BioRisk | 3 95- | 04 (2022) . aoe nae ie Seen e ae
doi: 10.3897/biorisk.| 7.77343 RESEARCH ARTICLE & B lO R IS kK

https://biorisk.pensoft.net

Assessment of PAHs accumulation in
Donax trunculus (Linnaeus, 1758) (Bivalvia,
Donacidae) from the Bulgarian Black Sea Coast

Stanislava K. Georgieva', Mona D. Stancheva', Zlatina V. Peteva',
Tsveteslava I. Ivanova”, Albena V. Alexandrova?

| Medical University-Varna, Faculty of Pharmacy. Department of Chemistry, Marin Drinov Str. 55, 9002
Varna, Bulgaria 2 Department of Ecology, Shumen University, Shumen, Bulgaria 3 Bulgarian Academy of Sci-
ences, Institute of Neurobiology, Laboratory of Free Radical Processes, 23 Acad G. Bonchev Str., Sofia, Bulgaria

Corresponding author: Stanislava K. Georgieva (stgeorgieva@mu-varna.bg)

Academic editor: Roumiana Metchev | Received 30 October 2021 | Accepted 20 January 2022 | Published 21 April 2022

Citation: Georgieva SK, Stancheva MD, Peteva ZV, Ivanova TI, Alexandrova AV (2022) Assessment of PAHs
accumulation in Donax trunculus (Linnaeus, 1758) (Bivalvia, Donacidae) from the Bulgarian Black Sea Coast. In:
Chankova S, Peneva V, Metcheva R, Beltcheva M, Vassilev K, Radeva G, Danova K (Eds) Current trends of ecology.
BioRisk 17: 95-104. https://doi.org/10.3897/biorisk.17.77343

Abstract

Anthropogenic pollution of marine ecosystems is one of the main sources of polycyclic aromatic hydro-
carbons (PAHs). Marine bivalves are often used as bioindicators of environmental pollution due to their
wide distribution and capability of xenobiotic bioaccumulation. The aim of the present study was to assess
the presence of PAHs in soft tissues of wedge clams Donax trunculus (Linnaeus, 1758), collected from
sublittoral sandy habitats at different locations off the Bulgarian Black Sea coast. Wedge clams from the
different locations showed variations in the content of accumulated PAHs’ compounds. ‘The concentra-
tions of PAHs were measured by gas chromatography system with mass spectrometry detection. The total
PAHs content (sum of 13 PAHs’ compounds) measured was in the range from 5.59 to 50.50 ng/g wet
weight and was comparable with other European studies. The compounds phenanthrene and fluorene
were most abundant in all analyzed samples. The results showed that low molecular weight (LMW) PAHs
(2 and 3 aromatic rings) were predominant, accounting for 91% of the total PAHs levels, while high mo-
lecular weight (HMW) PAHs (4—5- and 6- rings) presence was 8.9% on average. The ratio LMW/HMW
PAHs was higher than one, suggesting predominant pollution of petrogenic origin. The concentrations of
benzo (a)pyrene did not exceed the limit set in EC Regulation although it was detected in 20% of the ana-
lyzed samples. In conclusion, maximum overall PAHs content was found in clams from Arkutino, while
minimum PAHs content was present in samples from Elenite. The Sum PAH4 (sum of four polycyclic

aromatic hydrocarbons: benzo[a]pyrene, chrisene, benzo[aJanthracene, and benzo[b]fluoranthene) in the

Copyright Stanislava K. Georgieva 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.

96 Stanislava K. Georgieva et al. / BioRisk 17: 95-104 (2022)

wedge clams for all localities studied was below legislation limits. Data from the present research can be
used for assessing pollution levels in the marine environment and also risk of human exposure to PAHs

using D. trunculus as bioindicator species.

Keywords

bioaccumulation, Bulgaria, coastal marine ecosystems, polycyclic aromatic hydrocarbons, wedge clam

Introduction

Pollution with polycyclic aromatic hydrocarbons (PAHs) is becoming a serious prob-
lem 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 accumula-
tion of toxic substances in their tissues (Suarez 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 up-
per infralittoral subzone and feeds by filtration on phytoplankton and suspended par-
ticulate 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 pol-
lution affecting the Bulgarian Black Sea coastal area and the possible risks for humans.

