**1. Introduction**

A water supply system (WSS) is a set of civil infrastructures (tanks, pipes and others), hydro-mechanical facilities, electrical equipment and services that extracts, conveys and distributes water to consumers. This distribution must be compatible with the demand in both quantity and quality [1]. A sub-grid of a water supply network, also termed a "district metered area", is a part of the network between two nodes where the head pressure is controlled. This pressure is maintained constant or follows a certain schedule [2] to ensure that there is an optimal pressure distribution in all the branches downstream. This is normally ensured by pressure reducing valves (PRVs), but recent studies have proposed their replacement by turbines. Pumps operated as turbines and Kaplan turbines are the technologies proposed by [3,4] for low-head sites. The study presented in [4] has shown that there is an economical interest in micro-hydropower in WSSs. This study was performed at a regional scale, identifying existing infrastructure such as PRVs and reservoirs where there is potential for energy recovery. Moreover, there is potential for energy production within the urban network itself. Besides the locations where PRVs are installed, there are also areas within the sub-grids that have more pressure than necessary, even if not excessive, because of their connection to other higher areas [5].

The use of PRVs or even micro-turbines in a WSS may seem illogical, since most systems need pumping. Nevertheless, the use of operational pressure control has proven to be cost-effective for reducing leakage problems [1,6]. Water loss occurs due to the structural deterioration of the pipes [7]. Local energy dissipation is seen to reduce the frequency of pipe bursts [8], ensure the quality standards of water [9] and protect roads and building foundations from underground cavities originating from water losses [10]. From the perspective of hydropower, the implementation of production units in existing infrastructure has the economic advantage of low costs [11] given the synergies with the existing facilities of the WSS, of consumption dependent discharges, which are predictable and almost guaranteed, and of benefiting several countries with respect to legal and financial incentives for self-generation and self-consumption (e.g., feed-in tariffs provided for renewable energies). However, it is worth mentioning that the feed-in-tariff regulations vary considerably between countries and, for example, in the UK, a micro-hydropower plant (MHP) downstream from any pumping station is not eligible for the subvention [12]. In addition to the abovementioned synergies with the existing WSS facilities, the energy produced may justify the installation of a grid-connected generator or local consumption [13]; the latter provides financial savings by avoiding external consumption and expensive electrical connections [14]. The installation of these systems is highly dependent on the costs associated with the turbine, construction and grid connection [4].

Examples of solutions for the installation of MHPs in WSSs can be found in the literature. For example, the installation of PATs as replacement for a PRV in the an urban water distribution system of Pompei, with discharges between 20 and 50 L/s and heads between 35 and 90 m, would produce between 20 and 94 MWh/year [3]. In Portland, Oregon, a PRV was replaced by a 10" (approx. 250 mm) micro turbine that generates 150 MWh/year with 30 kW of installed power [15]. In Hong Kong, an eight blade spherical inline turbine is expected to produce 700 kWh/year in the city's water main pipes [16]. Finally, the installation of a turbine replacing a break pressure tank in Kildare in Ireland could generate approximately 237 MWh/year from 200 kPa to 17,910 m3/day [17].

Although the potential for energy production within urban WSSs exists, it is difficult to quantify unless focusing on a particular location. Distribution networks are complex systems, usually composed of multiple loops and asymmetric consumption in both time and space. The optimal location of turbines within such systems is a subject of research [5,18,19]. Moreover, before enduring deep analyses and simulations of turbine operation and water demand scenarios, a measure of the excess energy available within the city network, indicating the potential for that network to be used for hydropower, would be useful for WSS managers.

In this research, a method for the evaluation of the potential for hydropower in a city is presented. This method is then applied to a case study city and compared with the actual energy production with a proposed hydropower scheme. The energy production is estimated using an algorithm for the identification of optimal location of the micro-hydropower units considering the whole network where infrastructure may or may not exist. In Section 2, the two methodologies are presented, for potential assessment and for the actual energy production simulation. The case study, the city of Fribourg in Switzerland, and the hydropower scheme are described in Section 3. The results are presented and discussed in Section 4. Section 5 presents a straightforward methodology that is proposed for the presented hydropower scheme and the main conclusions are drawn in Section 6.


*3.3. Implementation of the Data Model*

The D-SI may be implemented in the language of choice of the user. Within the SmartCom project, Extensible Markup Language (XML) [17] has been used, but the use of alternative languages such as JavaScript Object Notation (JSON) [18] is equally valid. The following features have been imposed:

Within the XML implementation of the D-SI [23], to reflect the dependence on the SI, all structural elements have the prefix 'si', e.g., si:real.


**4. Synthesis and concluding statements**

The SSA countries in which we work exhibit a wide range of social and political systems, stages of economic evolution and cultural legacies. Yet in each, the need for collective action to address shared problems is evident, particularly in rural and resource-poor areas.

However, by organizing cooperatives and participatory land use planning approaches, farmers and entrepreneurs can mobilize capital, pool knowledge, achieve economies of scale and foster vertical integration. They can create greater leverage in the marketplace and policy arena, attract business service providers and more efficiently link to urban and export markets. Such disciplined groups not only catalyze local economic growth, they may serve as a vehicle for more equitable community land use planning and improve members income and level of productivity in the city economy. Likewise, they develop human capital, encourage the participation of women and youth in development activities including farmers' decision making, fostering democracy and entrepreneurship for organizational and membership development within the social solidarity economy context.

Whether working with legally registered agricultural and financial cooperatives, communitybased enterprises and private limited companies, financial institutions such as Banks seems they do not promote one standard model for enhanced cooperative development. Banks are interested in profit making and giving loans which they know later you will reimburse at high interest rate and impose exorbitant fees indiscriminately. The approach for enhancing cooperatives is to build on existing structures and to promote diversity and choice within itself. In this regard diagnostic tools for assessing the capacity and seriousness of existing cooperatives to serve the interests of their members in an effective, accountable and transparent manner are in high demand. Along with participatory subsector analysis, institutional mapping and competitiveness analysis tools, by engaging stakeholders in devising plans that address cooperative weaknesses and constraints.

To ensure cooperative sustainability, accountability, service orientation and surplus generation, members must contribute and monitor the progress of their group. There is a need for developing participatory methods and procedures in policy and legal context to allow group members to assess management, participation, organization, planning, economic performance, technical operations, financial operations and growth in both sectors of cooperative and community based initiated land use planning process. Some range of instruments–including memoranda of understanding, protocols, contracts and franchise agreements–to protect members from abuses of power and to discourage irresponsible groups from bringing the organizations into disrepute, can be adopted.

Community organized groups in their involvement in land use planning and cooperative development allows small-business owners and farmers to compete in the marketplace.

