Comparative Analysis of Heavy Metal and Radionuclide Contaminants In

Comparative analysis of heavy metal and radionuclide Technology & Science Water contaminants in Black Sea green and red macroalgae

A.S. Strezov and Tz.P. Nonova Institute for Nuclear Research and Nuclear Energy, Bulgarian Academy of Sciences, 72 Tzarigradsko shosse Blvd, Soﬁa 1784, Bulgaria, (E-mail: [email protected], [email protected])

Abstract A comparative analysis about the accumulation of heavy metal, natural and technogenic radionuclides from the Black Sea environment in different Bulgarian coastline regions is made. The possibilities to use Chlorophyta and Rhodophyta algae phylums as bioindicators in marine environment are 1–8 pp 11 No 51 Vol investigated. Environmental contamination in the Black Sea alga species (green and red) was studied from 1992 to 2003. Sampling sites were selected to cover the whole coastal region. Low level gamma spectrometry was used to determine the natural and technogenic nuclide concentrations. The heavy metal concentrations were measured by Atomic Absorption Spectrometry (AAS). The obtained data show that radionuclide and metal concentrations depend on the macrophyte type. Tendencies in the concentration of pollutant variations during the studied period are examined and all data give information about different Q macrophytic species’ ability to accumulate certain elements from one and the same sampling location. All W ulsig2005 Publishing IWA obtained results show that use of macroalgae in marine environmental monitoring reduces the need for complex studies on chemical speciation of aquatic contaminants and makes algae valuable indicators for seawater quality assessment. All data show the lack of serious pollution along the Bulgarian Black Sea coast. Keywords Black Sea; contamination; HM; macroalgae; monitoring; radionuclides

Introduction The anthropogenic contamination of aquatic ecosystems is a stress factor and deﬁnes the necessity for systematic monitoring and control of contaminants (heavy metals (HM) and radionuclides) that affect marine biota. In many cases contamination of the water phase can lead to a decrease in species number or total mass in the aquatic ecosystem and can cause its disintegration. Investigations of contamination of the marine environment are based on describing contaminant sources, pathways, dispersals and their concentration changes. Many authors have investigated biogeochemical migration of HM and radionuclides as well as biological effects on marine organisms in the Black Sea environment (Polikarpov et al., 1991; Gu¨ven et al., 1992; Topcuoglu and Gungor, 1999; Topcuoglu et al., 2001). Bioaccumulation studies are a relatively direct method for measuring the bioavailabi- lity of speciﬁc contaminants in aquatic media. The use of bioindicators reduces the need for complex studies on the chemical speciation of aquatic contaminants (Phillips and Segar, 1986). The macrophytes play a major role in the food chain in marine ecosystems and they can be used for studying the bioaccumulation because they are abundant and have adequate tissues for analysis. Algae are successfully used for long-term investigation of ecological status within aquatic ecosystems. Some species are widespread so they are appropriate for comparative assessment of seasonal and local contaminants concentration variations in different geographical areas. The Black Sea is one of the most isolated marine basins in the world. It is a unique ecosystem with special ecological conditions (inner sea with high primary productivity and low salinity and H2S accumulation below 150–200 m) and its hydrology and phyto- benthos content is different from the other seas in the same biogeographic region. One of the major anthropogenic pollutants in the Black Sea ecosystems is HM and radionuclides. 1 The main sources of pollution are atmospheric fallout, the big rivers run-off as well as local land-based outfalls (Mee, 1992; Tuncer et al., 1998). The Black Sea is located close to the Chernobyl power station and it is the most affected marine ecosystem after the accident (1986). Several biological and nonbiological factors (species, growth cycle, contaminant concentrations, complexing agents, tempera- ture, light, season, salinity, etc.) are known to affect HM and nuclide behavior resulting ..SrzvadT..Nonova Tz.P. and Strezov A.S. in contaminant accumulation by marine algae and sediments. The macrophytic species from phylums Rhodophyta (red) and Chlorophyta (green) are widely distributed in the coastal Black Sea ecosystems. Many studies of environmental pollution have demonstrated the use of green (Ulva lactuca, U. rigida and Enteromorpha intestinalis) and red (Ceramium rubrum) algae species. Ulva and Enteromorpha species, both belonging to the Ulvaceae family, have been extensively used to monitor marine pollution in various geographical areas (Favero et al., 1996; Muse et al., 1999). Ulva species have proved to be an indicator for, Mn, Cu, Pb and Cd and are one of the widespread green algae species not only in the Black Sea (Ho, 1990; Haritonidis and Malea, 1999). C. rubrum is one of the most studied red algae and its ability to accumulate some metals and radionuclides is well known too. The aim of this study is to give information about HM and radionuclide accumulation in green and red macroalgae species, which are widely distributed along the Bulgarian Black Sea coast. After comparison of local and seasonal concentration variations, the typical element values for the studied species are also determined from the created data- base (Strezov et al., 1998; 2001) about the monitoring of HM and radionuclides content at the Bulgarian Black Sea coastal ecosystems.