PAHs accumulation in wedge clams Donax trunculus OF

Materials and methods

Sampling and sample preparation

Wedge clams D. trunculus were collected manually or were obtained from commercial
providers from their sublittoral sandy habitats along the Bulgarian Black Sea coast
from May 2019 — September 2020 (Table 1). From each sampling location 2—3 kg of
adult wedge clams of similar shell length (mean 2.1 + 0.34 cm) were gathered, placed
in plastic bags, kept in ice and transported to the laboratory. Soft tissues of the indi-
vidual wedge clams were removed and 250 g samples were formed. ‘The tissues were
homogenized and stored at -20 °C until analysis.

Chemical analysis

Analytical procedures used for preparing soft tissue of mussels were described previously
(Georgieva et al. 2016) with some modification. Ten grams from the homogenized soft
tissue of clams were taken for extraction. Each sample was mixed with anhydrous so-
dium sulfate in a mortar and spiked with internal standards PCB 30 and PCB 204. The
compounds were extracted with hexane/dichloromethane (2:1; v/v) in Soxhlet Extrac-
tor for 16 hata rate of five cycles per hour. The extract was cleaned-up on a multilayer
glass column filled with neutral and acid silica. PAH compounds were eluted with n-
hexane followed by n-hexan/dichloromethane (4:1 v/v). The eluates were concentrated

Table |. Locations and period of D. trunculus collection along the Bulgarian Black Sea coast with GPS
coordinates, depth and distance from the shore.

Code Location Period GPS coordinates Depth [m] Distance from
the shore [m]

S1 Varna Bay May 2019 43.2667°N, 28.0266°E 1.50—2.00 50-100
$2 Kranevo July 2019 43.2667°N, 28.0266°E 3.00-3.50 50-100
$3 Shkorpilovtsi July 2019 42.9603°N, 27.8970°E 3.00—-3.50 20-50
S4 Ahtopol July 2019 42.1022°N, 27.9328°E 1.50—2.00 20-50
$5 Varna Bay October 2020 43.2365°N, 28.0160°E 1.50—2.00 50-100
S6 Shkorpilovtsi March 2020 42.9603°N, 27.8970°E 1.50—2.00 20-50
S7 Ahtopol March 2020 42.1140°N, 27.9243°E 1.50—2.00 20-50
S8 Sveti Vlas March 2020 42.7090°N, 27.7595°E 3.00-3.50 10-20
S9 _—_ Slanchev Bryag March 2020 42.6906°N, 27.7137°E 1.50—2.00 5-10
$10 — Irakli May 2020 42.7498°N, 27.8901°E 3.00-3.50 100-150
S11 Nessebar June 2020 42.6559°N, 27.7171°E 1.50—2.00 50-100
$12 Arkutino June 2020 42.331 1°N, 27.7368°E 2.50—3.00 50-100
$13. Duni June 2020 42.3684°N, 27.7094°E 1.50—2.00 5-10
$14 Sozopol June 2020 42.4148°N, 27.7004°E 1.50—2.00 5-10
$15 Primorsko June 2020 42.2530°N, 27.7533°E 1.50—2.00 50-100
S16 Cape Emine August 2020 42.7018°N, 27.8343°E 2.50—3.50 200-250
$17 Tsarevo September 2020 42.1666°N, 27.8533°E 2.50—3.00 50-100
$18 Elenite September 2020 42.7025°N, 27.8130°E 3.50—4.00 200-250
$19 Primorsko September 2020 42.2577°N, 27.7520°E 1.50—2.00 50-100
$20 = Arkutino September 2020 42.3282°N, 27.7496°E 3.00-3.50 100-150

98 Stanislava K. Georgieva et al. / BioRisk 17: 95-104 (2022)

to near dryness with a gentle stream of nitrogen and reconstituted in 0.5 cm? in hexane.
One microliter of extract was injected into the gas chromatography system in triplicate.

