Tetrodotoxins Occurrence in Non-Traditional Vectors of the North Atlantic Waters (Portuguese Maritime Territory, and Morocco Coast)
Abstract
:1. Introduction
2. Results and Discussion
2.1. Portuguese Continental Coast
2.2. Madeira Island (Madeira Archipelago)
2.3. São Miguel Island (Azores Archipelago)
2.4. Moroccan Coast
2.5. Statistical Analysis
3. Conclusions
4. Materials and Methods
4.1. Sampled Points and Selected Species
4.2. Reagents
4.3. Sample Extraction
4.4. UHPLC Conditions
4.5. MS Methods
4.5.1. Source Conditions
4.5.2. UHPLC-MS/MS Analysis: MRM Mode
4.5.3. UHPLC-MS/MS Analysis: PIS Mode
4.5.4. UHPLC-HRMS
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Structure | Analogue | R1 | R2 | R3 | R4 | Molecular Formula | Exact Mass |
---|---|---|---|---|---|---|---|
| TTX | H | OH | OH | CH2OH | C11H17N3O8 | 320.1088 |
4-epiTTX | OH | H | OH | CH2OH | C11H17N3O8 | 320.1088 | |
6-epiTTX | H | OH | CH2OH | OH | C11H17N3O8 | 320.1088 | |
11-deoxyTTX | H | OH | OH | CH3 | C11H17N3O7 | 304.1139 | |
11-norTTX-6(R/S)-ol | H | OH | H | OH | C10H15N3O7 | 290.0983 | |
6,11-dideoxyTTX | H | OH | H | CH3 | C11H17N3O6 | 288.1190 | |
11-oxoTTX | H | OH | OH | CHO | C11H17N3O9 | 336.1038 | |
| 5-deoxyTTX | H | OH | OH | CH2OH | C11H17N3O7 | 304.1139 |
5,11-dideoxyTTX | H | OH | H | CH3 | C11H17N3O6 | 288.1190 | |
5,6,11-trideoxyTTX | H | OH | H | CH3 | C11H17N3O5 | 272.1214 | |
| 4,9-anhydroTTX | --- | --- | OH | CH2OH | C11H15N3O7 | 302.0983 |
6-epi-4,9-anhydroTTX | --- | --- | CH2OH | OH | C11H15N3O7 | 302.0983 | |
| Tetrodonic acid | --- | --- | --- | --- | C11H17N3O8 | 320.1088 |
Suspicious TTXs | Multiple Reaction Monitoring (MRM) | Product Ions Scan (PIS) | |||
---|---|---|---|---|---|
Precursor Ion | Product Ions | [M+H]+ | [M+H-H2O]+ | Other Product Ions | |
oxoTTX | 336 | 318/162 | 336 | 318 | 300/176/162 |
TTX and epimers | 320 | 302/162 | 320 | 302 | 284/256/178/162 |
deoxyTTX | 304 | 286/162 | 304 | 286 | 268/176/162 |
anhydroTTX | 302 | 256/162 | 302 | 284 | 256/178/162 |
norTTX | 290 | 272/162 | 290 | 272 | 176/162 |
dideoxyTTX | 288 | 270/224 | 288 | 270 | 224/178/162 |
trideoxyTTX | 272 | 254/162 | 272 | 254 | 236/178/162 |
anhydrotrideoxyTTX | 254 | 236/162 | 254 | 236 | 178/162 |
Sampling Date | Sampling Site | Sample | Species | Trophic Level [Ref] | Edible | Code | Suspicious TTXs | RT (min) | Elemental Composition | Theoretical m/z | Experimental m/z | Error (mDa) | TTX eq (µg/Kg) |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
September 2011 | Porto Côvo | Limpet | Patella depressa | Grazer [27] | Yes | 225 | 11-oxoTTX | 7.2 | C11H17N3O9 | 336.1038 | 336.1000 | −3.8 | <LOQ |
Gastropod | Phorcus lineatus | Grazer [28] | Yes | 226 | trideoxyTTX | 7.7 | C11H17N3O5 | 272.1241 | 272.1232 | −0.9 | <LOQ | ||