Savings and Credit Cooperative organisations guarantee members' access to the city economy, in terms of capital outsourcing from relatives and friends, labour and exchange of goods and services, and strong communication action are common features, that triggers the transformation need of cooperative development and land use planning change in urban development. Thus, guiding urban development through proper land use planning is essential to ensure the increased urban investment, making a social justice city and therefore reduce

The SSA countries in which we work exhibit a wide range of social and political systems, stages of economic evolution and cultural legacies. Yet in each, the need for collective action

However, by organizing cooperatives and participatory land use planning approaches, farmers and entrepreneurs can mobilize capital, pool knowledge, achieve economies of scale and foster vertical integration. They can create greater leverage in the marketplace and policy arena, attract business service providers and more efficiently link to urban and export markets. Such disciplined groups not only catalyze local economic growth, they may serve as a vehicle for more equitable community land use planning and improve members income and level of productivity in the city economy. Likewise, they develop human capital, encourage the participation of women and youth in development activities including farmers' decision making, fostering democracy and entrepreneurship for organizational and

Whether working with legally registered agricultural and financial cooperatives, communitybased enterprises and private limited companies, financial institutions such as Banks seems they do not promote one standard model for enhanced cooperative development. Banks are interested in profit making and giving loans which they know later you will reimburse at high interest rate and impose exorbitant fees indiscriminately. The approach for enhancing cooperatives is to build on existing structures and to promote diversity and choice within itself. In this regard diagnostic tools for assessing the capacity and seriousness of existing cooperatives to serve the interests of their members in an effective, accountable and transparent manner are in high demand. Along with participatory subsector analysis, institutional mapping and competitiveness analysis tools, by engaging stakeholders in

To ensure cooperative sustainability, accountability, service orientation and surplus generation, members must contribute and monitor the progress of their group. There is a need for developing participatory methods and procedures in policy and legal context to allow group members to assess management, participation, organization, planning, economic performance, technical operations, financial operations and growth in both sectors of cooperative and community based initiated land use planning process. Some range of instruments–including memoranda of understanding, protocols, contracts and franchise agreements–to protect members from abuses of power and to discourage irresponsible

Community organized groups in their involvement in land use planning and cooperative development allows small-business owners and farmers to compete in the marketplace.

to address shared problems is evident, particularly in rural and resource-poor areas.

poverty among urban residents to implement the urban development agenda.

membership development within the social solidarity economy context.

devising plans that address cooperative weaknesses and constraints.

groups from bringing the organizations into disrepute, can be adopted.

**4. Synthesis and concluding statements**

There is therefore a need to strengthen the capacity of such groups to expand their trading options by scaling up production and applying quality-control standards. To this end, groups are assisted in accessing essential technical services, linking to higher-value markets, forming business alliances and advocating for a favorable regulatory environment for improved city productivity.

The potential for large-scale impact embodied in agriculture and financial cooperatives justifies the long-term commitment of resources required to build effective, independent and financially sustainable member organizations. As organizations mature, they generally expand their operations–diversifying products and services, branching into new geographic areas and addressing social concerns such as illiteracy, gender inequity or HIV/AIDS. Cooperatives play a significant role in nurturing the next generation of community leaders, and, with their extensive member networks, they are ideal vehicles for transferring knowledge, challenging social perceptions and poverty reduction tools.

In democratically run, member-owned organizations, smallholder farmers and small-scale business owners learn participatory decision making and develop ways to resolve conflicts. They enables members to demand accountability of elected leaders, present their views in public, claim ownership of the local political process, and on a daily basis they demonstrate the values of transparency and accountability.

The study shows that local community participation is important towards enabling residents in urban centers to come together and fight for their right of securing tenure system for improving small scale farmers' productivity as a response to food insecurity. However, linkages, synergy and partnerships in view to participation process and contribution of different resources appears to be an important ingredient in enhancing local community involvement in securing land tenure and therefore safeguard urban environment. Roles of different actors including the central government, local government and other planning and development institutions need to be clear and therefore contributory to enabling local community initiated project such as land use planning, cadastral survey and land registration to be implemented effectively and efficiently. This may be the case in most Sub Saharan African countries, where the need for having planned and secured formal landownership to residents is high in urban development agenda.

The economic characteristics entail how do they operate efficiently and attain high profit to cover operating costs as well as operate at low economic costs. In view of the social characteristics, all of them must involve people in its operations, must be competitive in the market economy, must have adequate capital investment from the membership and finally must be managed democratically, which shows the role of land use planning and cooperative development in improving city productivity.

The future outlook of cooperative and land use planning process should be based on community-centred and market-driven approach. Member-owned groups should increase their members' incomes, either directly, for example through bulk input purchases and collective marketing, or indirectly, such as through policy and advocacy initiatives. Members are motivated to participate in group activities because of profits and or benefits they obtain as a result of their engagement in the sector.

To move in the right direction, the Government may also prepare a transparent strategy for rebuilding the co-operative movement involving other interested parties like NGOs and

**14**

*Greece*

**The Evolution and Spatial**

Serafeim Polyzos and Dimitrios Tsiotas *Department of Planning and Regional Development,*

*University of Thessaly,*

**Dynamics of Coastal Cities in Greece**

The procedure of human evolution has been always connected with the concepts of space and time. Communities that existed in the same chronological period in different geographic locations, such as communities that existed in the same geographic location at different eras, present various patterns of development. This statement indicates, on one hand, the significance of the spatial parameter to the Economic and Regional Analysis and, on the other hand, the directions that the Economic Research should be oriented to, in order to provide more complete outcomes. From the amount of spatial attributes that a geographic location may possess, the present article deals with the coastal one. Coastal areas have played diachronically a leading role to the development of human civilization, ought to their geographical characteristics, focused on to the seaside location , which provides many transportation advantages, such as development of ports (Li, 2003), commodity (trade development) (Cori, 1999) and also environmental advantages, such as mild climate and natural beauty (Yeung, 2001). The purpose of this chapter is to study the evolution and the spatial dynamics of coastal cities, through a spatial, statistical and regional analytic approach, in order to recognize and to interpret the patterns that describe this evolution,

In particular, the chapter focuses on the case of Greek coastal cities (Polyzos et al., 2011), as it follows. First of all, this choice is considered by the authors to be a challenge, since the case of Greek coastal cities lies under the individuality of not presenting extended population coverage, agglomeration or demographic mega phenomena. This fact differentiates the performance of the Greek coastal system from other cases' and sets an inverse scaling transformation to the until now research findings of this field (Yeung, 2001; Sheng Han and Yan, 1999; Li, 2003; Sekovski et al., 2011). Secondly, the case of studying a recent Greek spatial and economic system is particularly up to date, by the time that Greece is currently being subjected to an economic crisis (considered from many to be an introducing crisis to the European economic system), fact that is expected to reveal some vital axes in order to comprehend the developmental potentials of the Country. Another reason is the authors' Greek parentage, which sets the Greek case to be a familiar research field. Finally, although the research on coastal cities' dynamics presents an efficient scientific matter (Miller & Auyong, 1991; Cori, 1999; Yeung, 2001; Li, 2003), so in Global as in

