Biomonitoring 2017; 4: 27–33

Research Article

Fazıl Özen*, Arda Acemi, Ezgi Gizem Pelin, Halim Aytekin Ergül Assessment of Macrophyte Plant Distribution and PAH Contamination in Selected Aquatic Habitats from an Industrialized City; Kocaeli, Turkey https://doi.org/10.1515/bimo-2017-0003 Received September 15, 2017; accepted: October 23, 2017 1 Introduction

Abstract: The macrophyte plant distribution in some Air and soil pollution by organic pollutants, especially aquatic habitats and their availability for monitoring the by persistent ones, are not only hazardous for the PAH contamination were investigated in Kocaeli Province environment but also may result in serious health issues of Turkey. Alisma plantago-aquatica L. was collected to for humans. It has always been a problem to keep these evaluate polyaromatic hydrocarbon (PAH) contamination pollutants under control in the cities which have mostly in four aquatic habitats located nearby or distant to the heavy industry-dependent economies. Among these industrial facilities and populated city center. Concentrations persistent organic pollutants (POPs), polyaromatic of eight PAH congeners were measured in A. plantago- hydrocarbons (PAHs) are widely spread chemicals all aquatica leaves using gas chromatography. PAH sources over the world. Their natural sources are known to be were estimated using the following ratios, Fluoranthene / incomplete combustion such as forest fires and volcanoes, (Fluoranthene + Pyrene), Benzo(a)anthracene / (Benzo(a) whereas wood burning, exhaust from gasoline and diesel anthracene + Chrysene) and Anthracene / (Anthracene + engine, tobacco smoke, municipal waste incineration Phenanthrene) and were found to be pyrogenic-sourced etc. are classified as anthropogenic sources [1]. PAHs in all stations. Since the ratios of congeners indicate the are considered toxic to almost all known life forms, and pyrogenic contamination, atmospheric deposition can be some PAH congeners are also known for their potentials considered as the main pathway for PAH transportation. to cause cancer [2, 3]. They can be spread by atmospheric The highest ∑PAH concentration (798 µg kg-1) was measured transport, adsorbed on the atmospheric particulate in the nearest station (Bıçkıdere Dam) to the city center matter and accumulated directly in soils or sunk in and highway, while the lowest concentration (183 µg kg-1) the water column. Also, PAHs can exist in particulate was determined in the Tahtalı Dam, which is relatively or vapor phase and they can be absorbed by plants. In distant from the industrial facilities, dense population aquatic ecosystems, humidity may affect the absorption and motorways. Present results indicate that incomplete of PAHs from the leaf surface of plants. After completion combustion of grass, wood and coal, as well as recreational of their vegetation periods, decaying plant bodies mingle and agricultural implementations and vehicular emissions, with the soil, thereby resulting in indirect deposition may cause pyrogenic PAH contamination. Also, broad of PAHs [4, 5]. Aquatic habitats, such as lakes, ponds basal leaves of A. plantago-aquatica can be considered as and wetlands, are attractive environments for many sampling material in further biomonitoring studies. vertebrate, invertebrate and plant species due to their advantages as nutrition sources for living organisms. Keywords: Alisma plantago-aquatica, Aquatic habitats, Also, those aquatic ecosystems are valuable destinations Bioaccumulation, Organic pollutants, Polyaromatic for human recreational activities. Therefore, these kinds hydrocarbons of valuable natural habitats and the organisms living within should be protected from POPs. Regular studies using widespread plant species that have larger leaves, *Corresponding author: Fazıl Özen, Kocaeli University, Science and such as macrophyte plants (aquatic plants that grow in Arts Faculty, Department of Biology, 41380 Kocaeli, Turkey or near water and are either emergent, submergent, or E-mail: [email protected] floating) may be suitable to monitor POPs around these Arda Acemi, Ezgi Gizem Pelin, Halim Aytekin Ergül, Kocaeli University, habitats. In this context, we investigated the application Science and Arts Faculty, Department of Biology, 41380 Kocaeli, Turkey