Methods Three green (Ulva rigida, Enteromorpha intestinalis and Cladophora vagabunda) and two red (Ceramium rubrum and Callithamnion corymbosum) alga species were studied for nuclides and HM concentrations. Samples were collected from same stations during the studied period at 0.5–2 m depth. All the plants were washed with seawater, transported to the laboratory. Sample preparation for HM and nuclide measuring was described elsewhere (Strezov et al., 1998, 2001). Sampling sites (Shabla, Tuzlata, Kaliakra, Ravda, Ahtopol, Sinemoretz and Rezovo) were selected along the Bulgarian Black Sea zone during the period 1992–2003 (Figure 1). The chosen algae species belong to the group of attached macrophytes – Ulva rigida is a perennial species and dominant through the whole year while the other four species are annual.

HM were determined after HNO3 digestion by AAS – Pye Unicam 1950 atomic absorption spectrophotometer with air–acetylene flame (Cu) and a Perkin-Elmer Zeeman 3030 spectrophotometer with graphite furnace (Pb, Cd). The g spectrometry measurements were carried out for 137Cs (technogenic), 226Ra and 210Pb (natural) radionuclides using HPGe detector (Ortec) with 35% counting efficiency and energy resolution 1.8 keV (at 1332 keV). Quality control was assured by use of certified material NIES-CRM-3 (Chlorella). The precision of the analytical methodology of HM examination is determined by the SR [%] values (McFarren et al., 1970) for each element in relation to standard reference materials. The assessment of reproducibility is presented as a relative standard deviation RSD [%]. The data show that Pb and Cd results in Chlorella species are excellent 2 (SR , 25%) and acceptable (25% , SR , 50%) for Cu. The RSD for Cd, Cu and Pb ROMANIA

Shabla h Tuzlata hhKaliakra Nonova Tz.P. and Strezov A.S.

Varna n

BLACK SEA

Ravdah h Burgas n

Ahtopol h Sinemoretz h h hRezovo

TURKEY

Figure 1 Map of sampling location was under 10%. From this it can be concluded that the performed analytical procedures lead to a good precision.

Results and discussion It is known that some metal ions are essential as trace elements for aquatic plants and their biological uptake is a function of the free metal concentration in seawater. The element Cu is one of the most biologically important metals and usually exists in the bio- sphere only in trace quantities. The determination of HM pollutants like Cd and Pb, in water ecosystems is of great importance because they are involved in the biological cycle and possess a high toxicity. The obtained data for Cu, Pb and Cd content was determined in all investigated alga species from three locations (see map) and is presented on Figure 2. HM concentration levels in alga tissues can be put in the following order: Cu . Pb . Cd. The obtained Cu content in the studied Black Sea algae varies between 0.6 and 15.4 mg/g. Minimal value was measured in green Ulva rigida (Sinemoretz, summer 2000) and the maximal value – in C. rubrum (Kaliakra, spring 1998). The mean Cu concentrations in all ﬁve species vary as follows: 7.4 mg/g in C. rubrum, 6.2 mg/g in Clad. vagabunda and Ent. intestinalis and 5.5 mg/g for Ulva rigida and Callith. corymbosum. The determined HM variations are small and the overall Cu mean is found to be 6 ^ 2 mg/g except for Ulva rigida at Kaliakra and Ent. intestinalis at Ravda. The obtained concentrations in all studied algae for Pb vary between 0.3 and 8.4 mg/g and from 0.2 to 3.2 mg/g for Cd. Minimal Pb contents were obtained in red alga C. rubrum from Ravda and Sinemoretz (autumn 1996) and from Ahtopol (autumn 2002). The maximal Pb value was measured in the same species from Tuzlata (summer 1992). Two Cd extremal concentrations were determined in green macroalgae species: the lowest – in Ulva rigida from Tuzlata (autumn 2002) and the highest – in Clad. vagabunda from Sinemoretz (spring 1998). 3 15 Cu 13 Pb 11 Cd 9 7 ..SrzvadT..Nonova Tz.P. and Strezov A.S. 5