**1. Introduction**

under the regional economic and policy perspective.

other sympathizers of co-operative enterprise. The tendency of the Government to dominate the co-operatives as in the past should be avoided, but will play a key facilitation role. The heart of the strategy should be a pro-poor participatory co-operative development and modernization education programs that may be drawn on the positive lessons of the wide range of participatory approach based pilot projects. This may bring fundamental change of attitudes and approaches of particularly the government policy makers and other interested stakeholders including extension officers, Co-operative support institutions, co-operative movement and other sectors in the economy. Likely they will succeed to introduce appropriate changes of the mindsets and governance practices. The study concludes that towards enhancing social-solidarity economy for improved livelihoods of members within city's neighborhoods or within cooperative associations, private-public partnership (PPP) may be an important combination to be considered in urban development. If the linkages and partnerships are not well created and addressed in policy and institutional settings, it may be difficult to retrofit and may enhance urban poverty in Sub-Saharan African cities and rural settings.

#### **5. References**


**8. Conclusions**

Overall, supplementing regular instruction with Read Like Me successfully accelerated the reading development of the second grader involved in the study. The stacked approach increased the student's word recognition automaticity, reading prosody, decoding skills, word knowledge, and reading comprehension. Although this study cannot be generalized, the results suggest that this may be a viable option for teachers or interventionists working with students who find reading difficult.

**Author Contributions:** Conceptualization, C.Y. and J.M.; methodology, C.Y.; validation, C.Y. and S.L.; formal analysis, C.Y. and S.L.; investigation, C.Y., S.L., and J.M.; resources, C.Y. and S.L.; data curation, S.L.; writing—original draft preparation, C.Y., S.L. and J.M.; writing—review and editing, C.Y., S.L. and J.M.; supervision, C.Y.; project administration, C.Y. and S.L.; funding acquisition, C.Y. and S.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by EURECA Center at Sam Houston State University, grant number 29021.

**Acknowledgments:** We'd like to thank the participant, his family, and his teacher. We appreciate your willingness to try out a new intervention.

**Conflicts of Interest:** The authors declare no conflict of interest.


**5. Conclusions**

From **Table 5** (upper part), one can see that the coefficients are significantly positive only in the recovery subperiod. This tells that for group A (five Asian emerging countries), the stock return can explain the economic growth only in the recovery period. **Table 5** (middle part) shows that the coefficients are significantly positive only in the depression subperiod, which indicates that for group B (the G7 countries), the stock markets will go down before the

The economic rationale behind this is as follows. Since the stock return and economic growth are positively correlated in the G7 countries, when they enter the depression subperiod, the stock market would go down to reveal the upcoming depressions. When the G7 countries enter the recovery subperiod, their production and consumption will increase. At this time, the G7 countries will place many orders on the Asian emerging markets, and this will help the Asian emerging markets grow and their firms perform well. These outcomes will be reflected by the stock markets in these emerging markets; with more foreign investments from the developed countries, these stock markets will stay in the bull status for a long time. This is why the stock market can significantly explain the economic growth in the recovery subperiod. Although both the Asian emerging markets and the G7 countries are all in the recovery subperiod, the economies will not grow as fast. Moreover, because developed countries tend to invest in high return foreign stock markets, there is no significant relationship between the stock return and

**Table 5** (lower part) reports the estimation result for group C (12 OECD members), the coefficients are significantly positive only in the depression subperiod, same as the result in **Table 5** (middle part). In addition, in the expansionary period, the stock return can significantly explain the economic growth, which is different from the results of other subperiod estima‐ tions. The results in **Table 5** (middle and lower parts) can be used to derive the results of **Table 4** that the stock return can explain the economic growth in both the depression and recovery

The results of **Tables 5** are not quite the same as the result of **Table 4** (the whole sample estimation), which indicates that one cannot apply the conclusion of **Table 4** to every case. Some of the effects may be "cancelled out" by pooling all the countries into one sample.

The empirical findings of this chapter could benefit the corporations, financial companies, as well as regular investors. The contribution of this chapter can be summarized as follows. First, the empirical findings could avoid corporations from misunderstanding the recession period. In the recession period, the government tends to reduce the interest rate and enhance the government spending to stimulate the economy. When making future operation and finance decisions, if the decision maker can seize the chance to adjust the factory size or to raise corporate debts in the recession period or to finance in the expansionary period, it would be beneficial to the corporation. Second, the findings help financial companies or financial supervisors better understand the business cycle. Macroeconomic analyses and business cycles are crucial factors to investment decisions to maximize capital gains and to minimize risks from market fluctuations. Third, the findings help regular investors better understand the business cycle. The business cycle information can help regular investors with medium or long term investment decisions and avoid capital loss from short term fluctuations in the market.

economic growth in the G7 countries in the recovery subperiod.

economies start to grow.

348 Nonlinear Systems - Design, Analysis, Estimation and Control

subperiods.

In this study, the existence of the subperiods of the recession period is observed by examine the business cycle plot of **Figure 1**, this chapter make up this gap by constructing a nonlinear DPDM to investigate the relationship between the stock return and economic growth in the subperiods. The finding of the present chapter can be summarized as follows.

First, the GMM estimation is adopted for the DPDM estimation to avoid possible bias from the OLS estimation. The NCDR proposed by Bradley and Jansen [12] is employed as the switched factor in the model. The empirical result shows that the stock market performance can be a leading indicator for the economic growth, especially in the recession period. This finding is consistent with the findings in previous studies.

Second, the empirical results show that in the whole sample estimation, the stock return can significantly explain the economic growth in the two subperiods of the recession period. In the estimation with different country development levels, it is found that in the Asian emerging markets, the stock return can significantly explain the economic growth only in the recovery period. The reason for this outcome is as follows. Generally speaking, the emerging markets have higher economic growth rates than the most of the developed countries do. When entering the recovery period, the emerging markets can attract more foreign funding into their stock markets. As to the developed countries, the stock return can significantly explain the economic growth only in the depression period. The reason for this result is the following. The developed countries lead the development of the world economy. When the developed countries enter the depression period, the investors will withdraw from the stock markets to avoid the risk of loss, which in turn, causes the stock markets to go down and results in a positive relationship between the depression and the down‐turn stock market. This result indicates that various development levels will have different impact on the relationship between the stock return and economic growth. If one would like to use the stock market information to predict the economic growth, one must first ascertain the country's develop‐ ment status.