The quantitative analysis of PAHs was performed on gas chromatograph (GC/MS)
GC FOCUS (Thermo Electron Corporation, USA) using POLARIS Q Ion Trap mass
spectrometer (MS), equipped with an AI 3000 autosampler and splitless Injector. The
experimental MS parameters, temperature of ion source and temperature of transfer
line, were 220 °C and 250 °C, respectively. The PAHs experimental oven temperature
was programed as follows: 40 °C (1 min), 40 °C/min to 130 °C (3 min), 12 °C/min to
180 °C, 7 °C/min to 280 °C, 10 °C/min to 310 °C with a final hold for 5.0 min. The
separation of compounds was achieved with a TG-5 ms capillary column with a length
of 30 m, 0.25 mm ID and a film thickness of 0.25 um (Thermo Fisher Scientific). The

carrier gas helium was used at a flow rate of 1 ml/min.

Quality control

Pure reference standard solution (EPA 525 PAH Mix B, 500 pg/mL) of each compo-
nent in acetone (Supelco) was used for instrument calibration, quantification of com-
pounds and recovery determination. Procedural blanks were analyzed between each 5
samples to monitor possible laboratory contamination.

In the prepared extracts thirteen PAHs compound were measured: acenaphthyl-
ene (ACL), anthracene (AN), benz[a]anthracene (BaA), benzo[b]fluoranthene (BbFA),
benzo[k]fluoranthene (BkFA), benzo[ghi]perylene (BghiP), benzo[a]pyrene (BaP),
chrysene (CHR), dibenzo[a,h]anthracene (DbahA), fluorene (FL), indeno[1,2,3-cd]
pyrene (IP), phenanthrene (PHE), pyrene (PY). Each sample was analyzed three times
and an average was taken.

Limits of detection (LOD) were estimated as 3 times the standard deviation, based
on the low concentrations of the analytes in the sample. LOD varied for individual
compounds from 0.075 to 0.294 ng/g wet weight (ww). For each PAHs were (ng/g
ww): ACL 0.185, FL 0.128, PHE 0.092, AN 0.294, PY 0.173, BaA 0.219, CHR
0.185, BbFA 0.155, BkFA 0.178, BaP 0.295, IP 0.175, DBahA 0.199, and BghiP
0.075. The concentrations were reported as MEAN+SD of three measurements. Dif-
ferences in means were determined by t-test (p < 0.05 considered significant).

Results

The mean concentrations of individual PAHs measured ranged from 0.05 (benzolg,h,i]
perylene) to 19.03 ng/g ww (phenanthrene) (Table 2). Acenaphthylene, anthracene,
benzo[aJanthracene, indeno[1,2,3-cd] pyrene and dibenzo[a.h]anthracene showed lev-
els 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). Phen-
anthrene (PHE) predominated (from 3.24 to 48.22 ng/g) in all samples.

PAHs accumulation in wedge clams Donax trunculus 99

Table 2. Individual PAHs concentrations (ME +SD ng/g ww) in D. trunculus from the representative
localities off the Bulgarian Black Sea coast in different sampling periods (nd — not detected, PAHs: poly-
cyclic aromatic carbons; BbFA: benzo[b]fluoranthene, BkFA: benzo[k]fluoranthene, BghiP: benzo[ghi]
perylene, BaP: benzo[a]pyrene, CHR: chrysene, FL: fluorene, PHE: phenanthrene and PY: pyrene)*.