3 1 –1 C. rubrum C. rubrum C. rubrum Ulva rigida Ulva rigida Ulva rigida Callithamnion Callithamnion Callithamnion Ent. intestinalis Ent. intestinalis Ent. intestinalis Clad. vegabunda Clad. vegabunda Clad. vegabunda

Kaliakra Ravda Sinemoretz

Figure 2 HM content (mg/g) in Black Sea algae from Kaliakra, Ravda and Sinemoretz

The mean Cu, Pb and Cd concentration values (mg/g dry weight) obtained during the whole studied period were calculated to compare the macroalgae accumulation ability. The results show that the ﬁve studied alga species can be put in the following orders: Cu: C. rubrum . Clad. Vagabunda < Ent. intestinalis . Ulva rigida < Callithamnion mg/g: 7.4 6.2 6.0 5.6 5.5 Pb: Clad. vagabunda . C. rubrum < Ent. intestinalis . Ulva rigida < Callithamnion mg/g: 3.9 3.0 2.9 1.80 1.78 Cd: Clad. vagabunda . Ent. intestinalis . C. rubrum . Ulva rigida . Callithamnion mg/g: 1.03 0.88 0.74 0.51 0.41 The narrow interval of Cu, Pb and Cd concentration variations (mean values) in marine macroalgae demonstrate weak dependence on alga type. The highest degrees of metal accumulation were found in red C. rubrum and green Clad. vagabuna and Ent. intestinalis species. Ulva rigida and Callith. corymbosum show the lowest HM concentrations. It can be concluded that the above three species can serve as better indicators for Cu, Pb and Cd for the whole Bulgarian coast. The yearly variations of HM concentration were evaluated by means of C. rubrum, Clad. vagabunda and Ulva rigida species, based on multiplicity of samples (Figure 3). The mean values vary in a narrow range and there is a weak change in Cu, Pb and Cd. There is an overall decrease of Cu, Pb and Cd content during the last three years in all species. All obtained data for HM content in red and green Black Sea algae in the period 1992–2003 show lack of serious pollution in the areas without direct anthropogenic impact along the Bulgarian coast. The technogenic and natural radionuclide concentrations (mean values), measured in all studied alga species are presented in Table 1. It should be pointed out that C. rubrum species accumulate all studied nuclides to the highest extent. Some authors (Topcuoglu et al., 1996) have reported similar behavior of C. rubrum species to accumulate radionuclides in the Black Sea region. The dominating artiﬁcial radionuclide in the biota is 137Cs (half-life of 30 years). 4 The levels of radiocaesium in marine biota are depending on the macrophyte type. Red ..SrzvadT..Nonova Tz.P. and Strezov A.S.

Figure 3 Seasonal variations of HM concentrations (mg/g) in three alga species

C. rubrum accumulates 137Cs approximately two times higher than other studied species. The obtained data show that 137Cs varies not only between different algae but within the same species too. Ent. intestinalis serves as an example for these variations (Table 1). This fact is a result of the impact of a complicated complex of factors (abiotic, biological, anthropogenic) that affects contaminant accumulation in algae. If we plot 137Cs mean values vs. location for Ceramium rubrum and Clad. vagabunda species we obtain the contamination dependency for Cs in all locations for the studied period. A tendency is observed for algae from north locations to accumulate higher 137Cs content, compared to the southern (Figure 4). A similar (even stronger) tendency is observed for 137Cs content in Black Sea sediment samples, collected in the same locations as the alga samples (Strezov et al., 1998, 2001). Yearly variations of mean 137Cs contents in the same alga species are presented on Figure 5. High cesium contents were determined during 1996 in all three analyzed species and during 1993 in Clad. vagabunda. Studying marine sediments and algae is an important stage in characterizing aquatic ecosystems and can be used for controlling the water quality, having in mind that seawater is the connecting factor.

