Research Article |
Corresponding author: Zlatina V. Peteva ( zlatina_peteva@mail.bg ) Academic editor: Stephka Chankova
© 2023 Zlatina V. Peteva, Stanislava K. Georgieva, Bernd Knock, Thomas Max, Mona D. Stancheva, Simona Valkova.
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.
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
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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 variable 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 period. 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 positive 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.
Domoic acid, monitoring, pectenotoxins, the Black Sea
Bulgarian Black Sea coastline comprises 432 km (
Black Sea mussel production and catchment has increased in recent years (
Microalgae are the primary food source for mussels (
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 importance. Furthermore, the determined levels will be compared to marine toxins levels from previous periods of investigation.
Phytoplankton samples (n = 21) (Table
№ | Sample № | Sampling site (Coordinates) | Type of the region | Sampling date |
---|---|---|---|---|
1 | ME6 | 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 | ME15 | 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 |
7 | 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 | ME58 | 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 | ME68 | 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 | 5.12.2021 |
20 | ME89 | Varna 43°12'42.2"N, 27°57'30.1"E | Areas with anthropogenic activities | 5.12.2021 |
21 | ME92 | Varna 43°11'36.8"N, 27°51'46.5"E | Areas with anthropogenic activities | 5.12.2021 |
Immediately after sampling, net haul concentrates were adjusted to a defined volume of 500–1000 ml (depending on the net tow volume) using 20 μm filtered seawater. After centrifugation (4000 × 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 μl 100% methanol for domoic 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 μm pore size, Ø25 mm, Minisart, Sartorius, Germany). Filtrates (1000 μl) 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
The quality control was performed by regular analysis of procedural blanks and certified reference material (National Research Council, Canada). Limits of detection (LOD) for lipophilic toxins and DA were determined based on 3:1 signal-to-noise ratio.
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.
In total, 21 plankton samples were collected in the studied period February-December 2021 along the Bulgarian coastline in accordance with sampling plan (Table
With the aim to analyse for the presence of selected marine biotoxins, appropriate retention times and LODs were achieved (Table
Investigated lipophilic toxins and domoic acid including associated standard solution concentrations, LODs, quantification transitions and retention times.
Marine toxins investigated | Concentration of standard solution pg/µl | LOD ng/NT | Quantification transition (m/z) | Retention time (min) |
---|---|---|---|---|
DA | 100 | 4.93 | 312→266 | 7.17 |
OA | 500 | 25.71 | 822→223 | 11.57 |
DTX2 | 500 | 36.59 | 822→223 | 11.87 |
DTX1 | 500 | 60.00 | 836→237 | 12.57 |
PTX2 | 100 | 3.73 | 876→213 | 12.14 |
PTX2-sa | – | – | 894→213 | 11.70 |
Epi-PTX2-sa | – | – | 894→213 | 11.90 |
GONA | 412.5 | 30.56 | 786→607 | 12.67 |
YTX | 1000 | 100.00 | 1176→981 | 13.00 |
OH-YTX | – | – | 1176→981 | 11.70 |
AZA1 | 100 | 0.92 | 842→824 | 12.62 |
GYMA | 500 | 1.50 | 508→490 | 10.33 |
SPX1 | 100 | 2.58 | 692→164 | 11.22 |
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
Sample № | DA, ng/NT | PTX2, ng/NT |
---|---|---|
ME6 | 170,94 | < LOD |
ME9 | < LOD | 9.23 |
ME15 | 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 protected region, respectively.
Comparison of the results with results obtained from previous studies in the same regions (
Comparison of DA levels with previous studies (n- indicates the number of positive samples).
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 former period of time, the PTX2 concentration ranges are much wider (Fig.
The Bulgarian coast is important for the development of the economy and tourism in the country (
Bulgaria is considered as a minor producer of seafood, responsible for 0.01 percent of world production and 0.4 percent of EU fishery and aquaculture products in terms of volume (
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 (
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 (
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 (
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 (
Quantitative and qualitative analysis of marine biotoxins was performed by applying 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 (
Interestingly, two other marine toxins were detected in previous periods – YTX and SPX1. Yesotoxins were registered in the samples from 2016–2018. The concentration 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 to determine in plankton samples (
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 abundance or even absence of A. ostenfeldii, as SPX 1 production is associated with this species (
This further reinforces the belief that toxin production by plankton is an unpredictable phenomenon (
Results obtained in this paper including the values below LOD indicate that abundance of marine biotoxins is not alarming. This suggests that good quality of mussel 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.
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. Project No BG14MFOP001-6.004-0006-C01, contract No МДР-ИП-01-13/25.01.2021 “Investigation of priority chemical pollutants and biotoxins and assessment of the state of the marine environment”.