BioRisk 20: 7 | -8 | (2023) Apeer-reviewed open-access journal doi: 10.3897/biorisk.20.97557 RESEARCH ARTICLE & 3 Ke) RP IS k https://biorisk.pensoft.net Occurrence of marine biotoxins on Bulgarian Black Sea coastal waters in 2021 Zlatina V. Peteva'’, Stanislava K. Georgieva', Bernd Knock’, Thomas Max?, Mona D. Stancheva!, Simona Valkova! | Medical University Varna, Tsar Osvoboditel 84, 9002 Varna, Bulgaria 2 Dobrudzha Technological College, Dobrotitsa 12, 9302 Dobrich, Bulgaria 3 Alfred Wegener Institut, Helmholtz Zentrum fir Polar- und Meeres- forschung, Chemische Okologie, am Handelshafen 12, 27570 Bremerhaven, Germany Corresponding author: Zlatina V. Peteva (zlatina_peteva@mail.bg) Academic editor: St. Chankova | Received 14 November 2022 | Accepted 23 December 2022 | Published 15 May 2023 Citation: Peteva ZV, Georgieva SK, Knock B, Max T, Stancheva MD, Valkova S (2023) Occurrence of marine biotoxins on Bulgarian Black Sea coastal waters in 2021. In: Chankova S, Danova K, Beltcheva M, Radeva G, Petrova V, Vassilev K (Eds) Actual problems of Ecology. BioRisk 20: 71-81. https://doi.org/10.3897/biorisk.20.97557 Abstract Marine biotoxins are produced by certain phytoplankton species and used to accumulate in filter-feeding marine organisms. The occurrence of marine biotoxins in all aquatic environments and latitudes is vari- able in time and space. Thus, it is an essentially natural phenomenon, but the occurrence of toxigenic phytoplankton cannot be completely avoided or eliminated. A serious concern appears if these substances accumulate at high levels in seafood. If it is consumed by mammals including humans, severe illness of consumers of intoxicated seafood may result. The aim of this study is to assess the presence of marine biotoxins in plankton samples taken in 2021 and to compare the determined levels with a previous pe- riod. Plankton samples (n = 21) were collected in 2021 along the whole Bulgarian coastline (Black Sea). The presence of hydrophilic (domoic acid (DA)) and lipophilic toxins (okadaic acid, dinophysis toxin — 1, dinophysis toxin -2, azaspiracid-1, goniodomin A, pectenotoxin-2 (PTX2), yessotoxin, spirolide-1 and gymnodimine A) was investigated via liquid chromatography — tandem mass spectrometry (LC-MS/ MS). Results indicated the presence of only DA in three samples and PTX2 in two samples. The posi- tive samples were sporadically distributed throughout the study period. During 2016-2019, LC-MS/MS analysis confirmed the presence of DA, PTX2, YTX, SPX-1 and GDA in plankton net samples collected from the same locations reported here. The matching toxins (DA and PTX2) were at comparable levels in both periods of investigation, thus lower than in other European waters where harmful algal blooms are registered. These results show the persistent appearance of some marine biotoxins in Bulgarian waters. Although levels were low in the monitored periods, a constant monitoring is required in order that toxic events by seafood consumption be avoided. Copyright Zlatina V. Peteva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. fies Ziatina V. Peteva et al. / BioRisk 20: 71-81 (2023) Keywords Domoic acid, monitoring, pectenotoxins, the Black Sea Introduction Bulgarian Black Sea coastline comprises 432 km (Stanchev et al. 2013). It is impor- tant for recreational, touristic (Stoyanova et al. 2019; Ihtimanski et al. 2020; Nikolo- va et al. 2021) and commercial (Raykov and Nicheva 2018; Stancheva et al. 2022) activities. The Black Sea is a commercial seafood source, including shellfish and fish, as well as providing popular recreational fisheries (General Doctorate for Internal Policies 2011; EAFA 2020). Black Sea mussel production and catchment has increased in recent years (EAFA 2021). Mussels have been documented to contain beneficial values of polyunsaturat- ed fatty acid, proteins, vitamins etc. and, therefore, are a preferred food worldwide (Hyung et al. 2018; Carboni et al. 2019; Yaghubi et al. 2021). Microalgae are the primary food source for mussels (Brown 2002; Pleissner et al. 2012), but some microalgal species are reported as toxic or harmful. These phyto- plankton species tend to produce potent toxins that accumulate in filter feeders. Yearly, potential producers of marine toxins (Pseudo-nitzschia, Alexandrium, Dinophysis) are registered in Bulgarian coastal waters (Dzhembekova et al. 2021; Dzhembekova