137 Figure 4 Cs contents in macroalgae – local variations 5 ..SrzvadT..Nonova Tz.P. and Strezov A.S.

Figure 5 137Cs contents in macroalgae – yearly variation

Macroalgae samples were analysed to obtain the content for natural (uranium) radionuclides as well. U minerals in the surrounding sediments are the source of natural nuclides, which can be accumulated by algae species via seawater. 226Ra concentrations in all studied alga species vary in a wide range (3–39 Bq/kg) and the mean measured concentrations are higher in C. rubrum species. The highest 226Ra content was measured in C. rubrum from Kaliakra (spring 1999) while the lowest was in Callith. corymbosum from Ahtopol (spring 1998) and Ravda (spring 1999) as well as in Ent. intestinalis from Tuzlata (spring 1999) and Ulva rigida from Sinemoretz (spring 1998). Judging from the 226Ra content in the red alga C. rubrum, the studied locations can be arranged in the following order: Ravda , Ahtopol < Sinemoretz , Shabla , Rezovo , Tuzlata , Kaliakra. The same order, obtained for Ulva rigida, differs from the C. rubrum one. The highest 226Ra values in Ulva were determined in both endmost locations Shabla (north) and Ahtopol (south), while the lowest were obtained in Kaliakra. It is interesting to classify the investigated sites by use of Ent. intestinalis species. The obtained mean values increase from Shabla to Rezovo (see Figure 1), according to its geographical position (north to south). It is clear that more detailed studies of the sites and environmental behavior of algae are necessary because of the different accumulation capacities of different species towards various contaminants. After careful analysis of the results for local variations obtained in all macrophytic species, it should be noted that northern and southern locations are characterised with considerably higher 226Ra content compared to the central ones.

Table 1 Mean radionuclide contents in Black Sea macroalgae – Bq/kg

137Cs 226Ra 210Pb

N Mean ^ SE Range Mean ^ SE Range 210Pb Range

Ceramium rubrum 23 9.7 ^ 0.8 4.8–18.0 21 ^ 2 5–39 17 ^ 2 4–41 Callith. 6 4.4 ^ 0.7 2.9–6.5 6 ^ 2 3–17 13 ^ 4 4–30 corymbosum Ulva rigida 14 3.9 ^ 0.7 1.5–10.0 9 ^ 1 3–19 6 ^ 1 3–15 Clad. Vagabunda 11 4.0 ^ 0.8 2.0–9.0 15 ^ 2 4–26 8 ^ 1 3–17 Ent. Intestinalis 9 3.8 ^ 0.8 1.5–13.0 10 ^ 2 3–22 11 ^ 2 3–20 6 ..SrzvadT..Nonova Tz.P. and Strezov A.S.

Figure 6 Radionuclide content in Black Sea algae – year variations

210Pb contents were measured in all samples and the obtained results show that red macrophytes accumulate this nuclide to a higher extent compared with the three studied green algae species. 210Pb levels vary in a wide range especially in C. rubrum, Callith. corymbosum and to a certain degree in Ent. intestinalis species (Table 1). Ulva rigida and Clad. vagabunda algae accumulate this nuclide less. It should be noted that 210Pb content in the studied Black Sea macroalgae is lower than its parent 226Ra (except in Callith. corymbosum). This long-term investigation gives information about the mean 210Pb levels at the studied sites, and our data show no clear tendency for local variations. The seasonal 226Ra and 210Pb variations were investigated after analyzing the data for three species (Figure 6). An increase of 226Ra content during the last three years can be seen in all three species. 210Pb concentration in the same period increases only in Ulva rigida. Despite this, the levels of these two nuclides are lower in Ulva during the whole period. Ceramium rubrum species contain high 226Ra and 210Pb amounts during the investigated period.