**18. Iminium catalysis: β-alkylation of enals and enones**

A major breakthrough in the field of asymmetric radical chemistry was represented by Melchiorre group; they achieved the first enantioselective radical conjugate addition (RCA) to β,β-disubstituted cyclic enones by a combination of photoredox catalysis and iminium-based organocatalysis. The organocatalyst and the primary amine moiety react with a α,β-unsaturated enone forming a chiral iminium ion as the reactive intermediate; the 1,3-benzodioxole present in the reaction medium upon irradiation by means of an UV light-emitting diode and in the presence of tetrabutylammonium decatungstate (TBADT) generates a carboncentered radical. Thus generated carbon-centered radical being short-lived and active, it reacts with the chiral iminium ion producing an α-iminyl radical cation. Then through an intramolecular SET process, the α-iminyl radical cation was reduced to enamine. A tautomerism reaction, that is, enamine-imine tautomerization, regenerates photocatalyst TBADT, and finally the work procedure gives rise to the product with excellent enantio- and diastereoselectivities, and the chiral amine is recovered [22] (**Figure 24**).

**Figure 24.** *Iminium catalysis: β-alkylation of enals and enones.*


**Author details**

Namera Thahaby1 \*, Afzal Hoque Akand1 , Abdul Hai Bhat1 , Shabeer Ahmed Hamdani1 and Mudasir Ali Rather2

1 Division of Veterinary and Animal Husbandry Extension, FVSc & AH, SKUAST-Kashmir, Srinagar, J&K, India

2 Division of Veterinary Public Health, FVSc & AH, SKUAST-Kashmir, Srinagar, J&K, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


*2.1. Modelled System and Study Conditions*

The photovoltaic system analysed in this article is located in the campus of the University of Salento in Monteroni di Lecce, Puglia, located in south Italy. The installation, fully described in [24], is grid-connected and covers a net area of 4710 m2 on the roofs of the parking lots of the aforementioned university. It is comprised of 3000 monocrystalline silicon modules connected in series, with a total nominal power of 960 kWp. The azimuth of all modules is −10◦, since they are oriented to the south east. However, two groups of modules can be differentiated according to their slope within the PV system: 3◦ (PV1); and 15◦ (PV2). Table 1 includes the main specifications of both groups.

The location of the present study matches the coordinates of the campus where the PV system is placed, i.e., 40◦19 32"16 N, 18◦5 52"44 E. This study area is characterised by a Mediterranean climate, with warm winters and dry summers. It is located 18 km from the nearest shore line, with an average elevation of 35 m above sea level. The starting and ending dates of this study are conditioned by the duration of the weather and PV power monitoring campaign done in [23] over said installation, as explained on the next subsection. Hence, the chosen temporal span of the study starts on 6th March 2012 at 00:00:00 UTC and ends on December 30th, 2013 at 23:59:59 UTC.


**Zhifu Zhang**

Zhifu Zhang graduated with a degree in Applied Geophysics from the China University of Mining and Technology in 2000, an M.Sc. degree in Earth Exploration and Information Technology in 2003 in Guilin University of Technology, and a Ph.D. degree in Solid Geophysics in 2006 from the Chinese Academy of Sciences. Zhifu Zhang is a vice professor at the School of Geophysics and Information Technology, China University of Geosciences (Beijing). Zhifu Zhang has participated in more than 20 scientific projects and has published more than 30 papers. Zhifu Zhang mainly engaged in research on surface wave imaging.


*Article* **An Update on Ciguatoxins and CTX-like Toxicity in Fish from Different Trophic Levels of the Selvagens Islands (NE Atlantic, Madeira, Portugal)**

**Pedro Reis Costa 1,2,\*, Pablo Estévez 3, Lucía Soliño 1,2, David Castro 3, Susana Margarida Rodrigues 1, Viriato Timoteo 4, José Manuel Leao-Martins 3, Carolina Santos 5, Neide Gouveia 4,†, Jorge Diogène 6,\* and Ana Gago-Martínez 3,\***

**Abstract:** The Selvagens Islands, which are a marine protected area located at the southernmost point of the Portuguese maritime zone, have been associated with fish harboring ciguatoxins (CTX) and linked to ciguatera fish poisonings. This study reports the results of a field sampling campaign carried out in September 2018 in these remote and rarely surveyed islands. Fifty-six fish specimens from different trophic levels were caught for CTX-like toxicity determination by cell-based assay (CBA) and toxin content analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Notably, high toxicity levels were found in fish with an intermediate position in the food web, such as zebra seabream (*Diplodus cervinus*) and barred hogfish (*Bodianus scrofa*), reaching levels up to 0.75 μg CTX1B equivalent kg−1. The LC-MS/MS analysis confirmed that C-CTX1 was the main toxin, but discrepancies between CBA and LC-MS/MS in *D. cervinus* and top predator species, such as the yellowmouth barracuda (*Sphyraena viridis*) and amberjacks (*Seriola* spp.), suggest the presence of fish metabolic products, which need to be further elucidated. This study confirms that fish from coastal food webs of the Selvagens Islands represent a high risk of ciguatera, raising important issues for fisheries and environmental management of the Selvagens Islands.

**Keywords:** ciguatoxins; Selvagens Islands; seafood safety; *Gambierdiscus*; ciguatera

**Key Contribution:** This article reports the first comprehensive evaluation of CTX in fish (*n* = 56) from the Selvagens Islands (Madeira, Portugal).

### **1. Introduction**

Ciguatera poisoning (CP) is one of the most common nonbacterial illnesses associated with fish and other seafood consumption worldwide. The illness has been known since the 16th century when ciguateric-like poisonings affected classic sea explorers, such as captains of the Spanish navy in the Gulf of Guinea in 1521 or the crew of captain James Cook in his

**Citation:** Costa, P.R.; Estévez, P.; Soliño, L.; Castro, D.; Rodrigues, S.M.; Timoteo, V.; Leao-Martins, J.M.; Santos, C.; Gouveia, N.; Diogène, J.; et al. An Update on Ciguatoxins and CTX-like Toxicity in Fish from Different Trophic Levels of the Selvagens Islands (NE Atlantic, Madeira, Portugal). *Toxins* **2021**, *13*, 580. https://doi.org/10.3390/ toxins13080580

Received: 10 July 2021 Accepted: 17 August 2021 Published: 20 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

second voyage to the South Pacific in 1772–1775 [1,2]. CP is widespread in the tropical and subtropical regions and particularly associated with the consumption of reef fish species that have bioaccumulated ciguatoxins (CTXs). These compounds are potent neurotoxins that act at the voltage-gated sodium channels (VGSC) increasing ion permeability and cell disruption leading to persistent neurological impairment [3]. The neurological symptoms include paraesthesia, dysesthesia, vertigo, and sensory abnormalities such as metallic taste, pruritus, arthralgia, myalgia, dental pain, and cold allodynia, a pathognomonic CP symptom that is characterized by burning pain in response to a cold stimulus [4–6]. Additionally, a variety of gastrointestinal symptoms including abdominal pain, nausea and vomiting, and cardiovascular symptoms, such as heart rhythm disturbances, may also affect poisoned patients. Some symptoms may become chronic and in extreme cases of severity CP may cause the death of patients [7].

