1.                   Introduction

The ecological monitoring of marine ecosystems can be performed by controlling marine organisms that have been successfully used as bio-indicators. The bio monitors are important indicators for evaluation of various contaminants impact on marine ecosystems. Macroalgae have been used as indicators for trace elements, because of their accumulation capacity and influence along the trophic chaining the marine environment [1-3]. Algae can bio concentrate amounts of biologically significant elements in their tissues. Some elements are essential for aquatic plant growth and their biological uptake is a function of the free element concentration in seawater. However, increase in element concentrations above certain values can be toxic to marine biota. Some algae have mutation capability so they can survive the ecological stress of the contamination. The determination of widespread bio monitors will allow comparison of accumulated element concentrations over large areas [4-5].

The Black Sea is a low salinity basin, half-isolated from the Mediterranean and its hydrology and phytobentos concentration is different from the other seas in the same area. Toxic elements (and nuclides) are introduced by airborne contaminants, rivers, oil pollution or by direct discharge of industrial wastes into the sea. Trace elements are transported via the seawater currents, coming from the Danube and other rivers. The ecological conditions at the coastal zone are diverse which results in element concentration variations in algae depending on many factors - species type, geographical location, season etc.

Green Macroalgae have been extensively used to monitor marine pollution in various geographical areas [6-9]. Green algae are among the widespread at the Bulgarian Black Sea coast, some of them (Ulva rigida, Enteromorpha intestinalis, Cladophora vagabunda) can be met at almost all areas, so they are appropriate for comparative assessment of contaminants concentration in different geographical areas. Green Macroalgae species have been used as bio-indicators in the Black Sea and other marine environments in neighboring seas [10-15]. Trace element concentrations in Black Sea green alga species have been determined by other authors [16-19] while data about heavy and toxic metals at the Bulgarian Black Sea coastal zone is scarce and insufficient. The purpose of this paper is to study the level and accumulation of 32 elements in eight macrophytes, collected from nine locations along the Bulgarian Black Sea coast during the period 1996 - 2011. It is important to determine whether these algae can be used as bio-indicators for some element pollution and evaluate the impact on the marine ecosystem habitat. Energy-dispersive X-Ray fluorescence method (EDXRFA) was used for trace element determination.[20-21]. This method was applied because of its Multielement capability, nondestructive analysis, simple sample preparation, relative little time consuming, sufficient sensitivity, accuracy and good reproducibility. Compared to ICP, the amount of analyzed sample is bigger (30 g), which reduces the influence of inhomogeneity error.

2.                   Materials and Methods

Sampling sites in this study were selected to cover the whole Bulgarian coastline from north to south - Shabla, Kaliakra, Tuzlata, Rossenetz, Ravda, Sozopol, Ahtopol, Sinemoretz and Rezovo and to possess abundant algae species. Eight green Macroalgae species (Ulva rigida, Ulva Lactuca, Enteromorpha  Enteromorpha compressa, Cladophora vagabunda, Cladophora coleotrix, Chaetomorpha gracilis, and Bryopsis plumosa) were collected during the period between spring 1996 and spring 2011.The algae were collected in the subtidal zone at 1 - 4 m depth and those abundantly covered with epiphyte were ejected. The samples were rinsed in clean seawater, followed by distilled water, dried to constant weight (85°C) and the grinded 0.5 grams of three subsamples.

Three spectrometric systems are available in EDXRFA Lab. The first one is equipped with Si(Li) detector with 12 mm Be window and 170 eV energy resolution at 5.9 KevMN-K_aline (PGT). The data are acquired with a multichannel analyzer (AX-1), interfaced to a personal computer, that applies specialized software for data acquisition and spectra processing. This system is combined with an exciting head, based on an annular source ²⁴¹Am (3.7 GBq) (AMERSHAM) and three types of secondary targets (Mo, Dy, Sn) in two excitation modes - transition and reflection in order to select the desired exciting energy. For determination of heavy elements, which are essential for biology the system uses secondary target of Dy, that allows detection of elements down to the Z = 50. This construction permits the measurement of great number of elements with a high sensitivity by achieving the desirable experimental conditions of low background level and at a high count rate.