Open Access. © 2017 Fazıl Özen et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution- NonCommercial-NoDerivs 4.0 License. 28 F. Özen, et al. of a large-leaved macrophyte as a bioindicator species km away from dense urbanization and industrialization for aquatic habitats. at an approximate altitude of 400 m, and it is also used In the present study, Alisma plantago-aquatica L. as a water-sports facility. The station SL is located in the was collected from selected aquatic habitats in order to Kartepe District which has intensively-used motor roads evaluate PAH contamination both at nearby and distant and industrial (mainly chemistry, automotive, and drug locations to the industrialized and populated city center factories) areas, while the station ThD is surrounded by in the Kocaeli Province. The plant has a wide distribution actively-used dense agricultural fields and some villages. in aquatic habitats of the Marmara Region in Turkey. Kocaeli is the most industrialized city in the region [5]. However, the city has numerous aquatic habitats which 2.2 Macrophyte Plant Distribution and have endemic and/or endangered plants living nearby. Selected Species Therefore, to screen the exposure and accumulation levels of PAHs, a widely-distributed plant should be sampled Identification of the sampled plant specimens was done and analyzed. In this context, it is hypothesized here that according to Davis’s Flora of Turkey [6]. Alisma plantago- A. plantago-aquatica may reflect possible PAH pollution aquatica L. (Alismataceae), usually known as European and exposure levels in aquatic habitats of the city. In water plantain, is a monocotyledonous, perennial, addition, the general macrophyte plant distribution cosmopolitan herb with some medicinal properties. was determined in four aquatic habitats for the future POP screening studies, and outcomes of these kinds of biomonitoring studies may help to build conservation strategies on valuable aquatic habitats.

2 Material and Methods

2.1 Study Area

Kocaeli Province is located on the eastern side of the Marmara Region. The city is known for its industry and dense population of around two million residents. The city Figure 1: General view of Alisma plantago-aquatica from Bıçkıdere hosts about two thousand industrial investments, of which Dam four hundred are heavy industrial facilities, including the major petrochemical, metallurgy and fertilizer factories. Moreover, the Trans-European Motorway passes through the middle of the city and is used daily by 100.000 motor vehicles alongside heavy marine traffic with around a thousand marine vessels annually. Those elements above can be considered as potential PAH sources. Therefore, a preliminary floristic study was conducted on the four pre-determined aquatic habitats both in the nearby and distant locations to the city center to monitor PAH contaminations and to find a potential bioindicator macrophyte species with large leaves that provide a total surface area to enable PAH uptake by aerial exposure. Alisma plantago-aquatica specimens (Fig. 1) were sampled from the aquatic habitats (Fig. 2) which are indicated as follows: Bayraktar Dam (BrD), Bıçkıdere Dam (BkD), Sapanca Lake (SL), Tahtalı Dam (ThD). The sampling station BkD is located in the Izmit district (the city center of the province), near an urbanized area. The other Figure 2: Locations of the sampling stations in Kocaeli province of sampling station BrD is located in the same district, but 20 Turkey Source assessment of PAH contaminations in Alisma plantago-aquatica 29

