were found to be unlikely to cause mercury toxicity in human consumption.

Multiplex Dipstick Technologies for Rapid and Simultaneous Screening of Analytes of Importance in Agri-Food-Nutrition and Health Care: A Review.

Dipstick test kits are being widely used for the rapid screening of a range of antigens or toxins in food, agriculture, and health care. They provide specific results on-site within 10 min with suitable accuracy and are, therefore, cost-effective. Multiplex dipsticks also provide the opportunity for simultaneous detection of multiple antigens in the target sample without using expensive instrumentation, minimizing the cost of analysis as well as the duration of assay. Because of these benefits, dipstick kits are widely being used in the simultaneous detection of several antigens/toxins in large number of samples and in high-throughput manner. This review focuses on the current status of developed multiplex strips and its working principles and future direction of the technology in the agriculture, food, nutrition, and health care sectors.

Review and analysis of occurrence, exposure and toxicity of cyanobacteria toxins in food

This report presents the extensive literature search conducted on 1) the occurrence of different cyanotoxins in food matrices; 2) the analytical methods for their detection; 3) their toxicological profile; 3) the environmental factors affecting toxicity of cyanobacterial population and 4) the combined effects of mixtures of cyanotoxins and other chemicals. It also includes a review of guidelines values or health-alert levels for cyanotoxins in food (or drinking water) adopted worldwide. The methodological aspects and the queries used in the extensive literature search, the collection and screening of retrieved papers and the inventory are briefly described in the report; all details are available in 3 supplementary appendices to this report. The analysis of collected papers indicated that most of them are focused on a single microcystin (MC) variant (MC-LR) out of the almost 100 MC known. Many studies on occurrence are affected by limited quality, due to analytical drawbacks in the detection methods and were not considered in the exposure assessment. Toxicity studies useful for the derivation of health based reference values are few, being many of them carried out using i.p. injection, which is poorly representative of actual human exposure. In addition, those toxicological studies carried out with poorly characterised cyanobacterial extracts or focused on single parameters, using a single dose, devoted to elucidation of mechanism of action, reporting qualitative description of effects were not used for data extraction. The relevant exposure scenarios are also described, although being the available data on exposure very limited, no definite conclusion on the health risks for the exposed population could be drawn. However, the possibility of risky exposure is evidenced for fish and shellfish consumers and for blue-green algae supplements (BGAS) as well in relation to MC contamination. Finally, many data gaps were identified.