October 2011 | Pedra de Jacó | Sea-urchin | Echinus esculentus | Grazer [29] | Yes | 229_2 | 11-oxoTTX | 7.2 | C11H17N3O9 | 336.1038 | 336.1020 | −1.8 | <LOQ |
Ovar | Gastropod | Charonia lampas | 3rd Level Predator [30] | Yes | 232_2 | trideoxyTTX | 7.7 | C11H17N3O5 | 272.1241 | 272.1230 | −1.1 | <LOQ | |
232_3 | trideoxyTTX | 7.7 | C11H17N3O5 | 272.1241 | 272.1251 | 1.0 | <LOQ | ||||||
November 2011 | Memória | Gastropod | Nucella lapillus | 1st Level Predator [27] | Yes | 236 | trideoxyTTX | 7.7 | C11H17N3O5 | 272.1241 | 272.1239 | −0.2 | <LOQ |
Phorcus lineatus | Grazer [28] | Yes | 239 | 11-oxoTTX | 7.2 | C11H17N3O9 | 336.1038 | 336.1000 | −3.8 | <LOQ | |||
Gibbula umbilicalis | Grazer [28] | Yes | 240 | trideoxyTTX | 7.7 | C11H17N3O5 | 272.1241 | 272.1238 | −1.1 | <LOQ |
Sampling Date | Sampling Site | Sample | Species | Trophic Level [Ref] | Edible | Code | Standard TTX | Suspicious TTXs | RT (min) | Elemental Composition | Theoretical m/z | Experimental m/z | Error (mDa) | TTX eq (µg/Kg) |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
June 2013 | Lagoa | Starfish | Ophidiaster ophidianus | Detritivorous [32] | No | 412 | norTTX | 3.1 | C10H15N3O7 | 290.0983 | 290.2489 | 150.6 | 30,902 | |
norTTX | 4.6 | C10H15N3O7 | 290.0983 | 290.2303 | 132.0 | 699 | ||||||||
June 2013 | Ilhéu de São Roque | Starfish | Ophidiaster ophidianus | Detritivorous [32] | No | 424 | norTTX | 3.1 | C10H15N3O7 | 290.0983 | 290.2475 | 149.2 | 42,604 | |
norTTX | 4.6 | C10H15N3O7 | 290.0983 | 290.2298 | 131.5 | 1638 | ||||||||
June 2013 | Caloura | Starfish | Ophidiaster ophidianus | Detritivorous [32] | No | 435 | norTTX | 3.1 | C10H15N3O7 | 290.0983 | 290.2478 | 149.9 | 20,853 | |
June 2013 | Caloura | Fish (Gonads) | Sphoeroides marmoratus | 3rd Level Predator [33] | Yes | 438 G | TTX | 8.1 | C11H17N3O8 | 320.1088 | 320.1088 | 0.0 | 352,886 | |
4-epiTTX | 7.8 | C11H17N3O8 | 320.1088 | 320.1082 | −0.6 | 3157 | ||||||||
deoxyTTX | 7.7 | C11H17N3O7 | 304.1139 | 304.1129 | −1.0 | 446 | ||||||||
11-deoxyTTX | 7.0 | C11H17N3O7 | 304.1139 | 304.1126 | −1.3 | 157,156 | ||||||||
deoxyTTX | 6.5 | C11H17N3O7 | 304.1139 | 304.1124 | −1.5 | 108,375 | ||||||||
4,9-anhydroTTX | 7.1 | C11H15N3O7 | 302.0983 | 302.1005 | 2.2 | 91 | ||||||||
dideoxyTTX | 6.4 | C11H17N3O6 | 288.1190 | 288.1157 | −3.3 | 22,232 | ||||||||
trideoxyTTX | 5.0 | C11H17N3O5 | 272.1241 | 272.1198 | −4.3 | 12,708 | ||||||||
trideoxyTTX | 4.8 | C11H17N3O5 | 272.1241 | 272.1228 | −1.3 | 37,367 | ||||||||
trideoxyTTX | 4.6 | C11H17N3O5 | 272.1241 | 272.1227 | −1.4 | 1849 | ||||||||
anhydrotrideoxyTTX | 4.7 | C11H15N3O4 | 254.1135 | 254.1122 | −1.3 | 364 | ||||||||
June 2013 | Caloura | Fish (Liver) | Sphoeroides marmoratus | 3rd Level Predator [33] | Yes | 438 F | TTX | 8.1 | C11H17N3O8 | 320.1088 | 320.1098 | 1.0 | 41 | |
4-epiTTX | 7.8 | C11H17N3O8 | 320.1088 | nd | 6 | |||||||||