The source of CTXs was linked to benthic dinoflagellates of the genus *Gambierdiscus* in the late 1970s [8,9]. *Gambierdiscus toxicus* was the first species described in the genus and, for a certain period, the only species known of this genus. Although the *Gambierdiscus* genus remains largely understudied, so far, at least 21 species have been described. *Gambierdiscus* and *Fukuyoa* are epibenthic dinoflagellates, which can be ingested by herbivorous fish. Some CTXs are produced by these dinoflagellates, but toxin congeners are products of fish metabolism. *Gambierdiscus* and *Fukuyoa* dinoflagellates are endemic to tropical and subtropical regions which are coincident with the world's highest incidence areas of ciguatera, namely the South Pacific, the Indian Ocean, and the Caribbean Sea [10]. Nevertheless, increasing occurrence and spread of *Gambierdiscus* to temperate regions has been reported and seems to be favored by the climate warming trends [11,12]. Coincident with CP outbreaks, *Gambierdiscus* have been detected in NE Atlantic subtropical-temperate regions, such as the Canary and Madeira Islands, and even in the Mediterranean Sea [13–17].

The first human poisonings in Europe due to the consumption of autochthonous fish occurred in 2004, in the Canary Islands, Spain [18]. Since then, several outbreaks have been reported in the Canary Islands and also in the nearby Portuguese islands, i.e., Madeira and Selvagens [19]. The first reported outbreak in Portugal dates from 2008 when 11 persons reported CP symptoms after consuming a 30 kg amberjack (*Seriola* sp) caught around the Selvagens Islands [20,21]. The symptoms reported by the crew members matched with symptoms previously reported by nature wardens of the natural park of the Selvagens Islands. CP was retrospectively diagnosed in the six wardens who had consumed fish caught locally [20]. In the following year, CP symptoms were observed in 20–30 people that ingested amberjack purchased in the Canary Islands but caught in the Selvagens Islands [22].

The Selvagens Islands are at the southernmost point of the Portuguese maritime area, located in the temperate-subtropical northeast Atlantic, closer to the Spanish Tenerife Island than to the Madeira Island, and 600 km from the African continental coast of Morocco (Figure 1). These small islands have a high fish species diversity and abundance, including important commercial species [23,24]. The total fish biomass has been estimated to be 3.2 times higher in the Selvagens Islands than in the Madeira habitat, and 10 times higher, when only top predators' biomass were considered [24]. The Selvagens Islands are a unique ecosystem, considered to be one of the last remaining intact ecosystems of the eastern Atlantic Ocean [25].

Despite several human poisonings associated with fish caught in waters surrounding the Selvagens Islands, only a limited number of studies have tried to assess and investigate the presence of CTX in fish from this potential ciguateric hotspot. Following the first human poisoning, in 2008, Otero and colleagues [21] analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) two amberjacks (*S. dumerili* and *S. fasciata*) caught in the Selvagens Islands and described a suite of toxins composed of several CTX, including CTX1B, CTX3C, and C-CTX1. At the same time, Boada et al. [22] reported the presence of C-CTX1 in amberjack specimens associated with human poisonings in Tenerife but that had been caught in the Selvagens Islands. A retrospective analysis carried out by

state-of-the-art LC-MS/MS instruments to these specimens confirmed that C-CTX1 was the main toxin analogue but also suggested the presence of several metabolites of this compound [26]. More recently, LC-MS/MS analyses have been performed to assess CTX in fish opportunistically obtained from the Selvagens and Madeira Islands [27]. The results suggested a higher risk of ciguatera for fish caught in the Selvagens Islands and raised the need to better understand the natural contamination of CTX in the coastal food web of this marine protected area. Therefore, a campaign was carried out, in September 2018, on these remote and rarely surveyed islands to collect fish specimens that were representative of several trophic levels to assess CTX-like toxicity by cell-based assay (CBA) and to identify the CTXs by LC-MS/MS.

**Figure 1.** Location of the Selvagens Islands, NE Atlantic, Madeira, Portugal.

#### **2. Results**

#### *2.1. Fish Toxicity by CBA*

CTX-like toxicity was not detected in specimens at the lowest trophic level, representative of primary consumers of the marine food web, namely Bermuda sea chub *Kyphosus sectatrix* and scribbled leatherjacket filefish *Aluterus scriptus*. However, toxicity was particularly found in fish species with an intermediate position in the food web, such as the parrotfish *Sparisoma cretense*, zebra seabream *Diplodus cervinus,* and barred hogfish *Bodianus scrofa* (Figure 2). From the 14 parrotfish *S. cretense* caught, CTX-like activity was detected in eight specimens ranging from 0.006 to 0.04 μg CTX1B equivalent kg−1. The single specimen of zebra seabream *D. cervinus* caught during the field campaign, revealed the second highest level determined, reaching 0.37 μg CTX1B equivalent kg−1, highlighting this trophic level of key importance to the CTX dynamics in the Selvagens marine environment. This finding is further supported by the high levels of CTX toxicity found in the 17 barred hogfish *B. scrofa* collected, which varied from 0.04 to 0.75 μg CTX1B equivalent kg−1. Although consistently found in each *B. scrofa* analyzed, CTX-like toxicity was not easily correlated with their size or weight (*p* > 0.05).

**Figure 2.** CTX-like toxicity (μg CTX1B equivalent kg−1) determined by CBA in fish species collected from the marine food web of the Selvagens Islands during 5–7 September 2018 (median, 25 and 75 quartiles, minimum and maximum, total *n* = 55). See Materials and Methods section for details on samples.

With a higher position in the food web, grey triggerfish *Balistes capriscus* showed a reduced CTX-like activity in CBA, but toxicity in blacktail comber *Serranus atricauda* was consistently determined in each sample, ranging from 0.006 to 0.02 μg CTX1B equivalent kg−1. Highly variable results were observed among the apex predators, the yellowmouth barracuda *Sphyraena viridensis*, and the amberjacks *Seriola* spp, ranging from not detected to considerably high levels (0.22 and 0.10 μg CTX1B equivalent kg−1 for *S. viridensis* and *Seriola rivoliana*, respectively).

