The alga Ulva lactuca (Ulvaceae, Chlorophyta) as a bioindicator of trace element contamination along the coast of Sicily, Italy

Science of the Total Environment 699 (2020) 134329

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

The alga Ulva lactuca (Ulvaceae, Chlorophyta) as a bioindicator of trace element contamination along the coast of Sicily, Italy Giuseppe Bonanno a,⁎, Vincenzo Veneziano b, Vincenzo Piccione b a b

Department of Biological, Geological and Environmental Sciences, University of Catania, Via Antonino Longo 19, 95125 Catania, Italy IRSSAT, Via del Fornaio 7, 95033 Biancavilla, CT, Italy

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• U. lactuca can accumulate similar levels of essential and non-essential metals. • U. lactuca metal content is correlated with trace elements in water and sediments. • U. lactuca metal content showed levels of environmental pollution. • U. lactuca acts as a bioindicator of trace elements in water and sediments.

a r t i c l e

i n f o

Article history: Received 16 July 2019 Received in revised form 2 September 2019 Accepted 5 September 2019 Available online 06 September 2019 Editor: Jay Gan Keywords: Biomonitoring Macroalgae Marine pollution Mediterranean Sea Metals Sea lettuce

a b s t r a c t The marine environment is subjected to ever-increasing levels of contamination, especially along the coastal areas with urban and industrial activities. Consequently, monitoring campaigns on large scales should be conducted on a regular basis for a better management of marine ecosystems. This study tested the capacity of the green alga Ulva lactuca to act as a bioindicator of trace elements along the coasts of Sicily (Italy). The concentrations of the metals Cd, Cr, Cu, Ni, Pb and Zn were analyzed in samples of water, sediments and U. lactuca thalli, which were collected in ten different sites at diverse levels of human impact. The results showed that U. lactuca can accumulate essential and non-essential elements at similar concentrations. The analysis of trace elements showed also that the metal content in U. lactuca is significantly correlated with the levels of trace elements in water and sediments. U. lactuca fits numerous features that make it one of the best bioindicators of marine pollution, also thanks to its worldwide distribution and capacity to accumulate trace elements under toxic conditions. The use of U. lactuca should be encouraged to set up large-scale monitoring programs, especially in highly impacted seas like the Mediterranean where U. lactuca is widespread. © 2019 Elsevier B.V. All rights reserved.

1. Introduction ⁎ Corresponding author. E-mail address: [email protected] (G. Bonanno).

https://doi.org/10.1016/j.scitotenv.2019.134329 0048-9697/© 2019 Elsevier B.V. All rights reserved.

The marine environment is globally subjected to an ever-increasing level of pollutants, among which trace elements have reached alarming

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G. Bonanno et al. / Science of the Total Environment 699 (2020) 134329

concentrations (Millennium Ecosystem Assessment, 2005; SánchezQuiles et al., 2017). Especially in the Mediterranean Sea, due to the high levels of trace elements detected (Zohra and Habib, 2016; Bonanno and Orlando-Bonaca, 2018), monitoring the environmental state of coastal habitats is of the utmost importance for the conservation of marine ecosystems and associated biodiversity. Metals are particularly significant for the ecotoxicology of the marine environment, since they are highly persistent and can be toxic in traces (Langston, 1990). Bioindicators are organisms that provide a measure of the degree of the environmental pollution (Bargagli, 1998), and to do this, such organisms need to be sensitive to specific pollutants and tolerate high concentrations of these pollutants in the environment (Malea and Kevrekidis, 2013). Since the analysis of total metal content in water and sediments does not predict the toxicity of contaminants to biota (Wang et al., 2010; Aly et al., 2012), the use of bioindicators allows also detecting the fraction of trace elements that has direct effects on living organisms. Bioindicators of aquatic environments have been regularly using since the early 1990s (Rainbow and Phillips, 1993), and especially for their high capacity of trace element bioaccumulation, algae are considered as good bioindicators and often used to assess marine ecosystems (Doshi et al., 2008). The species of the Ulva genus are a large class of macroalgae that can be found in waters with varying salinity in all zones of the Earth, except the Arctic (Bäck et al., 2000). The algae of the Ulva genus fit the general criteria for being good bioindicators because, apart from their worldwide distribution, they can also grow and reproduce in contaminated waters with high levels of trace elements and nutrients (Reed and Moffat, 2003; Ustunada et al., 2010). However, the validation of using Ulva species for monitoring contaminated waters requires information on trace element concentrations in surrounding water and sediments, analyzed across large spatial and temporal gradients (Villares et al., 2002; Rybak et al., 2012). Several studies documented the use of various Ulva species as bioindicators of trace elements in the Mediterranean (Conti and Cecchetti, 2003; Shams ElDin et al., 2014). Most of such studies, however, considered only narrow geographical areas, usually gulfs or stretches of coasts (Malea et al., 2015; Allam et al., 2016). Sea lettuce, Ulva lactuca Linnaeus 1753: 1163, is a common marine green alga with a worldwide distribution, living in littoral and sublittoral zones (Hurd, 2014; Rodríguez-Prieto et al., 2015; Guiry and Guiry, 2019). Although this alga is occasionally used as a bioindicator of metals (Caliceti et al., 2002; Chaudhuri et al., 2007; Diop et al., 2016), few studies have correlated the metal content in U. lactuca with surrounding water and sediments (Haritonidis and Malea, 1999). This makes the assessment of pollution levels relatively difficult. To provide new data on the presence of metals in the Mediterranean, this study conducted a large-scale monitoring campaign along the whole coastline of Sicily (c. 1600 km), by using U. lactuca as a bioindicator of trace elements. Specifically, this study analyzed the concentrations of the metals Cd, Cr, Cu, Ni, Pb and Zn in water, sediments and U. lactuca thalli. These essential and non-essential trace elements were selected to assess whether U. lactuca is able to accumulate them at similar levels. This study aimed to provide new data on the accumulation capacity of U. lactuca for selected trace elements; to correlate these data with trace elements in surrounding water and sediments; to ascertain to what extent U. lactuca reflects a pollution gradient of trace elements in water and sediments; to ascertain the level of metal pollution along the coasts of Sicily. 2. Materials and methods 2.1. Study areas and sampling The geographical distribution of the study areas is shown in Fig. 1, whereas the coordinates are reported in Table 1. Ten different sampling sites subjected to various levels of human impact were selected. The areas falling within two nature reserves (Vendicari and Plemmirio),