Site Location Period FL PHE PY CHR BbFA BkFA BaP BghiP Sum PAH4 Sum PAH13

S1 Varna Bay May-19 4.11 21.81 nd 0.92 nd nd nd_= nd 0.92 26.84
S2. Kranevo Jul-19 6.48 29.05 nd nd 0.50 nd 0.54 nd 1.04 36.56
$3 Shkorpilovtsi Jul-19 9.55 27.44 nd nd nd 0.46 nd nd 0.00 37.44
S4 Ahtopol Jul-19 0.43 3.24 nd 0.98 3.41 nd nd_ 0.07 4,39 8.13
S5 Varna Bay Oct-20 1.49 Nd nd nd 3.59 nd 2.34 0.07 5.93 7.49
S6 Shkorpilovtsi Mar-20 4.57 18.10 nd 0.92 0.16 nd nd _ 0.07 1.08 23.82
$7 Ahtopol Mar-20 1.56 8.92 nd 0.90 1.36 nd nd nd 2.26 12.74
S8 Sveti Vlas Mar-20 10.02 15.25 nd 0.53 0.30 0.54 nd _ 0.00 0.83 26.64
S9 Slanchev Bryag Mar-20 1.97 8.17 nd 0.91 1.46 nd 1.08 nd 3.45 13.58
$10 Irakli May-20 7.71 35.31 0.30 1.10 1.33 nd nd_ 0.08 2.43 45.83
S11 Nessebar Jun-20 14.21 31.19 nd 0.28 3.38 nd_ ond nd 3.66 49.07
$12 Arkutino Jun-20 0.21 48.22 nd 0.56 0.21 nd 1.22 0.07 2.00 50.50
$13 Duni Jun-20 21.00 22.68 nd 0.64 0.24 nd _ nd nd 0.89 44.57
S14 Sozopol Jun-20 4.51 nd nd 041 2.13 0.81 nd nd 2.54 7.86
S15 Primorsko Jun-20 6.12 7.17 nd 0.46 0.26 nd nd _ 0.10 0.72 14.11
S16 Cape Emine Aug-20 2.99 19.72 nd 0.47 1.18 0.42 nd _ 0.08 1.65 24.84
S17 Tsarevo Sep-20 9.38 73.81 nd nd 4.10 nd nd_ 0.00 4.10 87.29
S18 Elenite Sep-20 0.60 4.16 nd nd 0.78 nd nd_ 0.06 0.78 DIDS
$19 Primorsko Sep-20. nd 5.62 nd nd 41.76 nd = nd_ 0.00 1.76 7.38
S20 Arkutino Sep-20 0.63 20.38 nd 0.64 0.56 nd nd nd Fen 22.21

*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 and are not present in the Table.

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 (Shkorpi-
lovtsi (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 PAH 13) 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[aJanthracene, 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

100 Stanislava K. Georgieva et al. / BioRisk 17: 95-104 (2022)

—|North South

CHR BbFA BkFA BaP BghiP

0,00

Figure |. Individual levels of PAHs (ME+SD) in wedge clams (D. trunculus) sampled from the northern

and southern locations of the Bulgarian Black Sea coast.

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 signifi-
cant for autumn 2019) and Sum PAH 13 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).

Discussion

The results obtained in this study showed that in all wedge clams different PAHs were accu-
mulated. 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

PAHs accumulation in wedge clams Donax trunculus 101

= Sum 4PAHs
Sum 13PAHs
& LMW PAHs
= HMW PAHs

Sum PAHs, ng/g ww

Figure 2. Total PAHs levels in wedge clams (D. trunculus) sampled in different seasons from the studied

locations off the Bulgarian Black Sea coast.

reported earlier for bivalves from the Crimean Peninsula (Olenycz et al. 2015), Sevastopol
Bay (Shchekaturina et al. 1995) and the Turkish coast (Giiven and Coban 2012).

PAHs can originate from a variety of sources: LMW PAHs are defined as petro-
genic compounds (resulting from spillage of diesel and fuel oil), and HMW PAHs 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 hy-
drocarbons indicates the sources of PAHs. In our study the ratio LMW/HMW PAHs
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 ac-
cumulated composition of PAHs established by us was as follows: 91% were LMW
PAHs 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 PAH 13 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/HMW PAHs 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

102 Stanislava K. Georgieva et al. / BioRisk 17: 95-104 (2022)

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 re-
ported as the major contributor of total hydrocarbons (Readman et al. 2002).

The high content of LMW PAHs in the studied wedge clams, strongly indicated
that oil pollution was the major source of contamination. Phenanthrene has high lipo-
philicity 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 pres-
sure from the intensive maritime traffic and industrial activities in the area.