#### *2.2. Toxins Identification and Quantification by LC-MS/MS*

With the aim of confirming previous results and characterizing the profile of CTX in fish from the Selvagens Islands, selected samples were analyzed by LC-MS/MS taking into consideration the CTX-like activity measured with the CBA and species trophic level. The presence of Caribbean-CTX1 was identified in the analyzed samples with the highest concentration. The highest level, reaching 0.48 μg kg−1, was determined in a 3.0 kg barred hogfish *B. scrofa* specimen (Table 1). Quantification was performed, following the first detection method described in Section 4.3.3, which was based on the selection of the CTXs [M+Na]+ as precursor and product ion. On the one hand, C-CTX1 was consistently detected in all specimens of this species. On the other hand, C-CTX1 or any other CTX analogue was not found in any sample of *Seriola* spp. by LC-MS/MS under the applied conditions. C-CTX1 was also not found in *D. cervinus* and only one of the two barracuda specimens analyzed showed the presence of this toxin and at low levels.

A C-CTX1 methylated congener and additional C-CTX congeners previously detected in a sample of *Seriola fasciata* from the same region and related to a CP case were also monitored by LC-MS/MS [28,29]. The presence of these C-CTX congeners was assessed by monitoring water losses and C-CTX1 specific fragments *m/z* 191.1 and *m/z* 108.9 (Figure 3A). C-CTX1 and a compound at a retention time 4.94 min showing C-CTX1 water losses but not C-CTX1 specific fragments were detected in some *B. scrofa* samples (Figure 3B). Furthermore, a compound matching C-CTX-1157 was detected at a retention time of 7.64 min in the *D. cervinus* in which C-CTX1 was absent (Figure 3C). This compound was

detected in one of the fractions obtained after HPLC fractionation, which showed CTX-like activity as indicated in a previous study [29]. Further work is needed to characterize all these congeners, being necessary to obtain the adequate reference materials to be able to accomplish their full characterization.

**Table 1.** Concentration of Caribbean Ciguatoxin-1 (C-CTX1) determined by LC-MS/MS in selected fish species caught in the Selvagens Islands (NE Atlantic, Portugal) in September 2018.

LOQ, limit of quantification (0.015 μg kg−1).

**Figure 3.** LC-MS/MS chromatogram in MRM mode of: (**A**) Laboratory reference material containing C-CTX1 (8.79 min) and C-CTX1-Me (10.84 min), (A.1) Zoom of C-CTX1 less intense ion transitions: *m/z* 1123.6 [M+H-H2O]+ -> *m/z* 1069.6 [M+H-3H2O]+ (purple line), *m/z* 1123.6 [M+H-H2O]+ -> *m/z* 191.1 (blue line), *m/z* 1123.6 [M+H-H2O]+ -> *m/z* 108.9 (green line), (A.2) Zoom of C-CTX1-Me less intense ion transitions: *m/z* 1123.6 [M+H-CH3-H2O]+ -> *m/z* 1069.6 [M+H-CH3-3H2O]+ (purple line), *m/z* 1123.6 [M+H-CH3-H2O]+ -> *m/z* 191.1 (blue line), *m/z* 1123.6 [M+H-CH3-H2O]+ -> *m/z* 108.9 (green line); (**B**) C-CTX1 (8.79 min) and an unknown compound (4.94 min) detected in a *Bodianus scrofa*; (**C**) C-CTX-1157 (7.64 min) detected in the *Diplodus cervinus*.

#### **3. Discussion**

The Selvagens Islands are a unique ecosystem, listed among the least disturbed islands in the Atlantic Ocean. Despite their small size that implies a small coastal area and their distance to the continental mainland, these islands show high species richness in terms of ichthyofauna, as described by [23]. CTX-like toxicity, as measured by CBA in fish species representative of the several trophic levels, highlighted the presence of toxins in fish at intermediate levels as well as in the top predators. Toxicity was not detected in the two species with the lowest position in the food web, but herbivorous fish, such as the parrotfish *Sparisoma cretense*, and omnivorous species, such as *Diplodus cervinus* and *Bodianus scrofa*, showed notable high toxicity levels. These species commonly show positive relationships in fish assemblage structures and environmental variables, such as seaweed cover and productivity [30]. Further investigation on the presence and abundance of ciguateracausing dinoflagellates will be presented elsewhere, pointing out the Selvagens Islands as an archipelago with high abundances and biodiversity of marine life playing an important role on the growth and proliferation of *Gambierdiscus* in NE Atlantic (Godinho et al. in prep). The toxicity levels reported in the present study are similar or higher than that observed for groupers and moray eels in the nearby islands of the Canary archipelago [31], raising risks for their consumption.

Although the Selvagens Islands are a marine protected area where fisheries are interdicted, fish inhabiting these islands can migrate and spread CTX to regions in close proximity to the islands. In addition, some fisherman disrespecting fisheries interdictions are occasionally attracted by the abundance and high size fish inhabiting the Selvagens Islands. This was the case of a 4 kg red porgy (*Pagrus pagrus*), a fish species with an intermediate position in the food web, captured by Christmas 2016 that caused CP and was recently reported to contain levels of C-CTX1 as high as 0.76 μg kg−1, as determined by LC-MS/MS [26]. In the present study, higher levels of C-CTX1 were found in *Bodianus scrofa* ranging from 0.08 to 0.48 μg kg−1. These levels are among the highest determined in Macaronesia and exceeds the U.S. Food and Drugs Administration (FDA) guidance level of 0.1 μg C-CTX1 kg−1. Such high levels have been of key importance to fully characterize and confirm fragmentation pathways of C-CTX1 by high resolution mass spectrometry [32].

The present study suggests a higher prevalence and incidence of CTX in fish from the Selvagens Islands than that initially reported by [27]. In this study, two detection methods were used. CBA is a functional assay that provides a toxicological response and LC-MS/MS is a highly selective chemical method that can be used to identify the compounds in the fish matrices. Although a comparison of the advantages and specific issues of these methods is not within the scope of this study, it is important to pay attention to discrepancies regarding CTX toxicity found in the fish top predators, namely the yellow mouth barracuda and the amberjacks*,* and the results obtained by LC-MS/MS below the detection limit. These contrasting results indicate that CTX toxicity of top predators may derive from metabolic fish products of C-CTX1 that have not been elucidated yet. Furthermore, the absence of C-CTX1 and the detection of C-CTX-1157 in a toxic *D. cervinus* showed a possible new toxic profile in which C-CTX1 is not the major contributor to the toxicity. Again, it is important to remember that differences were obtained by these two methods Therefore, further investigations are needed to better understand CTX biotransformation processes, to assess the full suite of toxins in the fish top predators, which are key target species for fisheries in NE Atlantic, and to accurately assess the risk of ciguatera poisoning. This study provides a quite complete evaluation of CTXs in fish (*n* = 56). Nonetheless, for each species or trophic level, the number of fish may be modest and further work increasing number of fish may provide a better estimation of the incidence of CTXs in the different groups.