whose coastal waters are protected, were also included. These sites were selected as control sites. The other sites follow a growing gradient of human impact on the sea, ranging from the “Torre Verdura” site (tourist resort with low human impact) to the Port of Catania, which is supposed to be one of the most affected marine areas as a consequence of an intense maritime traffic. In order to include the main sources of marine pollution, the study areas included sites with high urbanization, massive maritime traffic, nearby refinery and heavy industries. These study areas were selected across the three main versants of the Sicilian coasts (north, south, east), in order to consider the whole coastline of Sicily (c. 1600 km) in the monitoring. Sampling was conducted twice in each site, specifically in April and June 2017, during the vegetative period of U. lactuca. Sampling was performed in three different matrices consisting of water, sediments and algal thalli. In each study site, 20 samples of each sample type were randomly collected. The depth of sampling was in the range of 0.5–5.0 m below surface. Water samples were collected with 1-L polyethylene bottles, and preserved in the field by adding 50% HNO3. Sediment samples were collected underneath the U. lactuca mats through plastic tube sediment samplers (5-cm diameter, 1.50-m length). Only the superficial layers of sediments (top 5 cm) were sampled for lab analysis. Sediments were put in sterilized plastic containers. The populations of U. lactuca were found either fragmented or forming dense stands. Thalli samples were collected manually from the center of the mats formed by U. lactuca. The weight of collected thalli varied according to the site, and was in the range of 0.5–1.5 kg. Once ashore, algal samples were washed with marine water to remove possible gross material, and then put in sterilized plastic containers. All samples of water, sediments and U. lactuca thalli were stored in cool conditions (3 °C ± 1), and taken to the laboratory on the same day of collection. 2.2. Chemical analysis In the laboratory, algal samples were further checked for the correct taxonomical identification. The specimens of U. lactuca were confirmed by professional algologists, who used mainly morphological criteria (Hayden and Waaland, 2004; Brodie et al., 2007; Škaloud et al., 2018). The water samples were filtered through a microbiological filter (0.45 μm pore size), poured into 100 mL plastic containers, and stored in a freezer at −20 °C. The sediment samples were preliminarily sieved through a sieve (1 mm mesh diameter) to remove gross particles and other extraneous materials (e.g., filamentous algae). The obtained sediment fraction was dried for 2 h 30 min. at 110 °C, and put in 100 mL plastic containers. The thalli of U. lactuca were first washed with running tap water to remove gross superficial particles, and then rinsed with bidistilled water to remove possible attached residual materials. Thalli were then dried for 45 min. on a “Whatman No. 42” quantitative filter paper, to retain extremely small particles, at ambient temperature and, after that, dried for 2 h 30 min. at 110 °C. The resulting dry substance was placed into a 100 mL plastic container. To extract the elements from the thalli of U. lactuca, algal samples were weighed at 0.5 ± 0.05 g and oven-digested in an acid solution (15 mL of 65% HNO3 and 5 mL of 30% H2O2), in Teflon bombs. Sample digestion was carried out at 90 °C overnight (microwave oven Mars 6, CEM Corporation). The labile fraction of sediments was obtained with 1 M HCl. The samples were stored overnight to allow the complete removal of CO2 exhaust fumes. Afterwards, these samples were shaken mechanically at ambient temperature for 1 h. The extracted fractions were then centrifuged at 5000 rpm for 10 min. For the element extraction, 0.5 g of sediment was digested. Regarding water, samples were preliminarily acidified with 63% HNO3 to pH ≤ 2, before being filtered through a 2.0-μm filter paper. Each batch of digests contained blanks, duplicates and CRMs. After the mineralization, the samples of water, sediment and thalli were transferred into 10 mL flasks, filled with distilled water. Subsequently, the concentrations of the following elements were analyzed: Cd, Cr, Cu, Ni, Pb and Zn. The analytical quantification of the elements

G. Bonanno et al. / Science of the Total Environment 699 (2020) 134329

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Fig. 1. Location of the study sites.

was carried out through ICP-MS (PerkinElmer Elan® 6000). Rhodium was the element used as internal standard. Quality control was performed through stability of instrumental recalibration, and using analytical blanks. The instruments were checked on a regular basis against low-level standards (once every five samples), and recalibrated when signs of drift were noted or after every 10 samples. All analyses were conducted in three replicates, and the detection limit was set at 0.01 mg/kg. The standard reference materials Ulva lactuca (B.C.R. reference material No. 279/504) and Virginia Tobacco leaves (CTA-VTL-2) were analyzed in order to ascertain the validity and accuracy of the analytical procedures. Student's t-test (α = 5%) was carried out to check good agreement between experimental and certified values. The

percent recovery fell within 10% of the certified values and ranged between 95 and 105% (Table 2). Temperature, pH and salinity were measured in the field through portable instruments. A Vernier salinity sensor was used to detect seawater salinity (range of measures: 0 to 50,000 ppm). Temperature and pH were measured with the same portable instrument (Hanna Instruments: accuracy ±0.1 °C and 0.1 pH). 2.3. Statistical processing The Kruskal-Wallis H-test was carried out to detect a possible relationship between trace elements in water, sediments and U. lactuca thalli. To identify significant differences between sample pairs, contrasts

Table 1 Description and coordinates of the study sites. No.