Most published dataon 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 con-
sidered 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).

Conclusion

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 indus-
trial 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.

Acknowledgements

This work was supported by grant KI1-06-H31/6 of National Science Fund, Bulgaria.

PAHs accumulation in wedge clams Donax trunculus 103

References

Banni M, Negri A, Dagnino A, Jebali J, Ameur S, Boussetta H (2010) Acute effects of
benzo[a]pyrene on digestive gland enzymatic biomarkers and DNA damage on mussel
Mytilus galloprovincialis. Ecotoxicology and Environmental Safety 73(5): 842-848. https://
doi.org/10.1016/j.ecoenv.2009.12.032

Barhoumi B, El Megdiche Y, Clérandeau C, Ameur WB, Mekni S, Bouabdallah S, Derouiche
A, Touil S, Cachot J, Driss MR (2016) Occurrence of polycyclic aromatic hydrocarbons
(PAHs) in mussel (Myzilus galloprovincialis) and eel (Anguilla anguilla) from Bizerte lagoon
Tunisia and associated human health risk assessment. Continental Shelf Research 124:
104-116. https://doi.org/10.1016/j.csr.2016.05.012

EC (2011) Commission Regulation (EC) n.835/2011 of 19 August 2011 amending. 201 1n.
Regulation (EC) No 1881/2006 as regards maximum levels for polycyclic aromatic hydro-
carbons in foodstuffs. Official Journal of the European Union, L 215: 4-8.

Ferrante M, Zanghi G, Cristaldi A, Copat C, Grasso A, Fiore M, Signorelli SS, Zuccarello P, Oliveri
Conti G (2018) PAHs in seafood from the Mediterranean Sea: An exposure risk assessment.
Food and Chemical Toxicology 115: 385-390. https://doi-org/10.1016/j.fct.2018.03.024

Georgieva S, Stancheva M, Makedonski L (2016) Investigation about the presence of organo-
chlorine pollutants in mussels from the Black Sea Bulgaria. Analele Universitatii Ovidius
Constanta. Seria Chimie 27(1): 8-12. https://doi.org/10.1515/auoc-2016-0006

Gumus MR, Todorova VR, Panayotova MD (2020) Recent Observations on the Size Structure
of Donax trunculus Linnaeus. 1758 and Chamelea gallina (Linnaeus. 1758) in the Bulgar-
ian Black Sea as Status Indicators of Commercially Exploited Shellfish under the Marine
Strategy Framework Directive (MSFD). Ecologia Balkanica. Special Edition 3: 63-71.
http://web.uni-plovdiv.bg/mollov/EB/2020_SE3/063-071_eb.20SE310.pdf

Giiven K, Coban B (2012) Oil pollution in Turkish Black Sea coast and input of oil from
Turkey to the Black Sea in 2004-2007. Fresenius Environmental Bulletin 21: 3711-3717.

IARC (2010) Monographs on the evaluation of carcinogenic risks to humans. some non-heter-
ocyclic polycyclic aromatic hydrocarbons and some related exposures. Lyon. http://mono-
graphs. iarc.fr/ ENG/Monographs/vol92/mono92.pdf

Ifegwu OC, Anyakora C (2016) Chapter Six - Polycyclic Aromatic Hydrocarbons: Part II,
Urine Markers In: Makowski GS (Ed.) Advances in Clinical Chemistry vol. 75. Elsevier,
159-183. https://doi.org/10.1016/bs.acc.2016.03.001

Kucuksezgin EK Gonul LT, Pazi I, Ubay B, Guclusoy H (2020) Monitoring of polycyclic aro-
matic hydrocarbons in transplanted mussels (Mytilus galloprovincialis) and sediments in
the coastal region of Nemrut Bay (Eastern Aegean Sea). Marine Pollution Bulletin 157:
e111358. https://doi.org/10.1016/j.marpolbul.2020.111358