The Selvagens Islands that are characterized by high abundances and biodiversity of marine life seem to play a role in incubation and proliferation of *Gambierdiscus* (Godinho et al. in prep) and ciguateric fish. Since fisheries, whether commercial or recreational, are forbidden, fish can grow and reach a greater size and can live longer. With a higher lifespan, fish have time to increasingly accumulate and metabolize ciguatoxins. The occurrence

of ciguatera in Europe may now pose a new challenge to the management of the marine protected area of the Selvagens Islands.

#### **4. Materials and Methods**

*4.1. Fish Sampling*

Fifty-six fish specimens belonging to 12 species within different trophic levels were caught between 5 and 7 September 2018 in Selvagem Grande and Selvagem Pequena (Table 2). Estimates of trophic levels were obtained from Fishbase that calculates trophic level from stomach contents data [33]. Fish were caught by means of spearfishing, performed by technicians of the Fisheries Department of Madeira Government, and after the required legal permission of the Madeira Natural Park.

**Table 2.** Length and weight of fish specimens caught in the coastal food web of the Selvagens Islands habitat.

\* Estimated trophic level (TL) as expressed in Fishbase (2021). TL = 2.0 is at the base of the consumers food web, with their diet composed mainly of detritus and plants, and TL = 5.0 is a top predator fish.

## *4.2. CTX-like Toxicity Measured by Neuroblastoma (Neuro-2a) Cell-Based Assay (CBA)*

#### 4.2.1. CTX Extraction for CBA

Fish samples for CBA screening assays were extracted at IPMA, according tothe protocol described elsewhere [34] and with some modifications. A 10 g portion of fish flesh was minced and boiled at 70 ◦C for 15 min. Once cooled, samples were homogenized with 20 mL of acetone in a Polytron® homogenizer (Fisher Scientific, Porto Salvo, Portugal) at approximately 18,000× *g* for 2 min. Samples were centrifuged for 15 min at 3000× *g* and the pellets extracted again with acetone. Then, the combined supernatants were filtered through a 0.2 μm pore PTFE filter (Whatman, Lisbon, Portugal) with a syringe and evaporated at 60 ◦C, under vacuum in a rotary evaporator (Fisher Scientific, Porto Salvo, Portugal). The resulting aqueous residue was transferred into a tube and partitioned twice with diethyl ether keeping the ratio 8:2 relative to the amount of residual water. The diethyl ether phases were pooled and evaporated. The dry residue was partitioned with 4 mL of n-hexane and 2 mL of aqueous methanol/H2O (4:1, *v*/*v*). The upper phase (hexane) was removed, and the partition repeated twice by adding 4 mL of hexane to the methanolic fraction. The methanolic phase was collected, evaporated, and stored at −20 ◦C until analysis.

#### 4.2.2. CBA Screening Assay

For cell maintenance, neuro-2a (N2a) cells (ATCC, CCL131) were cultured in 10% fetal bovine serum (FBS) RPMI medium with 1% sodium pyruvate solution (100 mM), 1% l-glutamine solution (200 mM), and 0.5% antibiotic solution (10 mg/mL streptomycin and 1000 U/mL penicillin) (Sigma-Aldrich, St. Louis, MO, USA). Cultures were maintained at 37 ◦C and 5% CO2 in a humid atmosphere incubator (Binder, Tuttlingen, Germany). The day prior to the assay, cells were seeded in a 96-well microplate in 200 μL of 5% FBS-

RPMI medium at a density of 35,000 cells per well. Cells were incubated under the same conditions as described for cell maintenance. Every standard (CTX-1B) and sample extract were assayed in triplicate. Half the wells of each microplate were pretreated with ouabain and veratridine corresponding to a final concentration of 1 and 0.1 mM, respectively. Standard solutions and sample extracts were reconstituted in RPMI 5%. Then, samples were serially diluted, and 10 μL added to each well, with (O/V+) and without (O/V-) ouabain and veratridine pretreatment. According to matrix effects estimated without (O/V-), concentrations for the different fish extracts were adjusted to avoid matrix effects. On the next day, cell viability was measured, by means of the MTT test [3-(4,5-dimethylthiadol-2 yl)-2,5-diphenyltetrazolium] (500 μg/mL) [35] and absorbance measured at 570 nm using an automated multi-well scanning. Samples were considered to be positive when cell viability was inhibited in O/V+ wells and unaffected in O/V- wells.

#### *4.3. Determination of CTX by Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) Analysis*

Sample pretreatment and LC-MS/MS analyses were carried out following the conditions described by [34] and modification performed by [26], and are briefly described below.

#### 4.3.1. Reference Materials

C-CTX1 pure standard (5 ng mL−1) was kindly provided by Dr. Robert Dickey (University of Texas, Austin, TX, USA) via Dr. Ronald Manger (Fred Hutchinson Cancer Research Center, Seattle, WA, USA).

The laboratory reference material (C-CTX1 and C-CTX1-Me) extracts from contaminated fish tissue were obtained after HPLC fractionation.

CTX1B (4466 ng mL−1) and a qualitative mixture of P-CTXs (CTX1B, 2,3-dihydroxy CTX3C, 51-hydroxyCTX3C, 52-epi-54-deoxyCTX1B/54-deoxyCTX1B, 49-epiCTX3C/CTX3C, and CTX4A/CTX4B) were kindly provided by Prof. Takeshi Yasumoto (Japan Food Research Laboratories, Tokio, Japan).

Calibration was performed by dilution of the stock solution of CTX1B (4466 ng mL−1) in methanol. The calibration range selected was from 0.45 to 27.88 ng mL−1 (*n* = 5).

C-CTX1 in samples was quantified as CTX1B in the calibration curve and transformed to C-CTX1 eq. with a correction factor previously obtained with C-CTX1 pure standard in the calibration curve.

#### 4.3.2. Sample Pretreatment

First, 15 g of homogenized raw fish muscle was extracted with acetone (2 × 45 mL) using an UltraTurrax®. Acetone extracts were combined and evaporated to an aqueous residue which was partitioned with diethyl ether (2 × 15 mL). Diethyl ether layers were combined and evaporated to dryness. The solid residue was dissolved in 90% methanol (4.5 mL) and defatted with hexane (9 mL). The resulting methanolic fraction was dried and resuspended in ethyl acetate (2 mL) for clean-up procedures. A Florisil cartridge (J. T. Baker, 500 mg, Center Valley, PA, USA) was conditioned with ethyl acetate (3 mL) and the sample loaded into the cartridge. The cartridge was washed with ethyl acetate (3 mL) and C-CTXs eluted with ethyl acetate/methanol (9:1, *v:v*) (5 mL) and ethyl acetate/methanol (3:1, *v:v*) (5 mL). The second fraction, containing most of the toxins was further dried under nitrogen at 50 ◦C, and then dissolved in 60% methanol (2 mL) for a second clean-up step. Sample was loaded into a C18 cartridge (SUPELCLEAN, Supelco, 500 mg, Bellefonte, PA, USA) previously conditioned with 60% methanol (3 mL). The cartridge was washed with 60% methanol (3 mL) and CTXs were eluted with 90% methanol (5 mL). The final eluate was dried under nitrogen at 50 ◦C and dissolved in 0.5 mL of MeOH LC-MS grade filtering (Syringe Driver filter Unit, Millex®-CV 0.22 μm, 13 mm, Millipore, Billerica, MA, USA) prior to LC-MS/MS analysis.