11-deoxyTTX | 7.0 | C11H17N3O7 | 304.1139 | nd | <LOQ | |||||||||
deoxyTTX | 6.9 | C11H17N3O7 | 304.1139 | nd | 4 | |||||||||
deoxyTTX | 6.5 | C11H17N3O7 | 304.1139 | 304.1153 | 1.4 | 136 | ||||||||
4,9-anhydroTTX | 7.1 | C11H15N3O7 | 302.0983 | nd | <LOQ | |||||||||
dideoxyTTX | 6.4 | C11H17N3O6 | 288.1190 | nd | 283 | |||||||||
trideoxyTTX | 5.0 | C11H17N3O5 | 272.1241 | nd | 31 | |||||||||
trideoxyTTX | 4.8 | C11H17N3O5 | 272.1241 | 272.1263 | 2.2 | 250 | ||||||||
trideoxyTTX | 4.6 | C11H17N3O5 | 272.1241 | nd | 14 | |||||||||
June 2013 | Caloura | Fish (Muscle) | Sphoeroides marmoratus | 3rd Level Predator [33] | Yes | 438 M | TTX | 8.1 | C11H17N3O8 | 320.1088 | 320.1088 | 0.0 | 9918 | |
4-epiTTX | 7.8 | C11H17N3O8 | 320.1088 | 320.1067 | −2.1 | 118 | ||||||||
deoxyTTX | 7.7 | C11H17N3O7 | 304.1139 | 304.1164 | 2.5 | 29 | ||||||||
11-deoxyTTX | 7.0 | C11H17N3O7 | 304.1139 | 304.1172 | 3.3 | 48 | ||||||||
deoxyTTX | 6.9 | C11H17N3O7 | 304.1139 | 304.1123 | −1.6 | 65 | ||||||||
deoxyTTX | 6.5 | C11H17N3O7 | 304.1139 | 304.1100 | −3.9 | 2085 | ||||||||
4,9-anhydroTTX | 7.1 | C11H15N3O7 | 302.0983 | 302.0958 | −2.5 | 45 | ||||||||
dideoxyTTX | 6.4 | C11H17N3O6 | 288.1190 | 288.1145 | −4.5 | 2953 | ||||||||
trideoxyTTX | 5.0 | C11H17N3O5 | 272.1241 | nd | 122 | |||||||||
trideoxyTTX | 4.8 | C11H17N3O5 | 272.1241 | 272.1217 | −2.4 | 507 | ||||||||
trideoxyTTX | 4.6 | C11H17N3O5 | 272.1241 | nd | 26 | |||||||||
anhydrotrideoxyTTX | 4.7 | C11H15N3O4 | 254.1135 | 254.1129 | −0.6 | 5 |
Sampling Date | Sampling Site | Sample | Species | Trophic Level [Ref] | Edible | Code | Standard TTX | Suspicious TTXs | RT (min) | Elemental Composition | Theoretical m/z | Experimental m/z | Error (mDa) | TTX eq (µg/Kg) |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
July 2013 | Mrizika | Gastropod | Onchidella celtica | Grazer [43] | No | 474 | TTX | 8.1 | C11H17N3O8 | 320.1088 | 320.1082 | −0.6 | 12 | |
4-epiTTX | 7.8 | C11H17N3O8 | 320.1088 | 320.1071 | 1.7 | 8 | ||||||||
TTX epimer | 7.5 | C11H17N3O8 | 320.1088 | nd | 4 | |||||||||
July 2013 | Oualidia | Gastropod | Aplysia depilans | Grazer [44] | No | 476 | TTX | 8.1 | C11H17N3O8 | 320.1088 | nd | <LOQ | ||
4-epiTTX | 7.8 | C11H17N3O8 | 320.1088 | nd | <LOQ |
Sampling Sites | Coefficents ± SE | z | p |
---|---|---|---|
São Miguel | 8.96 ± 0.001 | 5000.3 | <0.001 |
Madeira | −19.26 ± 21.38 | −0.9 | 0.37 |
Morocco | −10.67 ± 0.38 | −28.2 | <0.001 |
Continental Portugal | −19.26 ± 13.30 | −1.4 | 0.15 |
Date | Location | Sampling Site | Geographic Coordinates |
---|---|---|---|
September 2011 | Continental Portugal | Luz | 37°04′37″ N; 8°44′51″ W |
Camilo | 37°05′14″ N; 8°40′06″ W | ||
Porto Côvo | 37°53′33″ N; 8°47′38″ W | ||
Pelo Negro | 41°19′22″ N; 8°71′69″ W | ||