Site name

Site description

N

E

Temperature [°C]

pH

Salinity [g/kg]

Vendicari

Nature reserve, human impact negligible

36°49′48.60′′

15°06′28.80′′

13.4–25.3

7.96

38.15

Plemmirio

Nature reserve, human impact negligible

37°01′18.20′′

15°19′26.67′′

13.3–25.1

8.12

38.23

Port of Syracuse

37°03′43.03′′

15°16′47.85′′

13.2–25.2

8.27

38.35

37°29′02.26′′

15°05′33.61′′

13.7–25.0

7.89

38.40

38°13′13.69′′

15°16′03.17′′

13.5–24.9

8.40

38.17

37°58′51.57′′

13°43′16.76′′

13.6–25.3

8.37

38.48

San Vito Lo Capo

120,000 inhabitants, medium maritime traffic, anthropogenic wastes, light industrial activities, nearby river 310,000 inhabitants, high maritime traffic, ship unloading, anthropogenic wastes, welding activities 30,000 inhabitants, nearby refinery, medium maritime traffic, anthropogenic wastes 25,000 inhabitants, power plant, nearby ex car-factory, low maritime traffic, nearby river 5000 inhabitants, tourist resort, medium impact

38°11′27.94′′

12°43′20.29′′

13.4–25.4

8.08

38.25

Marsala

80,000 inhabitants, low maritime traffic, anthropogenic wastes

37°48′59.13′′

12°26′54.98′′

13.2–25.1

8.55

38.44

Torre Verdura

Tourist resort, low human impact

37°28′33.16′′

13°11′02.45′′

13.4–25.2

8.31

38.01

Licata

37,000 inhabitants, medium maritime traffic, ship unloading, nearby river

37°05′56.67′′

13°57′01.66′′

13.5–25.0

7.95

38.29

1 2 3 Port of Catania 4 Port of Milazzo 5 Termini Imerese 6 7 8 9 10

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Table 2 Analysis of certified reference materials (mean values ±95% confidence interval). Elements

Cd Cr Cu Ni Pb Zn a

Ulva lactuca (BCR-279) (mg/kg)

Virginia tobacco leaves (CTA-VTL-2) (μg g−1)

Certified

Experimental

Recovery (%)

Certified

Experimental

Recovery (%)

0.27 ± 0.02 10.7 ± 0.90a 13.1 ± 0.37 15.9 ± 0.40a 13.5 ± 0.40 51.3 ± 1.20

0.28 ± 0.02 10.3 ± 1.08 12.9 ± 0.40 16.7 ± 0.55 13.1 ± 0.52 50.1 ± 1.34

103.7 96.3 98.5 105.0 97.0 97.7

1.52 ± 0.17 1.87 ± 0.16 18.2 ± 0.9 1.98 ± 0.21 22.1 ± 1.20 43.3 ± 2.10

1.56 ± 0.24 1.80 ± 0.18 18.8 ± 1.10 1.89 ± 0.30 21.5 ± 1.36 42.6 ± 2.95

102.6 96.2 103.3 95.4 97.3 98.4

Indicative values. BCR: Community Bureau of Reference. CTA-VTL: Certified reference material of Virginia tobacco leaves.

were carried out with the Mann-Whitney U test. When performing multiple sample contrasts, the Type I error rate may become inflated. Consequently, the initial level of risk (α = 0.05) was adjusted according to the Bonferroni formula αB = α / k, where αB is the adjusted level of risk, and k is the number of comparisons. The Spearman's rankorder correlation test was used to check for significant monotonic correlations between trace elements in water/sediments and U. lactuca. The significance level was set at 0.05. Statistical calculi were carried out with the statistical package IBM SPSS Version 22.0. 3. Results and discussion Cadmium mean concentrations in water, sediments and U. lactuca thalli were respectively in the range of 0.65–31.2 μg/L, 0.13–25.6 mg/kg, and 0.06–0.26 mg/kg (Fig. 2). The levels of Cd showed a greater variability in water and sediments, whereas moderate variations were detected in U. lactuca. According to the Italian legal limits of trace elements in marine sediments (Gazzetta Ufficiale della Repubblica Italiana, 2011, Decree 260/2010), the admissible threshold of Cd = 0.30 mg/kg was significantly passed in all highly impacted study areas. Despite this, the range of Cd values in U. lactuca was in line with most studies conducted in Mediterranean sites with modest Cd presence (Table 3). The low accumulation of Cd seems typical for the Ulva genus, including those species with monostromatic tubullar and distromatic frondose thalli (Rybak et al., 2012). This study showed that Cd concentrations in U. lactuca reflect the general trend of Cd values in water and sediments across the study areas (Fig. 2). Significant correlations were indeed found between Cd concentrations in water/sediments and U. lactuca (Table 4), in line with previous findings (Rybak et al., 2012). Regarding chromium, mean values in water, sediments and U. lactuca were 4.02–12.27 μg/L, 3.18–11.89 mg/kg, and 0.25–2.21 mg/kg, respectively (Fig. 2). In general, Cr concentrations were significantly different in water, sediments and U. lactuca in the various study sites. The variations of Cr were within one order of magnitude in water, sediments and U. lactuca thalli. The concentrations of Cr in the study sites were close to and, in some cases, passed the admissible legal values of Cr in marine waters (7.00 μg/L). The admissible concentrations in sediments (50.0 mg/kg) were instead significantly lower in all study sites. In this study, the Cr concentrations in U. lactuca were overall comparable with previous surveys in the Mediterranean (Table 3). Significant correlation was found between Cr in water/sediments and U. lactuca (Table 4), suggesting that Cr presence in U. lactuca may reflect the general trend of concentrations across the various sites. Similar patterns were found in previous studies (KamalaKannan et al., 2008), where the variations of Cr in U. lactuca reflected the concentrations in water and sediments. Copper concentrations ranged within 3.12–16.4 μg/L (water), 3.15–64.4 mg/kg (sediments), and 1.48–10.4 mg/kg (U. lactuca) (Fig. 2). This study reported Cu values in U. lactuca in general agreement with previous investigations on Ulva species (e.g., U. linza, U. prolifera), in Mediterranean sites (Table 3). In literature, Cu levels of 200–300 mg/kg have been detected in species from polluted areas