#### 4.3.3. LC-MS/MS Analysis

An Agilent 1290 Infinity LC system coupled to an Agilent 6495 iFunnel Triple Quadrupole LC-MS (Agilent Technologies, Waldbronn, Germany) were used for the LC-MS/MS analyses. Two different methods of analysis were used in order to obtain the confident identification, quantification, and confirmation of the toxins responsible for the contamination:

The first method was used for the sensitive identification and quantification of the CTXs. The chromatographic separation was performed in a Poroshell 120 EC-C18 (3.0 × 50 mm, 2.7 μm, Agilent, Santa Clara, CA, USA) column set at 40 ◦C. The mobile phases were: (A) 0.1% formic acid and 5 mM ammonium formate in water (J. T. Baker, Center Valley, PA, USA) and (B) methanol (Merck KGaA, Darmstadt, Germany). The gradient of mobile phase started at 78% of B increasing to 88% of B in 10 min, and holding for 5 min to finally increase to the 100% of B at 15.01 min washing the column for 3 min before reducing to the initial conditions at 18 min equilibration for 4 min before the following injection. The injection volume was set at 1 μL and the flow rate 0.4 mL/min.

The MS instrument operated in ESI+ in multiple reaction monitoring (MRM) mode monitoring as precursor and product ion at 40 eV the sodium adduct [M+Na]+ of the different CTXs with reference material available: CTX1B (*m/z* 1133.6 [M+Na]+ -> *m/z* 1133.6 [M+Na]+), C-CTX1 (*m/z* 1163.7 [M+Na]+ -> *m/z* 1163.7 [M+Na]+), C-CTX1-Me (*m/z* 1177.6 [M+Na]+ -> *m/z* 1177.6 [M+Na]+), 2,3-dihydroxyCTX3C (*m/z* 1079.6 [M+Na]+ -> *m/z* 1079.6 [M+Na]+), 51-hydroxyCTX3C (*m/z* 1061.6 [M+Na]+ -> *m/z* 1061.6 [M+Na]+), 52-epi-54-deoxyCTX1B/54-deoxyCTX1B (*m/z* 1117.6 [M+Na]+ -> *m/z* 1117.6 [M+Na]+), 49-epiCTX3C/CTX3C (*m/z* 1045.6 [M+Na]+ -> *m/z* 1045.6 [M+Na]+), and CTX4A/CTX4B (*m/z* 1083.6 [M+Na]+ -> *m/z* 1083.6 [M+Na]+).

Source and interface settings were: Drying gas 15 L/min of N2 at 290 ◦C; sheath gas 12 L/min of N2 at 400 ◦C; nebulizer gas, N2 at 50 psi; capillary voltage, 5000 V; nozzle voltage: 300 V; fragmentor potential 380 V.

The second method was used to confirm the presence of C-CTX1 and specific C-CTX congeners. The chromatographic separation was performed in a Poroshell 120 EC-C18 (2.1 × 100 mm, 2.7 μm, Agilent USA) column set at 40 ◦C. The mobile phases were: (A) 0.1% formic acid and 5 mM ammonium formate in water (J. T. Baker, Center Valley, PA, USA); (B) acetonitrile (Merck KGaA, Darmstadt, Germany). The gradient of mobile phase started at 35% of B for 1 min increasing to 80% of B in 15 min, increasing in 1 min to a 95% of B and holding for 5 min to finally return to the initial conditions at 24 min equilibrating for 4 min before the following injection. The injection volume was set at 5 μL and the flow rate 0.4 mL/min.

The MS instrument operated in ESI+ in MRM mode monitoring C-CTX1 water losses ([M+H-nH2O]+) and specific fragments *m/z* 191.1 and *m/z* 108.9. The MS/MS ion transitions for the C-CTXs detected are summarized in Table 3. Source and interface settings were: Drying gas 16 L/min of N2 at 250 ◦C; sheath gas 12 L/min of N2 at 400 ◦C; nebulizer gas, N2 at 15 psi; capillary voltage, 5000 V; nozzle voltage 1000 V; fragmentor potential 380 V.

**Table 3.** MS/MS conditions for the confirmation of specific C-CTXs.

**Table 3.** *Cont.*

**Author Contributions:** Conceptualization, P.R.C., J.D. and A.G.-M.; methodology, L.S., S.M.R., P.E., D.C., J.D. and J.M.L.-M.; sampling P.R.C., L.S., N.G., V.T. and C.S.; formal analysis, L.S., J.D. and P.E.; resources, P.R.C., J.D. and A.G.-M.; data curation L.S., J.D., P.E., D.C. and J.M.L.-M.; writing—original draft preparation, P.R.C., L.S., S.M.R. and P.E.; writing—review and editing, P.R.C., J.D. and A.G.-M.; funding acquisition, P.R.C., J.D. and A.G.-M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the project EUROCIGUA, "Risk Characterization of Ciguatera Fish Poisoning in Europe" GP/EFSA/AFSCO/2015/03, co-funded by the European Food Safety Authority (EFSA), the project Cigua (PTDC/CTA-AMB/30557/2017) supported by the Portuguese Foundation for Science and Technology (FCT) and FEDER, and through FCT project (UID/Multi/04326/2020). Pablo Estevez (P.E.) acknowledges financial support from the Xunta de Galicia (Regional Government, Spain) under grant ED481A-2018/207. David Castro (D.C.) received financial support for PhD studies, obtained through EUROCIGUA project, Risk characterization of Ciguatera Fish Poisoning in Europe, framework partnership agreement GP/EFSA/AFSCO/2015/03, co-funded by the EFSA. The authors also acknowledge the CERCA Programme/Generalitat de Catalunya.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** Thanks are due to all technicians of Regional Fisheries Management—Madeira Government, Divisão de Serviços de Investigação—Direção Regional de Pescas (DSI-DRP), to the Natural Park of Madeira, and to the Nature's Wardens of Selvagens Islands for their efforts and contribution obtaining samples in the Selvagens Islands. The authors are also in debt to Takeshi Yasumoto (Japan Food Research Laboratories) who kindly provided the standards of Pacific ciguatoxins and Robert W. Dickey (University of Texas at Austin Marine Science Institute, Port Aransas, TX, USA) via Ronald Manger (Fred Hutchinson Cancer Research Center, Seattle, WA, USA) who kindly provided the standard of C-CTX1.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**