Lehtonen K, d’Errico G, Korpinen S, Regoli F, Ahkola H, Kinnunen T, Lastumaki A (2019)
Mussel Caging and the Weight of Evidence Approach in the Assessment of Chemical Con-
tamination in Coastal Waters of Finland (Baltic Sea). Frontiers in Marine Science 6: e688.
https://doi.org/10.3389/fmars.2019.00688

Machado AA, Hoff ML, Klein RD, Cordeiro GJ, Lencina Avila JM, Costa PG, Bianchini
A (2014) Oxidative stress and DNA damage responses to phenanthrene exposure in the

104 Stanislava K. Georgieva et al. / BioRisk 17: 95-104 (2022)

estuarine guppy Poecilia vivipara. Marine Environmental Research 98: 96-105. https://
doi.org/10.1016/j.marenvres.2014.03.013

Mercogliano R, Santonicola S, De Felice A, Anastasio A, Murru N, Ferrante MC, Cortesi ML
(2016) Occurrence and distribution of polycyclic aromatic hydrocarbons in mussels from
the gulf of Naples. Tyrrhenian Sea. Italy. Marine Pollution Bulletin 104(1—2): 386-390.
https://doi.org/10.1016/j.marpolbul.2016.01.015

Okay O, Karacik B, Giingordii A, Yilmaz A, Koyunbaba NC, Yakan SD, Henkelmann B, Sch-
ramm K-W, Ozmen M (2017) Monitoring of organic pollutants in marine environment by
semipermeable membrane devices and mussels: Accumulation and biochemical respons-
es. Environmental Science and Pollution Research International 24(23): 19114-19125.
https://doi.org/10.1007/s11356-017-9594-0

Olenycz M, Sokolowski A, Niewinska A, Wolowicz M, Namiesnik J, Hummel H, Jansen J
(2015) Comparison of PCBs and PAHs levels in European coastal waters using mussels
from the Mytilus edulis complex as biomonitors. Oceanologia 57(2): 196-211. https://doi.
org/10.1016/j.oceano.2014.12.001

Panteleeva M, Chamova R, Radeva N, Romanova H (2021) Anthropogenic Disasters on Bulgar-
ian Territory: Chemical Accidents on Land and at Sea. Journal of IMAB—Annual Proceed-
ing Scientific Papers 27(2): 3718-3722. https://doi.org/10.5272/jimab.2021272.3718

Perugini M, Visciano P, Giammarino A, Manera M, Di Nardo W, Amorena M (2007) Polycyclic
aromatic hydrocarbons in marine organisms from the Adriatic Sea. Italy. Chemosphere
66(10): 1904-10. https://doi.org/10.1016/j.chemosphere.2006.07.079

Readman JW, Fillmann G, Tolosa I, Bartocci J, Villeneuve JP, Catinni C, Mee LD (2002)
Petroleum and PAH contamination of the Black Sea. Marine Pollution Bulletin 44(1):
48-62. https://doi.org/10.1016/S0025-326X(01)00189-8

Shchekaturina TL, Khesina AL, Mironov OG, Krivosheeva LG (1995) Carcinogenic polycyclic
aromatic hydrocarbons in mussels from the Black Sea. Marine Pollution Bulletin 30(1):
38-40. https://doi.org/10.1016/0025-326X(94)00070-P

Srogi K (2007) Monitoring of environmental exposure to polycyclic aromatic hydrocarbons:
A review. Environmental Chemistry Letters 5(4): 169-195. https://doi.org/10.1007/
s10311-007-0095-0

Suarez P, Ruiz Y, Alonso A, San Juan F (2013) Organochlorine compounds in mussels cul-
tured in the Ria of Vigo: Accumulation and origin. Chemosphere 90(1): 7-19. https://doi.
org/10.1016/j.chemosphere.2012.02.030

Tanabe S, Subramanian A (2006) Bioindicators Suitable for Monitoring POPs in Developing
Countries. Kyoto University Press, Kyoto, 190 pp.

Tlili S, Mouneyrac C (2019) The wedge clam Donax trunculus as sentinel organism for Medi-
terranean coastal monitoring in a global change context. Regional Environmental Change

19(4): 995-1007. https://doi.org/10.1007/s10113-018-1449-9