(Hawk et al., 1974). Significant contamination of Cu should be excluded along the Sicilian coasts given the relatively low levels of Cu found in this study. Under natural conditions, Cu is more abundant in seawater than Cd, thus resulting in lower Cd accumulation in algae (Rybak et al., 2012; Ismail and Ismail, 2017). Previous studies showed that Cu may affect the uptake of toxic metals (e.g., Cd, Pb) in microalgae (Franklin et al., 2002; Sánchez-Marín et al., 2014), but no further physiological evidence is available. Both Cd and Cu are toxic when present at excessive concentrations, but Cu shows generally a higher toxicity than Cd in algae (Štork et al., 2013). In this study, the values of Cu detected in U. lactuca followed a similar pollution gradient in water and sediments, in agreement with the Spearman's coefficient that showed a significant relationship between Cu in water/sediments and U. lactuca. Nickel mean concentrations ranged within 6.04–45.4 μg/L in water, 11.7–55.4 mg/kg in sediments, and 0.97–8.12 mg/kg in U. lactuca thalli (Fig. 3). The legal admissible concentrations of Ni in marine waters (20.0 μg/L) and sediments (30.0 mg/kg) were significantly passed in the most impacted sites. This study found Ni concentrations in U. lactuca fairly in line with the range of values found in U. lactuca and other Ulva species from Mediterranean locations (Table 3). To date, the highest concentrations of Ni have been detected in monostromatic tubular thalli of Ulva species (identified as Enteromorpha sp.), from the coast of NW Spain, in the range values of 7.64–339 mg/kg (Puente, 1992). Previous studies showed possible correlations between Ni values in water, sediments and Ulva (Rybak et al., 2012). In agreement with these results, this study showed that the content of Ni in U. lactuca can reflect the concentrations in water and sediments, according to the Spearman's coefficient, which found significant correlations between water/sediments and U. lactuca (Table 4). Lead mean values were within 0.15–0.77 μg/L in water, 2.69–8.91 mg/kg in sediments, and 0.67–5.77 mg/kg in U. lactuca thalli (Fig. 3). All study sites showed Pb concentrations below the legal admissible concentrations of Pb in marine waters (7.20 μg/L) and sediments (30.0 mg/kg). This study found Pb concentrations in U. lactuca within the range of values detected in those Mediterranean sites where Pb pollution is relatively low (Table 3). Concentrations N10 mg/kg are considered as a threshold between contaminated and uncontaminated species (Lozano et al., 2003). Consequently, this study found Pb concentrations in U. lactuca lower than the expected limits of Pb-unpolluted areas. The correlations of Pb between water/sediments and U. lactuca were significant in the study sites, thus suggesting that the concentrations of Pb in U. lactuca follow a pollution gradient in water and sediments. This result is in line with previous studies that found significant relationship between Pb content in U. lactuca, water and sediments (e.g. Rybak et al., 2012). Zinc mean concentrations in water, sediments and U. lactuca thalli were within the ranges of 6.81–34.2 μg/L, 7.49–51.4 mg/kg and 8.21–85.1 mg/kg, respectively (Fig. 3). The levels of Zn detected in U. lactuca were in general agreement with previous studies (Table 3). In particular, the maximum concentrations of Zn found in this study were among the highest values so far detected in the Mediterranean (Storelli et al., 2001; Favero and Frigo, 2002; Malea et al., 2015). According to Moore and Ramamurti (1987), however, Zn levels not exceeding

G. Bonanno et al. / Science of the Total Environment 699 (2020) 134329

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Fig. 2. Concentrations of Cd, Cr and Cu in water [μg/L], sediments [mg/kg] and U. lactuca thalli [mg/kg] (same letters were used for statistically similar concentrations between trace elements present in the same sample type, Mann-Whitney U test, α = 0.05, N = 20).

100 mg/kg in benthic macrophytes are considered as background values for non-polluted areas. In this study, significant correlations were found between Zn in water/sediments and U. lactuca (Table 4). This study corroborated previous findings on the significant capacity of U. lactuca to accumulate trace elements. In coastal ecosystems, metals may exist either in dissolved state in the water column or deposited on bottom sediments, according to the nature of the chemical species and physico-chemical factors (Hagan et al., 2011; Lim et al., 2012). Specifically, trace elements become associated with organic matter through biological uptake and adsorption with subsequent incorporation into

resistant organic degradation products, such as humic substances (Alagarsamy, 1991). Algae, in particular, bind only free metal ions whose concentrations depend on the nature of the suspended particulate matter, which in turn include both organic and inorganic complexes (Luoma, 1983; Volterra and Conti, 2000). Metals in U. lactuca may be adsorbed at the algal surface or internalised within the cytosol of component cells (Turner et al., 2008). Metal adsorption relies on surface complexation, whereas internalisation may occur subsequent to surface complexation, or independently as a passive mechanism if metal complexes are sufficiently lipophilic (Mason et al., 1996).