The second system is equipped especially for light element analysis with Si pin diode detector, Peltie cooling, with 7 mm Be window and 160 eV (KETEK). The determination is performed with three radionuclide sources ⁵⁵Fe(AMERSHAM) by means of an exciting head, especially designed for low Z elements. In this case the construction, the distances between the source and sample and sample-detector are greatly reduced and optimized, which allows elements with atomic number Z = 13 to 25 to be analyzed.

The third system is equipped with SDD detector with 0.7 mm Be window and 170 eV energy resolution at 5.9 KevMN-Kline (KETEK). An exciting head with source of ²³⁸Pu is specially designed for analyzing of elements with Z between 17 and 35. This head is very suitable for analyzing very low concentrations of these elements. The calibration procedures for each spectrometer are based on set of international standards like Bowen’s kale, orchard leaves, grass (IAEA - V10), fish flesh (IAEA), copepod (IAEA), Nettle etc. The results were checked with IAEA standard - lichen. Lichen was chosen because of the similar matrix with the green algae due to their similar biochemistry. The Lab has also taken part in two International Inter compression Projects - Worldwide Proficiency Test for X-Ray Laboratories PTXRFIAEA/05 and 06 IAEA (2009). The LLD for elements with Z between 13 and 14 is about 1000 µg/g, for Z between 15 and 20 - about 500 µg/g, for Z between 22 and 56 about 1 to 2 µg/g. The LLD for elements Hg, Pb and U is about 5 µg/g.

3.                   Results and Discussion

There is a dependence between microelement bio sorption and their function in the organism. Most intensively accumulated are those microelements that play a major part in the metabolism. A considerable number of elements form stable complexes with proteins, phosphates and lipids. Fe, MN, and Co take part in enzyme formation and as complex compounds participate in exchange reactions. The enriched in elements lipids from algae play a major biogeochemical role for the processes of fixation, transport and sedimentation in the hydrosphere.

The selected green Macroalgae species are suitable for application because they possess potential qualities to be used as bio-indicators, the algae are sufficiently widespread, characteristic for the studied areas, possess enough biomass and are easily collected; and they react to the environmental changes. 32 elements have been measured using EDXRFA in 121 green algae samples - 25 for Ulva rigida, 20 for E17 for C. vagabunda, 14 for Ulva lactuca etc. in the period 1996 - 2011. Data for 19 elements are presented in this paper. The concentrations of some elements are below the LLD limits of the method and the specific spectrometric systems. The concentrations of the most interesting macro and trace elements are presented in tables and graphs.

It is interesting to evaluate the impact of elements accumulation in a certain algae species in all locations along the Black Sea coast. This evaluation can be done at sites where multiple sampling has been performed during the years and where most of most abundant algae were collected. For this reason, we have chosen Ulva rigida, E. intestinalis and C. vagabunda species to assess the ecological status of the coast. The results for 19 detected elements (Figure 1) concentrations in the three most widespread green Macroalgae species from the Bulgarian Black Sea coast, are presented in (Table 1) for Ulva rigida (Figure 2-6) (Table 2) for E. intestinalis (Figure 7-11) and in (Table 3) for C. vagabunda (Figure 12 -16). The measured high element concentrations in algae (2000 - 50000 µg/g) are those for the microelements - Al, Si, P, S Cl, K, Ca, the highest of which are for Cl and S. Cl is essential for the algae metabolism and these high concentrations are understandable. The amount of S in marine algae is much higher, then the terrestrialplants, which is probably due to the dissolved sulfates in the sea water which are sorted by the algae. Fe, Br and Sr concentrations are with one or two orders of magnitude higher than the other elements in all algae species.

The overall Fe mean value obtained for Ulva rigidia 510 mg/g and the variation range is wide (300 – 1100 mg/g ). The concentration of Fe in E. intestinalis is twice higher, compared to Ulva rigida, the mean value being about 1100 µg/g (except Sizemore Autumn’96 - 1840 µg/g and Rossenetz Spring’05 - 2885 µg/g). The mean Fe concentration in Cladophora vagabunda is close to E. intestinalis with exception of Tzarevo Spring`96 - 6680 mg/g. It is clear that the two green Cladophora and Enteromorpha species accumulate twice higher Fe concentration than Ulva species.