et al. 2022). Marine toxins are transferred through the food chain to the higher trophic levels and may cause severe illness in them. A wide range of symptoms, from dizziness, digestive (nausea and vomiting) to nervous complaints, are associated with human intoxication by biotoxins, characterising different and specific syndromes, called shell- fish poisonings. The risk assessment of the occurrence of toxigenic phytoplankton is complicated by the fact that toxin levels of plankton samples do not always correlate with biomass and abundance of potentially toxigenic species. The aim of this study is to evaluate the levels of marine biotoxins in plankton samples of the year 2021 collected from areas of different function and economic im- portance. Furthermore, the determined levels will be compared to marine toxins levels from previous periods of investigation. Materials and methods Sampling plan Phytoplankton samples (n = 21) (Table 1) were hauled vertically from depths between one and five metres from the surface with a conical plankton net (20 um mesh size, 40 cm outer diameter) along the Bulgarian coast in the period March-December 2021. Occurrence of marine biotoxins on Bulgarian coastal waters in 2021 re: Table |. Collected plankton samples. Ne Sample Ne Sampling site (Coordinates) Type of the region Sampling date 1 MEG North 43°32'348"N, 029°18'224"E Intensive fishing activities 31.03.2021 2 ME8 South 42°26'296"N,027°41'360"E Mussel farming site 25.05.2021 3 ME9 North 43°21'276"N, 028°27'053"E Intensive fishing activities 31.03.2021 4 MEI15 North 43°39'961"N, 029°39'793"E Intensive fishing activities 13.04.2021 5 ME17 North 43°21'885"N, 028°20'528"E Mussel farming site 13.04.2021 6 ME38 North 43°01'23.5"N, 27°53'22.0"E Protected area 18.07.2021 E ME47 North 43°24'14.3"N, 28°21'11.8"E Mussel farming site 26.07.2021 8 ME48 North 43°23'58.9"N, 28°09'34.5"E Intensive fishing activities 27.07.2021 9 ME49 South 42°38'14.3"N, 27°40'26.5"E Area with anthropogenic activities 11.08.2021 10 ME57 North 43°07'09.8"N, 28°02'52.1"E Protected area 08.10.2021 11 MES58 Varna 43°13'31.9"N, 28°02'12.3"E — Areas with anthropogenic activities 08.10.2021 12 ME59 Varna 43°16'52.5"N, 28°07'03.9"E — Areas with anthropogenic activities 08.10.2021 13 ME66 South 42°33'19.4"N, 27°38'19.4"E Mussel farming site 1.11.2021 14 MEG68 South 42°39'58.6"N, 27°43'06.5"E Protected area 1.11.2021 15 ME74 North 43°21'01.7"N, 28°28'49.8"E Protected area 4.11.2021 16 ME75 North 43°23'49.9"N, 28°19'36.1"E Mussel farming site 4.11.2021 17 ME76 South 42°43'15.0"N, 27°55'26.1"E Protected area 4.11.2021 18 ME83 South 43°01'23.5"N, 27°53'22.0"E Protected area 29.11.2021 19 ME86 Varna 43°10'28.3"N, 27°54'60.0"E — Areas with anthropogenic activities 5.122021 20 ME89 Varna 43°12'42.2"N, 27°57'30.1"E — Areas with anthropogenic activities 5.122021 21 ME92 Varna 43°11'36.8"N, 27°51'46.5"E — Areas with anthropogenic activities 5.12.2021 Sampling sites close to mussel farming areas, areas used for harvesting of wild mussels (including areas of intensive fisheries and areas with anthropogenic activities), as well as protected areas, were included in the sampling plan. Experimental plan Immediately after sampling, net haul concentrates were adjusted to a defined volume of 500-1000 ml (depending on the net tow volume) using 20 um filtered seawater. After centrifugation (4000 x g, 10 min at 10 °C), the supernatant was discarded. The cell pellets were stored in at -20 °C until further processing. Plankton pallets were suspended washed with 1000 pl 100% methanol for domo- ic acid and lipophilic toxins extraction. The methanolic acid suspensions were than sonicated (40 Hz, 15 min) and centrifuged by 4000 x g for 10 min at 10 °C. The supernatant was filtered through syringe filters (0.45 um pore size, @25 mm, Minisart, Sartorius, Germany). Filtrates (1000 ul) were transferred into chromatographic vials and kept at -20 °C until further analysis. The hydrophilic domoic acid (DA) and lipophilic toxins — goniodomin A (GDA), okadaic acid (OA), dinophysistoxins -1 and 2 (DTX1,2), pectenotoxins (PTX2, PTX2-sa, epi-PTX-sa), yessotoxins (YTX, OH-YTX), azaspiracid-1 (AZA1), spirolides (SPX1) and gymnodimine A (GYMA) were analysed according Krock et al. (2008) on 74 Zilatina V. Peteva et al. / BioRisk 20: 71-81 (2023) a LC-MS/MS system. It consists of liquid chromatograph (model 1100 