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Table 3 Metal concentrations in Ulva ssp. from the Mediterranean Sea (mg/kg dry weight) (Note: ranges are expressed with “–”). Taxa

Ulva lactuca Linnaeus Ulva lactuca Linnaeus

Ulva laetevirens J.E. Areschoug Ulva linza Linnaeus (as Enteromorpha linza)

Ulva prolifera O.F.Müller Ulva rigida C.Agardh

Ulva spp. (as Enteromorpha spp.)

Ulva spp.

Coast of Sicily (Italy) Thermaikos (Greece) Crete (Greece) Apulian coast (Italy) Gulf of Gaeta (Italy) El-Mex Bay, Alexandria coast (Egypt) Eastern Harbour, Alexandria coast (Egypt) Ouled Saleh, coast of Honaïne (Algeria) Tartous (Syria) Abu-Qir Bay (Egypt) Venice Lagoon (Italy) Thermaikos Gulf (Greece) Chaldiki (Greece) Thermaikos (Greece) Ghazaouet (Algeria) Beni-Saf (Algeria) Apulian coast (Italy) Rovinj (Croatia) Thermaikos Gulf (Greece) Thermaikos Gulf (Greece) Tartous (Syria) Evros Delta (Greece) Gulf of Thessaloniki (Greece) Piran Bay (Slovenia) Rovinj (Croatia) Portoscuso (Sardinia, Italy) San Pietro (Sardinia, Italy) Çanakkale/Dardanos (Turkey) Izmir/Foça (Turkey) Izmir/Bostanli (Turkey) Çanakkale/City (Turkey) Çanakkale/Dardanos (Turkey) Izmir/Foça (Turkey) Izmir/Bostanli (Turkey) Gulf of Thessaloniki (Greece)

References Cd

Cr

Cu

Ni

Pb

Zn

0.06–0.26 0.42 0.42–1.10 0.20 ± 0.23 0.18 ± 0.06 0.73 ± 0.24 1.84 ± 0.93 0.15 ± 0.08 11.0 ± 0.33 1.27 0.03–0.47 No data 0.77 0.61 0.36 ± 0.35 1.92 ± 1.05 0.72 ± 0.36 0.3 No data 0.10–2.50 0.09 ± 0.01 0.36–1.97 0.03 ± 0.003 0.10–0.30 0.40–0.90 19.2 4.62 0.04 ± 0.008 0.01 ± 0.003 0.03 ± 0.002 0.05 ± 0.01 0.06 ± 0.02 0.01 ± 0.002 0.02 ± 0.002 0.02–2.22

0.25–2.21 No data No data No data 1.63 ± 0.60 No data No data No data 4.71 ± 0.02 No data 0.50–6.94 3.73 ± 0.64 No data No data No data No data No data No data 2.60 ± 0.54 No data 1.69 ± 0.09 No data 9.38 ± 1.49 No data No data No data No data 9.63 ± 1.86 4.45 ± 1.72 6.95 ± 0.47 0.78 ± 0.20 3.74 ± 0.83 1.59 ± 0.66 2.82 ± 0.22 4.13–104

1.48–10.4 7.40 7.00–14.5 12.1 ± 7.12 5.80 ± 1.10 7.24 ± 2.30 14.5 ± 4.70 0.45 ± 0.10 5.48 ± 0.28 2.96 4.91–19.2 No data 3.4 6.70 2.41 ± 2.32 10.9 ± 7.92 10.3 ± 3.48 8.00 No data 1.10–4.30 3.60 ± 0.04 9.86–36.8 7.39 ± 0.89 3.00–6.00 3.00–5.00 41.3 3.80 8.89 ± 0.03 10.7 ± 0.96 13.2 ± 0.14 8.51 ± 0.86 6.49 ± 0.22 13.9 ± 3.11 8.29 ± 0.21 2.50–15.4

0.97–8.12 9.20 8.70–13.7 No data No data No data No data 0.82 ± 0.17 No data 3.64 1.21–3.99 4.87 ± 0.43 6.9 10.3 No data No data No data No data 3.88 ± 0.43 No data No data No data 6.44 ± 0.55 No data No data No data No data No data No data No data No data No data No data No data 3.00–23.9

0.67–5.77 0.02 0.02 0.84 ± 0.34 1.94 ± 0.38 No data No data 1.44 ± 0.36 0.55 ± 0.09 2.84 1.48–27.5 No data 0.02 0.02 19.4 ± 4.94 28.3 ± 16.9 1.15 ± 0.46 No data No data 6.30–29.8 0.18 ± 0.02 1.66–43.3 3.06 ± 0.67 2.00–9.00 6.00–8.00 386 115 b 0.01 b 0.01 b 0.01 b 0.01 b 0.01 b 0.01 b 0.01 2.41–14.2

8.21–85.1 43.7 16.4–56.3 127 ± 60.2 45.0 ± 12.0 27.4 ± 8.30 63.1 ± 3.20 2.81 ± 0.54 11.0 ± 0.33 12.6 8.50–94.0 No data 45.0 67.5 29.5 ± 13.1 34.3 ± 0.71 58.8 ± 22.3 81.0 No data 39.0–82.5 35.9 ± 0.46 42.0–90.0 122 ± 14.11 14.0–48.0 31.0–38.0 722 88.2 68.8 ± 2.26 74.8 ± 15.3 107 ± 15.3 50.1 ± 6.00 41.8 ± 3.29 67.8 ± 16.0 40.4 ± 6.09 82.9–240