The species lives in shallow water of lakes, rivers, and acetone/hexane mixture overnight and then ultrasonically brackish water bays, especially on grazed shores, ditches extracted. The supernatant was concentrated to 2 mL and ponds [6]. The species has large basal leaves and a volume, using pure nitrogen stream. The clean-up was well-developed root system. Their leaves and roots have performed using deactivated alumina–silicic acid and antibacterial, anticholesterolemic, antiplasmodial, florisil columns. Dichloromethane and petroleum ether diaphoretic, diuretic, hypoglycemic and hypotensive were used for infiltration. After solvent exchange into effects [7, 8]. hexane, the final sample volume was adjusted to 1 mL by nitrogen blow-down. Gas chromatography (Agilent 7890A) with a flame ionization detector was used in 2.3 Sample Collection and Analytical quantification of Phenanthrene (Phe), Anthracene Procedure (Ant), Fluoranthene (Fla), Pyrene (Pyr), Chrysene (Chr), Benzo(a)anthracene (BaA), Benzo(a)pyrene (BaP) and Macrophyte plant samples were collected within a distance Benzo(ghi)perylene (BghiP); these have three (Phe and of 100 m coastline from each site. The sample size consisted Ant), four (Fla, Pyr, Chr and BaA), five (BaP) and six of 10 individual plants per habitat, in order not to harm (BghiP) rings in their molecular structure respectively. the plants’ natural populations. Samples were wrapped ∑PAH concentrations were determined using the sum of with aluminum foil, placed in labeled polyethylene bags these eight PAH concentrations as µg kg-1 dry weight. To and put into an icebox for immediate transportation to the calculate recovery values, a standard reference sample laboratory. The samples were gently washed and cleaned (NIST-SRM-2974-a) was used (Table 1). with distilled water to remove visible particles from the A five-point calibration was made using PAH Mix leaf surface. The leaves were sliced into little particles solution (SUPELCO®) and the calibration curves showed and freeze-dried for 48 hours at -55 °C. Then, the samples linear regression (r2>0.99). The reported detection limits were ground with a porcelain mortar and pestle. The plant were calculated based on a signal-to-noise ratio of 3:1 for material from the 10 collected individual plants was pooled each sample and the lowest values were given as the limit for the measurement, to limit the possible variability of detection. due to plant size/uptake surface and specific properties To estimate potential sources of the PAHs, the of their individual microhabitats. Five grams dried and diagnostic ratios (Fla / [Fla + Pyr], BaA / [BaA + Chr] homogenized samples were analyzed to determine PAH and Ant / [Ant + Phe]) were used. These ratios mainly concentrations using the modified EPA 8100 method [9]. help us to understand whether the PAH sources are of Samples were placed into a 40 mL amber glass vial with petroleum (i.e. petrogenic) or combustion (i.e. pyrogenic) 0.5 mL surrogate standard (2-fluorobiphenyl) and 0.5 g of origin. Some of these ratios are also used to discriminate anhydrous sodium sulfate. The samples were kept in 1:1 combustion sources, as given in Table 2.

Tabe 1. Recoveries of the investigated PAH compounds (NIST-SRM-2974a) PAH congeners Certified ± Std. Dev. Measured ± Std. Dev. Recovery

(µg/kg) (µg/kg) (%)

Phenanthrene (Phe) 74.4 ± 4.70 59.6 ± 3.80 80.1

Anthracene (Ant) 2.46 ± 0.20 2.02 ± 0.30 82.1

Fluoranthene (Fla) 287 ± 34.0 247 ± 21.1 86.2

Pyrene (Pyr) 166 ± 21.0 157 ± 14.3 94.6

Benzo(a)anthracene (BaA) 31.1 ± 3.90 22.8 ± 4.20 73.3

Chrysene (Chr) 124 ± 2.90 119 ± 2.20 96.4

Benzo(a)pyrene (BaP) 9.73 ± 0.43 8.40 ± 0.60 86.3

Benzo(ghi)perylene (BghiP) 23.7 ± 2.20 15.0 ± 1.30 63.3 30 F. Özen, et al.