LC, Agilent, Waldbronn, Germany) coupled to a triple quadrupole mass spectrometer (API 4000 QTrap, Sciex, Darmstadt, Germany), equipped with a Turbo Spray interface. The quality control was performed by regular analysis of procedural blanks and certified reference material (National Research Council, Canada). Limits of detec- tion (LOD) for lipophilic toxins and DA were determined based on 3:1 signal-to- noise ratio. Calculations Contents of the toxin are expressed as nanograms per net tow (ng/NT) in order to be compared with previous results and other literature data. Results In total, 21 plankton samples were collected in the studied period February-December 2021 along the Bulgarian coastline in accordance with sampling plan (Table 1). With the aim to analyse for the presence of selected marine biotoxins, appropriate retention times and LODs were achieved (Table 2). The huge efforts for the toxin profile revealed a scarce presence of marine biotoxins in the plankton samples. Amongst the investigated toxins - DA, GDA, OA, DTX1, DTX2, PTX2, PTX2-sa, epi-PTX-sa, YTX, OH-YTX, AZA1, SPX1) and GYMA, only DA and PTX2 were detected (Table 3). Table 2. Investigated lipophilic toxins and domoic acid including associated standard solution concen- trations, LODs, quantification transitions and retention times. Marine toxins | Concentration of standard LOD ng/NT Quantification Retention time investigated solution pg/ul transition (m/z) (min) DA 100 4.93 312—266 alsy OA 500 25.71 822223 157 DTX2 500 36.59 822223 11.87 DTX1 500 60.00 836237 Le PTX 100 i Ase) 876213 12.14 PTX2-sa - - 894-213 LILO Epi-PTX2-sa c. . 894-9213 11.90 GONA 412.5 30.56 786—607 12.67 YTX 1000 100.00 1176981 13.00 OH-YTX - - 1176-981 11.70 AZA1 100 0.92 842824 12.62 GYMA 500 1.50 508-490 10.33 SPX1 100 2.58 692— 164 11.22 Occurrence of marine biotoxins on Bulgarian coastal waters in 2021 75 Table 3. Levels of detected toxins in plankton samples. Sample Ne DA, ng/NT PTX2, ng/NT MEG 170,94 < LOD ME9 < LOD 9:93. MEI15 138.48 < LOD ME58 13.38 < LOD ME76 < LOD 6.51 Results obtained in this study showed that only 14% of the samples were positive for DA and only 9% for PTX2. DA was present in spring and autumn samples from areas with intensive fishing and anthropogenic activities. Pectenotoxin-2 was detected in a spring and autumn sample from an area with anthropogenic activities and a pro- tected region, respectively. Comparison of the results with results obtained from previous studies in the same regions (Peteva et al. 2018; Peteva et al. 2020) showed that, in 2016, DA was detected in 57% of the samples, in 2017 in 41%, in 2018 — 17% of samples and in 2019, in none of the samples. In 2017 and 2018, plankton samples investigated were many more than in 2016 and in 2021. Comparison of the concentration range of the positive sam- ples showed a great variability between the different periods of investigation (Fig. 1). N=6 2017 2018 Emin ®@max Figure |. Comparison of DA levels with previous studies (n- indicates the number of positive samples). 76 Ziatina V. Peteva et al. / BioRisk 20: 71-81 (2023) 2017 2018 min @max Figure 2. Comparison of PTX2 levels with previous studies. The two PTX2 positive samples from 2021 represent 10% of all samples. Thus, in previous studies, the portion of positive samples was much higher — 86% in 2016, 48% in 2017, 47% in 2018 and in 2019 — 67% of the samples. Moreover, in this for- mer period of time, the PTX2 concentration ranges are much wider (Fig. 2). Discussion The Bulgarian coast is important for the development of the economy and tourism in the country (Dimitrov and Rangelov 2018; Mooser et al. 2022). Bulgaria is considered as a minor producer of seafood, responsible for 0.01 per- cent of world production and 0.4 percent of EU fishery and aquaculture products in terms of volume (EUMOFA 2020). Recent investigations showed a persistent value of the catch (for 2015—2020 — 8476 tonnes), as well as a visible peak in 2019 Occurrence of marine biotoxins on Bulgarian coastal waters in 2021 ris (Shivarov 2021). Fishing activities are performed almost throughout the whole year and along the whole coastline. Thus, it is known that fishing activities change the environmental parameters in the regions where they are undertaken (Stier et al. 2020; Gissi et al. 2021). In Bulgarian mariculture farms, Mediterranean mussel (Mytilus galloprovincialis Lamarck) is dominant