This study Sawidis et al. (2001) Sawidis et al. (2001) Storelli et al. (2001) Conti and Cecchetti (2003) Abdallah and Abdallah (2008) Abdallah and Abdallah (2008) Allam et al. (2016) Al-Masri et al. (2003) Shams El-Din et al. (2014) Favero and Frigo (2002) Haritonidis and Malea (1995) Sawidis et al. (2001) Sawidis et al. (2001) Benguedda et al. (2011) Benguedda et al. (2011) Storelli et al. (2001) Munda and Hudnik (1991) Haritonidis and Malea (1995) Haritonidis and Malea (1999) Al-Masri et al. (2003) Boubonari et al. (2008) Malea et al. (2015) Munda and Hudnik (1991) Munda and Hudnik (1991) Schintu et al. (2009) Schintu et al. (2009) Akcali and Kucuksezgin (2011) Akcali and Kucuksezgin (2011) Akcali and Kucuksezgin (2011) Akcali and Kucuksezgin (2011) Akcali and Kucuksezgin (2011) Akcali and Kucuksezgin (2011) Akcali and Kucuksezgin (2011) Malea and Kevrekidis (2014)

G. Bonanno et al. / Science of the Total Environment 699 (2020) 134329

Ulva lactuca Linnaeus (as Ulva fasciata)

Location

G. Bonanno et al. / Science of the Total Environment 699 (2020) 134329 Table 4 Spearman's rank-order correlation coefficient between metal concentrations in Ulva lactuca, seawater and sediments (N = 20; p b 0.05). Sites 1 2 3 4 5 6 7 8 9 10 a

Correlations U. U. U. U. U. U. U. U. U. U. U. U. U. U. U. U. U. U. U. U.

lactuca lactuca lactuca lactuca lactuca lactuca lactuca lactuca lactuca lactuca lactuca lactuca lactuca lactuca lactuca lactuca lactuca lactuca lactuca lactuca

– seawater – sediments – seawater – sediments – seawater – sediments – seawater – sediments – seawater – sediments – seawater – sediments – seawater – sediments – seawater – sediments – seawater – sediments – seawater – sediments

Cd

Cr

Cu

Ni

Pb

Zn

0.672a 0.648a 0.535a 0.644a 0.721a 0.683a 0.595a 0.675a 0.751a 0.610a 0.656a 0.585a 0.633a 0.741a 0.571a 0.644a 0.667a 0.587a 0.710a 0.675a

0.589a 0.670a 0.742a 0.593a 0.656a 0.773a 0.569a 0.706a 0.665a 0.687a 0.621a 0.594a 0.661a 0.681a 0.587a 0.653a 0.621a 0.596a 0.651a 0.612a

0.573a 0.653a 0.781a 0.733a 0.580a 0.643a 0.719a 0.621a 0.596a 0.706a 0.567a 0.628a 0.745a 0.599a 0.592a 0.662a 0.583a 0.634a 0.608a 0.626a

0.722a 0.607a 0.675a 0.722a 0.645a 0.679a 0.590a 0.647a 0.621a 0.669a 0.593a 0.733a 0.588a 0.731a 0.596a 0.631a 0.569a 0.660a 0.682a 0.641a

0.629a 0.597a 0.656a 0.714a 0.675a 0.723a 0.642a 0.572a 0.656a 0.711a 0.756a 0.573a 0.663a 0.641a 0.579a 0.674a 0.607a 0.572a 0.762a 0.645a

0.688a 0.614a 0.742a 0.651a 0.756a 0.685a 0.721a 0.631a 0.569a 0.644a 0.679a 0.652a 0.683a 0.593a 0.625a 0.592a 0.727a 0.588a 0.638a 0.590a

Significant correlation at significance threshold = 0.05.

Environmental conditions characterizing the habitats of marine macroalgae, including physical and chemical factors (e.g. temperature, pH, salinity, organic matter, nutrients, etc.), all play a role in the processes of metal accumulation (Ariza et al., 1999; Tabudravu et al., 2002; Abdallah et al., 2005). Several studies, in particular, focused mainly on the role of salinity in influencing the capacity of metal accumulation in algae (e.g., Koelmans et al., 1996; Worms et al., 2006). However, these studies of algal metal uptake as a function of salinity are not always in agreement. Various authors showed indeed that an increasing gradient of salinity may contribute to reduce the biouptake of trace elements in Ulva species (e.g., Wang and Dei, 1999; Mamboya et al., 2009). In turn, Turner et al. (2008) found in a lab experiment that an increase in salinity can favor the uptake of Pb in U. lactuca. Similarly, the values of pH exert an influence on the metal uptake by macroalgae (Vigneault and Campbell, 2005). The marine pH in this study showed comparable values within the range of 7.89–8.55, which indicates moderately alkaline seawaters. A pH range of 6.5–8.5 is generally acceptable according to the quality standards suggested by WHO (1993). The temperature of seawaters is also among the main factors that influence the seasonal patterns of metal concentrations in macroalgae (Haritonidis and Malea, 1995, 1999). The values of seawater temperature were similar among the different study sites, namely 13.2–13.7 °C in April (2017), and 24.9–25.4 °C in July (2017). Overall, the measurement of salinity, pH and temperature showed relatively constant values, suggesting that such parameters may have influenced secondarily the diverse levels of trace elements accumulated by U. lactuca in the different sites of this study. Various laboratory studies showed that other factors (e.g., light intensity, organic matter, particle size, P and N levels, etc.) exert an influence at a different degree on the metal uptake in macroalgae (Ariza et al., 1999; Abdallah et al., 2005). Similarly, the algal growth rate affects the seasonal variations in the metal concentrations, for example in macroalgae the metal concentrations decrease during growing periods and increase during the winter period (Phillips, 1994). However, what matters in biomonitoring it is that the metal content in algae reflects the temporal variations of metal concentrations in water and sediments. Consequently, the correlation of metal levels in algae and water/sediments should be considered as one of the main guiding principles in testing the capacity of species as bioindicators. Macroalgae have been also mainly studied for their capacity of metal bioaccumulation rather than for their physiological responses (Kumar et al., 2010; Mellado et al., 2012; Jiang et al., 2013). One of the main signs of metal toxicity