Table 2. Diagnostic ratio formulas to estimate possible PAH sources were Alisma plantago-aquatica, Equisetum telmateia and Cyperus longus. However, E. telmateia and C. longus have Formula Range Possible Source of PAHs very narrow leaves and surfaces, which are not suitable for PAH accumulation and sampling. Therefore, A. plantago- Fla / (Fla + Pyr) * < 0.4 Petrogenic aquatica was selected for the present study. 0.4 – 0.5 Fossil fuel combustion > 0.5 Grass, wood, coal combustion The calculated PAH concentrations in the leaves of the BaA / (BaA + Chr) ** 0.2 – 0.35 Coal combustion plant were given in Table 4. The highest concentrations > 0.35 Vehicular emission of all investigated PAH congeners were detected in the < 0.2 Petrogenic samples from BkD station while the samples from ThD Ant / (Ant + Phe) *** < 0.1 Petrogenic station had the lowest concentrations. The highest ∑PAH > 0.1 Pyrogenic concentration was found in the samples collected from * De La Torre-Roche et al., 2009; ** Akyüz and Çabuk, 2010; *** Pies et BkD station while the lowest ∑PAH concentration was al., 2008 found in the samples from ThD station. Based on the ∑PAH contamination levels, the stations are ordered as BkD > SL > BrD > ThD. 3 Results The Ant, Phe, Fla, Pyr, BaA, Chr and ∑PAH concentrations were determined in the leaves of A. Twenty macrophyte species were identified. The list of plantago-aquatica, and diagnostic ratios were calculated most abundant macrophytes of the investigated aquatic for all stations to estimate its potential sources (Table 5). habitats is given in Table 3. Also, risk categories of The highest Fla/(Fla+Pyr) ratio was calculated from the these species according to the International Union for ThD station whereas the highest BaA/(BaA+Chr) and Ant/ Conservation of Nature (IUCN) are indicated. All species (Ant+Phe) ratios were calculated from the BkD station. were found to be in the category of Least Concern (LC). The highest ∑PAH concentration was also determined in In total, 20 macrophyte species were observed during the BkD station. Calculated Fla/(Fla+Pyr), BaA/(BaA+Chr) the preliminary field studies. The most abundant species and Ant/(Ant+Phe) ratios were higher than 0.4, 0.35, and

Table 3. The list and distribution of the dominant species in the macrophyte flora of investigated aquatic habitats Species name Family IUCN category Distribution

Alisma plantago-aquatica L. Alismataceae LC BrD, BkD, SL, ThD Cyperus longus L. Cyperaceae LC BrD, BkD, SL, ThD Callitriche stagnalis SCOP. Callitrichaceae LC BrD Equisetum telmateia EHRH. Equisetaceae LC BrD, BkD, SL, ThD Iris pseudacorus L. Iridaceae LC SL Juncus acutus L. Juncaceae LC BkD, ThD Lemna minor L. Lemnaceae LC BkD Lycopus europaeus L. Lamiaceae LC BrD, BkD Lysimachia nummularia L. Primulaceae LC BrD, BkD Nasturtium officinale R. BR. Brassicaceae LC BkD Nuphar lutea (L.) SM. Nymphaeaceae LC SL Nymphaea alba L. Nymphaeaceae LC SL Phragmites australis (CAV.) TRIN. EX STEUDEL Poaceae LC BkD Plantago major L. ssp. major Plantaginaceae LC BrD, SL Polygonum hydropiper L. Polygonaceae LC BrD, SL Potamogeton lucens L. Potamogetonaceae LC BkD Ranunculus sphaerospermus BOISS. ET BALANCHE Ranunculaceae LC BrD, ThD Salvinia natans (L.) ALL. Salviniaceae LC SL Typha latifolia L. Typhaceae LC BkD, SL Veronica anagallis-aquatica L. Scrophulariaceae LC BrD, ThD Source assessment of PAH contaminations in Alisma plantago-aquatica 31