The results for MN in  Ulva vary in very narrow limit (the mean value 70 ± 3mg/g). The same is true for the results MN in E. intestinalis which are also close (the mean value 70 ± 3mg/g) like in Ulva rig. except two samples, (Rezovo Summer’00 – 38 µg/g, Sizemore Spring’03 - 140 µg/g) which means that MN concentration is almost constant, independent of sampling time and location. The situation for MN in Cladophora is quite different – in one third of the samples the concentration is about 50 µg/g, while in the other samples the concentration is four times higher except Tuzlata Spring`99 - 371 µg/g and Ahtopol Spring`05 - 430 µg/g. The element Cu and Zn belong to the group of biologically important metal ions and their compounds can be found as trace metals in the biosphere. Cu and Zn are essential for organisms’ life and its toxicity is connected with the concentrations of other essential elements and some marine algae show an ability to accumulate Cu and Zn. Trace metals should be monitored because they play an important role in metabolism and their high or low concentrations can be equally harmful to the living organisms.

Cu and Zn concentration in Ulva rigida, are presented in The Cu values in Ulva, Enteromorpha and Cladophora are close – many of the samples are in the range of 5 - 7 µg/g (while the mean Zn value is 38 ±3 µg/g) except several samples where the concentrations are three or four times higher. Cu and Zn concentrations are almost constant in the uncertainty limit of the method and are also independent of sampling time and location. Two sampling sites (Bjala and Pomorie) have considerably higher Cu and Zn concentration. The case with the Rossenetz location (Sping’05 - 111 µg/g) is completely different – there is a copper mine in the vicinity and the concentration of all elements is completely different than all other sites due to the human mining activity.

Copper concentration determined in algae during the present investigation is comparable with those reported by for algae collected from Crimean coast at the Black Sea, while for lead (nonessential element, but it is present in all biomaterials) the concentrations are lower than those found by Burdin. Pb is found in seawaters mainly in the form of different organic compounds. As it is seen from Pb and Sn data for the three green algae in the highest lead concentration is measured in spring 1996 - 1998, while the concentrations decrease during the next sampling period. The measured concentration for Pb is close to typical Pb obtained for the terrestrial plants.

The concentration of Sn in Ulva are relatively constant (mean 11 ± 3 µg/g), except Bjala (28 µg/g). The Sn values for E. intestinalis vary more and are higher than the other two green algae samples and are close to those measured in marine sediments.  Cd concentration in Ulva (~4 µg/g) was measured only in four samples from Tuzlata (2), St. Constantine & Elena and Ahtopol. Cd is not measured in all samples for the other green algae (in Enteromorpha - 4, and C. vagabunda only two sites). The overall concentration of Pb, Sn and Cd in the sample Tuzlata Spring 98 is three times higher than all other measured algae samples.

It should be pointed out that some samples differ substantially from the others, which can be attributed to the various factors affecting elements accumulation and the specific conditions of studied locations especially depending on the sampling time (mainly spring). Another example for great deviation of elements values is the data (mg/g) for Ulva rigida in Bjala location Spring 2004 and C. vagabunda in Tzarevo Spring`1996. The concentration of halogen elements Cl, Br and I, which are biologically important for the algae and marine biota, were measured also in Ulva rigida, E. intestinalis and C. vagabunda. The obtained average value for Br and I are close for both green algae Ulva rigida, E. intestinalis (360 µg/g - for Br and 47 µg/g for I. while the Br value in Cladophora is twice lower. Lower values were measured for E. intestinalis from Tuzlata Autumn 2005, Sinemorez Autumn 1996, Sinemorez Autumn 2002, suggesting seasonal dependence. The measured Cl values in E. intestinalis and C. vagabunda are higher (mean value 30800 µg/g) compared to those of Ulva r. (22400 µg/g). The values in half of the samples in Cladophora are twice higher than the other samples.