October 2011 | Continental Portugal | Pedra de Jacó | 41°17′14″ N; 8°70′78″ W |
Ovar | 40°52′58″ N; 8°40′31″ W | ||
Esposende | 41°29′5″ N; 8°46′45″ W | ||
November 2011 | Continental Portugal | Memória | 41°13′50″ N; 8°43′18″ W |
December 2011 | Continental Portugal | Esposende | 41°29′5″ N; 8°46′45″ W |
Valadares | 41°5′29″ N; 8°39′27″ W | ||
January 2012 | Continental Portugal | Póvoa do Varzim | 41°22′41″ N; 8°46′7″ W |
Angeiras | 41°15′50″ N; 8°43′37″ W | ||
September 2012 | Madeira Island | Reis Magos | 32°39′16″ N; 16°49′05″ W |
Caniçal | 32°44′20″ N; 16°44′17″ W | ||
June 2013 | São Miguel Island | Cruzeiro | 37° 50′31″ N; 25° 41′33″ W |
Étar de Ponta Delgada | 37°44′19″ N; 25°39′38″ W | ||
São Roque | 37°45′15″ N; 25°38′31″ W | ||
Mosteiros | 37°53′25″ N; 25°49′14″ W | ||
Lagoa | 37°44′42″ N; 25°19′47″ W | ||
Caloura | 37°42′49″ N; 25°29′54″ W | ||
July 2013 | Morocco Coast | Casablanca corniche | 33°36′01″ N; 7°39′57″ W |
El Jadida Haras | 33°14′42″ N; 8°28′37″ W | ||
El Jadida Sâada | 33°14′42″ N; 8°32′26″ W | ||
Sidi Bouzid | 33°13′57″ N; 8°33′20″ W | ||
Mrizika | 32°57′21″ N; 8°46′53″ W | ||
Oualidia | 32°43′55″ N; 9°02′57″ W |
Compound | Precursor Ion | Product Ion | CE |
---|---|---|---|
11-oxo-TTX | 336 | 318 | 24 |
178 | 40 | ||
TTX 4-epiTTX TTX epimers Tetrodonic acid | 320 | ||
302 | 24 | ||
161.9 | 36 | ||
11-deoxyTTX deoxyTTX | 304 | 286 | 24 |
176 | 40 | ||
4,9-anhydro-TTX anhydroTTX | 302 | 256 | 28 |
161.9 | 40 | ||
11-norTTX-6(R/S)-ol | 290 | 272 | 24 |
162 | 40 | ||
dideoxyTTX | 288 | 270 | 24 |
224 | 40 | ||
hydroxytrideoxyTTX | 288 | 270 | 24 |
162 | 40 | ||
trideoxyTTX | 272 | 254 | 24 |
162 | 40 | ||
hydroxyanhydrotrideoxyTTX | 270 | 252 | 24 |
162 | 40 | ||
anhydrotrideoxyTTX | 254 | 236 | 24 |
162 | 40 |
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Silva, M.; Rodríguez, I.; Barreiro, A.; Kaufmann, M.; Neto, A.I.; Hassouani, M.; Sabour, B.; Alfonso, A.; Botana, L.M.; Vasconcelos, V. Tetrodotoxins Occurrence in Non-Traditional Vectors of the North Atlantic Waters (Portuguese Maritime Territory, and Morocco Coast). Toxins 2019, 11, 306. https://doi.org/10.3390/toxins11060306
Silva M, Rodríguez I, Barreiro A, Kaufmann M, Neto AI, Hassouani M, Sabour B, Alfonso A, Botana LM, Vasconcelos V. Tetrodotoxins Occurrence in Non-Traditional Vectors of the North Atlantic Waters (Portuguese Maritime Territory, and Morocco Coast). Toxins. 2019; 11(6):306. https://doi.org/10.3390/toxins11060306
Chicago/Turabian StyleSilva, Marisa, Inés Rodríguez, Aldo Barreiro, Manfred Kaufmann, Ana Isabel Neto, Meryem Hassouani, Brahim Sabour, Amparo Alfonso, Luis M. Botana, and Vitor Vasconcelos. 2019. "Tetrodotoxins Occurrence in Non-Traditional Vectors of the North Atlantic Waters (Portuguese Maritime Territory, and Morocco Coast)" Toxins 11, no. 6: 306. https://doi.org/10.3390/toxins11060306