0.1, respectively for all stations. Therefore, PAH derivation there is no standard PAH diagnosis method, various is estimated as pyrogenic for all stations, and the main methodologies are suggested to discriminate the PAH transportation pathway is supposed to be atmospheric. source (petrogenic or pyrogenic), based on the ratios However, the results showed that different emission between specific congeners [11, 12, 13]. Some of these sources exist for the PAHs in all stations (Table 2 and 5). ratios help us to estimate the type of the material (e.g., fossil fuel, grass, wood, coal, etc.) which produces PAHs after combustion; another one can discriminate between 4 Discussion coal combustion and vehicular emissions (Table 2). Among the sampling stations, the highest ∑PAH This is the first macrophyte-targeted field study conducted concentration (798 µg kg-1) was determined in the A. in Kocaeli, since there is no record found in the literature plantago-aquatica leaves collected from Bıçkıdere Dam regarding the distribution of macrophyte species in (BkD) station. Since BkD station is the closest station to the aquatic habitats, to date. In a review article on the flora populated and industrialized center of Kocaeli Province, of Kocaeli, Özen and Acemi [10] indicated the industrial combustion-derived emissions may have an effect on threats towards the natural protection areas. Therefore, the PAH accumulation. Beside industrial facilities, the this study may be useful to shape possible conservation Trans-European Motorway is very close to this station (3 strategies in protected habitats, emphasizing the sources km), and both combustion and vehicular emissions are of POPs which could be hazardous to the species. expected. The highest values for BaA/(BaA+Chr) and To make a comprehensive environmental risk Ant/(Ant+Phe) ratios (0.42 and 0.18, respectively) were assessment, an appropriate biomonitor organism and calculated from this station. These results indicate the proper methodologies are required. PAH diagnosis may existence of pyrogenic PAHs derived from incomplete provide valuable data to estimate contamination sources; combustion of organic matter [11, 13]. thus it can play role in the prevention and solution of Bayraktar Dam (BrD) is a relatively distant location ongoing and/or future environmental problems. Although from the city center. The lowest BaA/(BaA+Chr) and

Table 4. Concentrations of PAH congeners in A. plantago-aquatica specimens

Congeners Stations

BkD BrD SL ThD LOD

Phenanthrene 50 39 40 36 36 Anthracene 11 6.0 8.0 5.0 10 Fluoranthene 91 63 70 28 24 Pyrene 99 42 57 7.0 12 Benz(a)anthracene 68 33 41 11 22 Chrysene 95 83 78 26 22 Benzo(a)pyrene 171 77 91 20 20 Benzo(ghi)perylene 213 130 137 50 25 ∑PAH (µg kg-1 dw) 798 473 522 183

LOD : Limit of detection; the data are expressed as µg kg-1 dw

Table 5. Calculated diagnostic ratios according to different formulas for estimating PAH sources and total PAH concentrations for the samp- ling locations Formula Stations

BkD BrD SL ThD

Fla / (Fla + Pyr) 0.48 0.60 0.55 0.81 BaA / (BaA + Chr) 0.42 0.28 0.35 0.30 Ant / (Ant + Phe) 0.18 0.11 0.16 0.12 32 F. Özen, et al.

Ant/(Ant+Phe) ratios were calculated as 0.28 and 0.11, PAH concentrations in their sources may be higher than respectively (Table 5) in the leaves of A. plantago-aquatica the measured concentrations from A. plantago-aquatica, from BrD station. These results indicate coal combustion- and calculated ratios may be slightly different because originated PAH contamination rather than vehicular of atmospheric degradation. Also, it is suggested that the emissions [11, 13] (Table 2). This location is known as application of correction factors in measuring the PAHs a relatively clean habitat of the Kocaeli Province, and on the particles in the air and water samples would be inhabitants prefer to buy agricultural and/or horticultural beneficial [17, 18]. Therefore, it is concluded that, in order products cultivated around Bayraktar district. However, to better evaluate whether A. plantago-aquatica is a proper the ∑PAH concentration was determined as 473 µg kg-1, biomonitoring organism, future research is needed for and the calculated ratios indicate pyrogenic PAH analysis of PAHs in air and water samples, besides leaves. contamination for the region. However, the plant’s broad leaves can be considered as Based on all of the used ratios [11, 12, 13], the sampling materials in further biomonitoring studies. possible PAH sources for the station Sapanca Lake (SL) were estimated as combustion of grass, wood and coal for heating purposes, and from vehicular emissions References

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