The concentration of alkaline earth elements Ca, Sr and Ba was obtained for Ulva rigida, E.intestinalis and C. vagabunda The data for Ulva and Enteromorpha showed no specific dependence on location or time of sampling while the Cladofora values varied to a greater extent. The average value for Sr in Ulva rigida is lower (~150 µg/g) than those for Enteromorpha (~340 µg/g) and Cladophora(~280 µg/g) with several samples with much higher values. The average value for the barium concentration for the three algae is close (~40 µg/g) also with few exceptions. The Ba value in Tzarevo Sping`96 is exceptionally high 300 µg/g but the sampling was performed close to the area of the port. We have investigated several other green algae - Ulva lactuca, Bryopsis plumosa, Enteromorpha compressa, Cladophora coleotrix, Chaetomorpha gracilis. The data obtained for Ulva lactuca species were from locations Rusalka, Bjala, Tzarevo and Sizemore at the Bulgarian Black Sea coast. The determined element concentrations are similar to those, obtained for Ulva rigida. The concentrations of the most of elements in Bjala location are higher compared to the other sites (like Ulva rigida) – for Fe – 1125 ± 50 µg/g, Cu – 14 ± 2 µg/g, Zn – 78 ± 5 µg/g, Br - 807 ± 20 µg/g, Ba - 84 ± 5 µg/gSn - 28 ± 2 µg/g, Rb – 23 ± 2 µg/g. MN concentration is twice higher in two locations (Sinemorez Spring`98, Tzarevo Autumn`02) than the rest sites. Unlike Ulva rigida species, in Ulva lactuca Cd was measured only in two locations - Tzarevo Autumn`02 - 4 ± 1 µg/g, Sinemorez Spring`05 - 6 ± 1 µg/g.

The data for Enteromorpha compressa are similar to those of  E. intestinalis. The same is true for both the Cladophora algae species (e.g. higher concentration of I). The measured values for the different elements in Chaetomorphagracilis are closer to C. vagabunda, than to the two green Ulva rigida and Enteromorphavaga. Special attention should be about the green alga Bryopsis plumosa. The elements from alkaline earth group -Sr and Ba are accumulated to much higher extent – two to four orders of magnitude (~30000 µg/g for Ba (~50 µg/g for the other green algae); ~4000 µg/g for Sr (~200 for the others). The concentrations of I is ~2000 µg/g (~50 µg/g for the others). We have measured [13] radio metrically the Ra daughter isotope concentrations and also found three orders of magnitude higher concentration compared to all other algae. This comes to confirm the specific accumulation of alkaline earth elements by the green alga Bryopsis plumosa.

4.                   Conclusion

The concentrations of 32 elements have been determined in eight Black Sea green Macroalgae from nine locations along the Bulgarian Black sea coast during the period 1996-2011. If Fe data during the monitoring period are compared, no clear seasonal or location dependence can be observed. It has been shown that Ulva rigida and Bryopsis plumosa species accumulate studied elements less than the other species and the element concentration in green macrophytes from Tuzlata, Ravda and Sinemoretz is higher compared to the other locations.

The obtained data show that no serious artificial pollution along our shore and element concentrations in algae during the studied period demonstrate considerably constant accumulation pattern. The studied green algae species are suitable for assessment of element behavior in Black Sea marine ecosystems (especially Ulva, Enteromorpha and Cladophora) and can be used as bio-indicators species for the ecological status of the ecosystem, together with the abiotic element concentration in the ecosystem (water, sediment). This work contributes to the evaluation of for heavy metal concentration in green Macroalgae and their ecological impact on marine ecosystems along the Bulgarian Black Sea coast. 

Figure 1:   Map of sampling locations.

Figure 2:  Fe concentration in Ulva species.

Figure 3: MN, Cu, Zn concentration in Ulva species.

Figure 4: Sn and Pb concentration in Ulva species.

Figure 5:Sr and Ba concentration in Ulva species.

Figure 6:  Br and I concentration in Ulva species.

Figure 7: Fe concentration in Enteromorpha species.

Figure 8: MN, Cu and Zn concentration in Enteromorpha species.

Figure 9: Cd, Sn and Pb concentration in Enteromorpha species.

Figure 10: Br and I concentration in Enteromorpha species.

Figure 11: Cd, Sn and Pb concentration in Enteromorpha species.

Figure 12: Fe concentration in Cladophora species.

Figure 13: MN, Cu and Cu concentration in Cladophora species.

Figure 14: Cd, Sn and Pb concentration in Cladophora species.

Figure 15: Ba and Sr concentration in Cladophora species.

Figure 16: Br and I concentration in Cladophora species.

Table 1:  Element concentratioin in Ulva rigida.