Pollution with polycyclic aromatic hydrocarbons (PAHs) is becoming a serious problem in marine ecosystems. PAHs are a group of organic compounds that consist of two, three or more condensed aromatic rings. They are highly persistent and widespread in the marine environment. PAHs are produced primarily from organic combustion and also from other anthropogenic activities and can enter into the marine environment in two main ways - by chronic pollution (associated with boat traffic) or by acute pollution due to oil spills (Kucuksezgin et al. 2020). Different marine bivalves (clams, oysters, mussels, scallops) have been recommended as suitable bioindicators of contamination in the sea due to their wide geographical distribution, filter feeding and rapid accumulation of toxic substances in their tissues (Suárez et al. 2013; Tlili and Mouneyrac 2019). Reliable data exist on the bioavailability and accumulation of marine environmental pollutants in bivalve tissues (such as PAHs, polychlorinated biphenyls, pesticides and heavy metals) and their toxic effects (Tanabe and Subramanian 2006; Georgieva et al. 2016; Lehtonen et al. 2019). However, the accumulation of organic pollutants depends not only on the physico-chemical characteristics of contaminants, but also on bivalve physiology and their lipid content (Barhoumi et al. 2016; Lehtonen et al. 2019). The potential of PAHs to cause adverse effects as endocrine- and reproductive disruption, genotoxicity and oxidative damage in marine organisms has been previously reported (Banni et al. 2010; Machado et al. 2014) and it was also demonstrated that acute benzo[a]pyrene exposure significantly depressed acetylcholinesterase (AChE) activity in gills and digestive gland of mussels (Mytilus galloprovincialis) (Banni et al. 2010).

The wedge clam (D. trunculus) is a species inhabiting fine sandy habitats of the upper infralittoral subzone and feeds by filtration on phytoplankton and suspended particulate matter. In the Bulgarian Black Sea coastal zone D. trunculus dominates usually between 1.0 and 6.5 m depth and is exposed to intense wave action and fluctuations of abiotic environmental factors (Gumus et al. 2020). Although local people do not traditionally consume wedge clams, D. trunculus are increasingly being collected with dredges for export due to the high prices on the foreign markets (Gumus et al. 2020). Data on the accumulation of PAHs in tissues of marine bivalve species is scarce, which determines the importance of research of PAHs’ content accumulated in wedge clams inhabiting the Bulgarian Black Sea area.

The aim of the present study was to carry out the first comprehensive assessment of the levels of bioaccumulated PAHs in tissues of D. trunculus as an indicator of the pollution affecting the Bulgarian Black Sea coastal area and the possible risks for humans.

The mean concentrations of individual PAHs measured ranged from 0.05 (benzo[g,h,i]perylene) to 19.03 ng/g ww (phenanthrene) (Table 2). Acenaphthylene, anthracene, benzo[a]anthracene, indeno[1,2,3-cd]pyrene and dibenzo[a.h]anthracene showed levels below the instrumental detection limit in all samples (not present in Table 2). Four of the 13 target PAHs were found in almost all samples. BkFA and BghiP were detected in clams with very low values, close to the limit of quantitation (LOQ). Phenanthrene (PHE) predominated (from 3.24 to 48.22 ng/g) in all samples.

The sum of 4 PAHs: benzo[a]pyrene, chrisene, benzo[a]anthracene, and benzo[b]fluoranthene (Sum PAH4) in the studied wedge clams from the Bulgarian Black Sea coastal zone varied in the range from 0.72 to 5.93 ng/g ww (Table 2). Relatively low Sum PAH4 levels were observed in wedge clams from 3 sampling locations (Shkorpilovtsi (S3), Primorsko (S15) and Elenite (S18)) which are situated far from highly urbanized and industrial areas off the Black Sea coast (Table 2). In contrast, the Sum PAH4 in D. trunculus from Varna Bay (S5) (industrial city and harbor area) was found to be higher (5.93 ng/g ww) (Table 2). The total PAHs levels (sum of 13 individual PAHs, Sum PAH13) in samples from Elenite (S18) (5.59 ng/g ww) and Tsarevo (S17) (87.29 ng/g ww) differed significantly (p < 0.05). Relatively high total PAH levels were observed also in wedge clam samples from Arkutino (S12), Nessebar (S11), Irakli (S10), and Duni (S13).

Among the four PAHs (benzo[a]pyrene, benz[a]anthracene, benzo[b]fluoranthene and chrysene), chrysene was the most abundant compound (Table 2). As a whole, chrysene and benzo (b)fluoranthene were detected in more than 60% of the samples, while benzo[a]pyrene, known to be the most carcinogenic, was found in only 4 samples. Chrysene was present in 67% of the wedge clam samples (from 0.41 to 1.10 ng/g ww) while benzo[a]pyrene was found in 20% of the analyzed samples with (mean 1.30 ng/g ww). Benzo[k]fluoranthene was found (mean 0.56 ng/g ww) only in wedge clams from Shkorpilovtsi (S3), Sveti Vlas (S8), Sozopol (S14) and Cape Emine (S16) (Table 2).

The mean individual levels of PAHs in D. trunculus sampled from the northern (north of Cape Emine) and southern (south of Cape Emine) locations of the Bulgarian Black Sea coast are presented in Fig. 1.