farmed species. The total marine aquaculture production of 2,531 t in 2018 consists mainly of this mollusc (Klisarova et al. 2020). Recent state reports showed that, since 2008, Bulgaria is one of the important suppliers of Mediter- ranean mussels in the Black Sea region. Nowadays, Bulgarian mussel farms produced over than 1.5% of the cultivated mussels in the world (Ministry of Agriculture and Food Bulgaria 2019). A number of factors of natural and predominantly anthropogenic nature have a negative impact on the state of the environment of this region of the country (Kotsev and Prodanov 2020, Kotsev et al. 2021). Natural factors, superimposed in a number of cases by anthropogenic activity, are mainly abrasion, landslides, floods of the coast from the sea and climate change (Penchev 2019). Anthropogenic activities and technological advances are commonly pointed out to justify the increasing occurrence, frequency and intensity of harmful algal blooms and the detection of new toxins or emergence of toxins in regions where they were previously not known (Costa 2019; Otero and Silva 2022). In this regard, investi- gation and comparison of the toxin profiles of plankton samples from locations of fishing and anthropogenic activities, as well as mussel farming sites, seem meaningful and informative. Coastal protected areas in Bulgaria are established by national policy instruments/ laws and EU Directives to protect a wide range of natural and cultural resources (Stancheva et al. 2016). In these areas, any catch and industrial activities are banned by the law (Ministry of Regional Development and Public Works 1998). Accordingly, protected areas are considered control sites in this study. Quantitative and qualitative analysis of marine biotoxins was performed by apply- ing liquid chromatography coupled with mass spectrometry which is acknowledged by the scientific community as one of the most powerful analytical tools able to identify multiple toxins (Visciano et al. 2016; Estevez et al. 2019). Results indicate that, in 33% of samples from the areas with anthropogenic activities and in 50% of the sam- ples from the areas of intensive fishing, marine biotoxins were detected. No toxins were detected in the samples from the mussel farms and in one sample from a protected area. Further interpretation of the results would be possible if the investigation is re- peated in a future period. Interestingly, two other marine toxins were detected in previous periods — YTX and SPX1. Yesotoxins were registered in the samples from 2016-2018. The concen- tration range is very large — 0.001 — 1.959 ng/NT. In the present study, no YTXs were detected. The small number of positive samples in the previous period, as well as the absence hereby, is most likely due to the fact that yesotoxins are exotoxins. Once synthesised, they are rapidly released into the environment and, therefore, difficult 78 Ziatina V. Peteva et al. / BioRisk 20: 71-81 (2023) to determine in plankton samples (Hess and Aasen 2007). For example, Krock et al. (2013) also investigated the levels of lipophilic toxins along the German and Danish coasts, but yesotoxins were not determined. Our previous investigation showed that SPX1 was registered in the samples from summer-autumn 2018 in a concentration range from 0.054—0.245 ng/NT. No spirolides were registered in this study. This result might be associated with low abun- dance or even absence of A. ostenfeldii, as SPX 1 production is associated with this species (Van Wagoner et al. 2011; Guinder et al. 2018). This further reinforces the belief that toxin production by plankton is an unpre- dictable phenomenon (Kremp et al. 2019) and studies on it should be continued. Conclusions Results obtained in this paper including the values below LOD indicate that abun- dance of marine biotoxins is not alarming. This suggests that good quality of mus- sel meat might be expected. Monitoring of harmful phytoplankton composition and biotoxins should be continued in future, so it can provide the opportunity to react in good time in order to prevent negative consequences which can be caused by HABs and biotoxins. Acknowledgements This work was supported by the Maritime Affairs and Fisheries Program 2014-2020 co-financed by the European Union through the European Maritime Affairs and Fisheries Fund. 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