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is oxidative stress, which leads to the damage of biomolecules including fatty acids (Pinto et al., 2011; Machado and Soares, 2016). Trace elements may have a remarkable impact on algae. For example, Kováčik et al. (2018) found that in Ulva compressa, high concentrations of Cd and Cu significantly stimulate reactive oxygen species (ROS) and deplete nitric oxide (NO) formation. These authors showed also that a higher Cu dose depleted proline, ascorbic acid, and phenol levels more than Cd, whereas Cd elevated nonprotein thiols and ascorbic acid in combined treatments. By considering as negative an increase in ROS and decrease in NO, studies showed that Hg has a more negative impact than Pb (Kováčik et al., 2017), and Cd has a more negative impact than Ni (Kováčik et al., 2016). As far as biomonitoring is concerned, rather than physiological responses to trace elements, a prominent role is especially played by ecological factors such as abundance, persistence and distribution. The results of this study found further evidence of the bioindicator nature of U. lactuca, whose metal concentrations showed correlations with the levels of trace elements in water and sediments. Good bioindicator macroalgae should show a strong linear relationship between metal levels in cells and water/sediments (Phillips, 1990; Villares et al., 2002). Several laboratory studies confirmed these correlations for various species of the Ulva genus (Tabudravu et al., 2002; Chan et al., 2003; Chojnacka, 2008). However, the relationship between metal content in algae and water/sediments in the field is not as well defined as found in laboratory studies under controlled conditions. Indeed, on the one hand, different studies found no significant correlation between metal concentrations in algae and water/sediments (e.g., Malea and Haritonidis, 2000; Kamala-Kannan et al., 2008). On the other hand, several authors reported positive linear correlations between metals in Ulva species, water and sediments (Boubonari et al., 2008; Akcali and Kucuksezgin, 2011; Chakraborty et al., 2014). Given the absence of roots, significant correlations between metal levels in macroalgae and sediment may seem unusual. Although the uptake by seaweeds is mainly of metals in solution, to a certain level, metal concentrations in sediments reflect the degree of pollution in the water column. Consequently, significant correlations between metal concentrations in sediments and algae may be possible, also because parts of such trace elements may have been scavenged from sediments by algae (Luoma et al., 1982). Bioindicators should be sensitive to specific pollutants and tolerate large concentrations of these pollutants in the environment (Rainbow and Phillips, 1993). The use of bioindicators can prove very useful in the study of coastal areas subjected to anthropogenic impact. Macroalgae are considered as promising bioindicators for metal pollution in seawater due to their sedentary nature, considerable biomass, and easy identification (Chaudhuri et al., 2007). Although several internal and external factors determine metal uptake by macroalgae, seaweeds are still considered to provide qualitative information of metal contamination degree and environmental quality of an area. Ulva species may also grow in heavily polluted waters with high concentrations of trace elements and other nutrients (Reed and Moffat, 2003). In particular, the green alga U. lactuca should be considered as a useful bioindicator of trace elements given its capacity of high accumulation, cosmopolitan distribution, easiness of sampling and taxonomical identification, as well as availability all year round. However, although the scientific evidence suggests the use of U. lactuca as a bioindicator of marine metal pollution, cases of large-scale monitoring are overall scarce and generally limited to small geographical areas and short temporal periods (Malea and Haritonidis, 1999; Orduña-Rojas and LangoriaEspinoza, 2006; Akcali and Kucuksezgin, 2011). The standardization of sampling protocols and chemical analysis, the creation of regularly updated and public databases as well as transnational collaborations, will all contribute to prompt the use of marine macroalgae as a wellestablished methodology for the biomonitoring of trace elements. Macroalgae have the capacity to accumulate metal concentrations several times more than marine water (Doshi et al., 2008). As a result,

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Fig. 3. Concentrations of Ni, Pb and Zn in water [μg/L], sediments [mg/kg] and U. lactuca thalli [mg/kg] (same letters were used for statistically similar concentrations between trace elements present in the same sample type, Mann-Whitney U test, α = 0.05, N = 20).

it should be also stressed that many species of Ulva are also a food source for invertebrates and fish (Gerking, 1994; Kamermans et al., 2002), which are consumed as seafood by humans in many countries. Therefore, investigating the levels of metals in algae is not only important for biomonitoring purposes but also for food safety and for understanding the transfer of toxic metals across the marine trophic web. Because of the ever-increasing urban and industrial activities along the coastal areas worldwide, the amount of trace elements entering the marine environment is destined to rise significantly. As such, research should be intensified to consolidate the routine use of algae in trace element biomonitoring, and a preliminary step to do this is to better clarify the patterns of metals accumulation in marine algae.

Biomonitoring is basically an applied discipline, thus bioindicator species need further field studies to validate their suitability as detectors of pollution. 4. Conclusions This study corroborated previous findings on the capacity of U. lactuca to reflect pollution levels due to trace elements in the marine environment. In particular, this study found that trace element concentrations in U. lactuca are significantly correlated with the element levels in surrounding water and sediments. The values of trace elements detected in U. lactuca were in general agreement with previous studies

G. Bonanno et al. / Science of the Total Environment 699 (2020) 134329

in the Mediterranean Sea. U. lactuca showed also the capacity to accumulate essential and non-essential elements at comparable levels. Other studies are necessary to shed further light on the role played by chemical, physical and biological factors on trace element accumulation in U. lactuca.