In general, the mean concentrations of CHR, BbFA, and BaP were higher in wedge clams from the northern coastal zone (Fig. 1). The concentration of PHE in wedge clam samples from locations in the northern part of the Black Sea coast was significantly higher (23.44 ng/g ww) than from the southern coast samples (17.00 ng/g ww). Only the mean concentration of FL was significantly higher in wedge clams from the southern coastal locations, compared to the northern ones.

The level of PAHs, accumulated in the studied wedge clams, displayed some seasonal variation (Fig. 2). The Sum PAH4 was significantly higher in autumn (difference significant for autumn 2019) and Sum PAH13 was significantly higher in summer compared to spring (2020) and autumn (2019 and 2020). The low molecular weight (LMW) PAHs accumulation was lower in autumn, whereas the accumulated high molecular weight (HMW) PAHs were higher in autumn (difference significant for autumn 2019).

The results obtained in this study showed that in all wedge clams different PAHs were accumulated. This is a strong indication that seawater and sediments along the Bulgarian Black Sea coast are contaminated with organic pollutants. Similar findings for the Black Sea were reported earlier for bivalves from the Crimean Peninsula (Olenycz et al. 2015), Sevastopol Bay (Shchekaturina et al. 1995) and the Turkish coast (Güven and Coban 2012).

PAHs can originate from a variety of sources: LMWPAHs are defined as petrogenic compounds (resulting from spillage of diesel and fuel oil), and HMWPAHs as having pyrolitic origin (products of the incomplete combustion of organic matter) (Srogi 2007; Mercogliano et al. 2016). The molecular ratio of LMW and HMW hydrocarbons indicates the sources of PAHs. In our study the ratio LMW/HMWPAHs was higher than one (mean 17.61), suggesting that PAHs pollution of the Bulgarian Black Sea coastal region was predominantly of petrogenic origin. The pattern of accumulated composition of PAHs established by us was as follows: 91% were LMWPAHs with 3-rings (PHE and FL); 8.7% were PAHs with 4- and 5-rings -; < 1% were the PAHs with 6-rings.

In this study, the highest levels of Sum PAH13 were found in wedge clams from Tsarevo (S17), followed by Arkutino (S12) and Nessebar (S11). Given that the PAHs have mainly pyrolytic and petrogenic origin (Mercogliano et al. 2016), it seemed most likely that the high content of PAHs, present in wedge clams from these locations, resulted from local spills or currents that carried these pollutants. Relatively high levels of Sum PAH13 were also found in the wedge clams from the southern locations Duni (S13) and Arkutino (S12), but they were most probably the result of the oil spill in 2018 from the sunken ship SS Mopang near the coast of Sozopol (Panteleeva et al. 2021). This was further confirmed by the high ratio LMW/HMWPAHs established in D. trunculus from these locations, which strongly suggested a petrogenic source of the pollution. Although all the mentioned localities are situated in the southern part of the Bulgarian Black Sea coast, there were no significant differences present in the accumulated PAHs in wedge clams from the northern and southern coastal regions. As an exception, statistically higher levels of accumulated phenanthrene were measured in wedge clams from the northern coastal areas. This could be associated with discharges from the Danube River, containing degraded petroleum and fresh oil which are reported as the major contributor of total hydrocarbons (Readman et al. 2002).

The high content of LMWPAHs in the studied wedge clams, strongly indicated that oil pollution was the major source of contamination. Phenanthrene has high lipophilicity that makes it readily absorbed from the gastrointestinal tract and transferred to the tissues (Ifegwu and Anyakora 2016). The highest benzo[a]pyrene contend was observed in wedge clams from Varna Bay (S5), indicating significant ecological pressure from the intensive maritime traffic and industrial activities in the area.

Most published data on PAHs accumulation concerned mussels (M. galloprovincialis) (Perugini et al. 2007; Mercogliano et al. 2016) and the only more comprehensive study of PAHs in wedge clams was from the Mediterranean Sea (Ferrante et al. 2018). The content of PAHs in D. trunculus found in our study (4.75 ng/g ww) was significantly lower than the PAHs’ levels in the wedge clams reported from the Mediterranean Sea (247.4 ng/g ww) (Ferrante et al. 2018).

Admissible limit was set for Sum PAH4 in bivalve species by EC n.835 regulation (EC 2011). The established in our study values of Sum PAH4 in wedge clams were well below the regulation limit (35 ng/g ww). With regard to benzo[a]pyrene, which is considered to be highly carcinogenic according to the International Agency for Research on Cancer (IARC Group 1) (IARC 2010), we also did not establish values exceeding the admissible limits (6 ng/g ww) (EC 2011).

Results from this study showed the presence of accumulated PAHs in wedge clams from all the 15 locations along the Bulgarian Black Sea coast, which strongly indicated the presence of contamination with organic pollutants. The level of PAHs content in the wedge clams showed variations depending on local conditions. The maximum PAHs content was found in wedge clams from locations near to urbanized and industrial areas, or areas with highly intensive tourism. In general, there were no significant seasonal variations in the level of total PAHs, although some exceptions were observed. Our data did not show the presence of PAHs values exceeding admissible limits set by national and EC regulations. The data from the present research can be useful in further studies for assessing PAHs pollution levels and risks for human exposure using D. trunculus as bioindicator species.