Conclusions In conclusion it can be pointed out that Chlorophyta and Rhodophyta algae phylums can be used as bioindicators for monitoring of the ecological state of the Black Sea environment. A comparative analysis of contaminants in different Bulgarian coastline regions leads to the following conclusions. The data obtained for red and green macroalgae illustrate the level of contamination by HM and nuclides at seven locations of the Black Sea Coast. The possibility of Black Sea macroalgae application for marine environmental studies was conﬁrmed. There is no strict seasonal or local dependence of hazardous element content. All results seem to depend on the biological speciﬁcity of the algae. All data show a lack of serious pollution in areas without direct human impact along the Bulgarian Black Sea coast. All obtained results show that use of macroalgae in marine environmental monitoring reduces the need for complex studies on chemical speciation of aquatic contaminants and makes algae valuable indicators for the seawater quality assessment. 7 References Favero, N., Cattalini, F., Bertaggia, D. and Albergoni, V. (1996). Metal accumulation in a biological indicator (Ulva rigida) from the Lagoon of Venice (Italy). Arch. Environ. Contam. Toxicol., 31, 9–18. Gu¨ven, K.C., Topcuoglu, S., Kut, D., Esen, N., Erentu¨rk, N., Saygi, N., Cevher, E., Gu¨vener, B. and O¨ ztu¨rk, B. (1992). Metal uptake by Black Sea algae. Bot. Mar., 35, 337–340. Haritonidis, S. and Malea, P. (1999). Bioaccumulation of metals by the green alga Ulva rigida from Thermaikos Gulf, Greece. Env. Poll., 104, 365–372. ..SrzvadT..Nonova Tz.P. and Strezov A.S. Ho, Y. (1990). Ulva lactuca as bioindicator of metal contamination in intertidal waters in Hong Kong. Hydrobiology, 203, 73–81. McFarren, E., Lishka, R. and Parker, J. (1970). Criterion for judging the acceptability of analytical methods. Analytical Chemistry, 42, 358–360. Mee, L.D. (1992). The Black Sea in crisis: A need for concerted international action. Ambio, 2(4), 278–286. Muse, J.O., Stripeikis, J.D., Fernandez, F.M., d’Huicque, L., Tudino, M.B., Carducci, C.N. and Troccoli, O.E. (1999). Seaweeds in the assessment of heavy metal pollution in the Gulf San Jorge, Argentina. Environ. Pollut., 104, 315–322. Polikarpov, G.G., Kulebakina, L.G., Timoshchuk, V.I. and Stokozov, N.A. (1991). 90Sr and 137Cs in surface waters of the Dniepr River, the Black Sea and the Aegean Sea in 1987 and 1988. J. Env. Radioac., 13, 25–34. Phillips, D. and Segar, D. (1986). Use of bio-indicators in monitoring conservative contaminants: program design imperatives. Mar. Poll. Bull, 17, 10–17. Strezov, A., Milanov, M., Mishev, P. and Stoilova, T. (1998). Radionuclide accumulation in near-shore sediments along the Bulgarian Black Sea Coast. Appl. Radiat. Isot., 12, 1721–1728. Strezov A., Nonova Tz., Ayranov M., Stoilova T. and Mitev K. (2001). Assessment of radionuclide and heavy metal pollution in Black Sea sediments and macroalgae, Preprints. Third Black Sea Int. Conference, 6–8 June 2001, Varna, Bulgaria, p. 44–55. Topcuoglu, S. and Gungor, N. (1999). Radionuclide concentrations in macroalgae and sediment samples from the Bosphorus. Turk. J. Mar. Sci., 5, 19. Topcuoglu, S., Gu¨ven, K.C., Ku¨cu¨kcezzar, R., Kut, D. and Esen, N. (1996). The natural depuration of 137Cs radionuclide in the Black Sea macroalgae. J. Radioan. Nucl. Chem., 214(4), 319–325. Topcuoglu, S., Kut, D., Esen, N., Gungor, N., Olmez (Egilli), E. and Kirbasoglu, C. (2001). 137Cs in biota and sediment samples from Turkish coast of the Black Sea, 1997–1998. J. Radioan. Nucl. Chem., 250(4), 381. Tuncer, G., Karakas, T., Balkas, T.I., Gokcay, C.F., Aygun, S., Yurteri, C. and Tuncel, G. (1998). Land- based sources of pollution along the Black Sea coast of Turkey: Concentrations and annual loads to the Black Sea. Mar. Poll. Bull., 36(6), 409–423.