Declaration of competing interest None. Acknowledgements This study was supported by EcoStat Srl – Spin-off of Catania University (Italy), and by IRSSAT (Istituto di Ricerca, Sviluppo e Sperimentazione sull'Ambiente e il Territorio). The authors are thankful to all the people involved in data collection, processing and proof reading. The authors wish also to thank the three anonymous referees whose constructive and insightful comments improved significantly the quality of this article. References Abdallah, A.M.A., Abdallah, M.A., Beltagy, A.I., 2005. Contents of heavy metals in marine seaweeds from the Egyptian coast of the Red Sea. Chem. Ecol. 21, 399–411. Abdallah, M.A.M., Abdallah, A.M.A., 2008. Biomonitoring study of heavy metals in biota and sediments in the south eastern coast of Mediterranean Sea, Egypt. Environ. Monit. Assess. 146, 139–145. Akcali, I., Kucuksezgin, F., 2011. A biomonitoring study: heavy metals in macroalgae from eastern Aegean coastal areas. Mar. Pollut. Bull. 62, 637–645. Alagarsamy, R., 1991. Organic carbon in the sediments of Mandovi estuary, Goa. Indian J. Mar. Sci. 20, 221–222. Allam, H., Aouar, A., Benguedda, W., Bettioui, R., 2016. Use of sediment and algae for biomonitoring the coast of Honaïne (far west Algerian). Open J. Ecol. 6, 159–166. Al-Masri, M.S., Mamish, S., Budier, Y., 2003. Radionuclides and trace metals in eastern Mediterranean Sea algae. J. Environ. Radioact. 67, 157–168. Aly, W., Williams, I.D., Hudson, M.D., 2012. Metal contamination in water, sediment and biota from a semi-enclosed coastal area. Environ. Monit. Assess. 185, 3879–3895. Ariza, M.E., Bijur, G.N., Williams, M.V., 1999. Environmental Metal Pollutants, Reactive Oxygen Intermediaries and Genotoxicity. Kluwer Academic Publishers, Boston, USA. Bäck, S., Lehvo, A., Blomster, J., 2000. Mass occurrence of unattached Enteromorpha intestinalis on the Finnish Baltic Sea coast. Ann. Bot. Fenn. 37, 155–161. Bargagli, R., 1998. Trace Elements in Terrestrial Plants. An Ecophysiological Approach to Biomonitoring and Biorecovery. Springer, Berlin (324 pp.). Benguedda, W., Dali, Y.N., Amara, R., 2011. Trace metals in sediments, macroalgae and benthic species from the western part of Algerian coast. J. Environ. Sci. Eng. 5, 1604–1612. Bonanno, G., Orlando-Bonaca, M., 2018. Chemical elements in Mediterranean macroalgae. A review. Ecotoxicol. Environ. Saf. 148, 44–71. Boubonari, T., Malea, P., Kevrekidis, T., 2008. The green seaweed Ulva rigida as a bioindicator of metals (Zn, cu, Pb and cd) in a low-salinity coastal environment. Bot. Mar. 51, 472–484. Brodie, J., Maggs, C.A., John, D.M., 2007. Green Seaweeds of Britain and Ireland. pp. [i-v], vi-xii, 1–242, 101 figs. British Phycological Society, London. Caliceti, M., Argese, E., Sfriso, A., Pavoni, B., 2002. Heavy metal contamination in the seaweeds of the Venice lagoon. Chemosphere 47, 443–454. Chakraborty, S., Bhattacharya, T., Singh, G., Maity, J.P., 2014. Benthic macroalgae as biological indicators of heavy metal pollution in the marine environments: a biomonitoring approach for pollution assessment. Ecotoxicol. Environ. Saf. 100, 61–68. Chan, S.M., Wang, W., Ni, I., 2003. The uptake of Cd, Cr, and Zn by the macroalga Enteromorpha crinita and subsequent transfer to the marine herbivorous rabbitfish, Siganus canaliculatus. Arch. Environ. Contam. Toxicol. 44 (3), 298–306. Chaudhuri, A., Mitra, M., Havrilla, C., Waguespack, Y., Schwarz, J., 2007. Heavy metal biomonitoring by seaweeds on the Delmarva Peninsula, east coast of the USA. Bot. Mar. 50, 151–158. Chojnacka, K., 2008. Using biosorption to enrich the biomass of seaweeds from the Baltic Sea with microelements to produce mineral feed supplement for livestock. Biochem. Eng. J. 39 (2), 246–257. Conti, M.E., Cecchetti, G., 2003. A biomonitoring study: trace metals in algae and molluscs from Tyrrhenian coastal areas. Environ. Res. 93, 99–112. Diop, M., Howsam, M., Diop, C., Goossens, J.F., Diouf, A., Amara, R., 2016. Assessment of trace element contamination and bioaccumulation in algae (Ulva lactuca), mussels (Perna perna), shrimp (Penaeus kerathurus), and fish (Mugil cephalus, Saratherondon melanotheron) along the Senegalese coast. Mar. Pollut. Bull. 103, 339–343. Doshi, H., Seth, C., Ray, A., Kothari, I.L., 2008. Bioaccumulation of heavy metals by green algae. Curr. Microbiol. 56 (3), 246–255. Favero, N., Frigo, M.G., 2002. Biomonitoring of metal availability in the Southern Basin of the Lagoon of Venice (Italy) by means of macroalgae. Water Air Soil Pollut. 140, 231–246.

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