Ecotoxicology and Environmental Safety 112 (2015) 231–237

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Effects of lead on tolerance, bioaccumulation, and antioxidative defense system of green algae, Cladophora De-ju Cao n, Xiao-dong Shi, Hao Li, Pan-pan Xie, Hui-min Zhang, Juan-wei Deng, Yue-gan Liang School of Resource and Environment, Anhui Agricultural University, Hefei 230036, People’s Republic of China

art ic l e i nf o

a b s t r a c t

Article history: Received 9 June 2014 Received in revised form 6 November 2014 Accepted 11 November 2014

Effects of various concentrations (0.5, 1.0, 2.5, 5.0, 7.5, and 10.0 mg/L) of lead (Pb2 þ ) on the growth, bioaccumulation, and antioxidative defense system of green algae, Cladophora, was investigated. Low concentrations of Pb2 þ accelerated Cladophora growth, but concentrations of 10.0 mg/L and above inhibited the growth because of the hinderance to photosynthesis. The total soluble sugar content of Cladophora was affected by Pb2 þ treatment, but the protein content showed no significant changes. The malondialdehyde (MDA) content and peroxidase(POD) activity of Cladophora gradually increased whereas superoxide dismutase(SOD) decreased with Pb2 þ concentrations. Catalase (CAT) activity exhibited no significant changes following Pb2 þ treatment. Pb2 þ accumulated in Cladophora and that the lead content in Cladophora was correlated with POD growth, MDA, and Metallothionein (MT). POD and MT play a role in the survival of Cladophora in Pb-contaminated environments. This study suggests that Cladophora can be a choice organism for the phytoremediation of Pb-polluted coastal areas. & 2014 Elsevier Inc. All rights reserved.

Keywords: Cladophora Pb2 þ Growth Physiological characteristic Bioaccumulation Antioxidative defense

1. Introduction Heavy metal pollution is a worldwide problem (Chehregani et al., 2009). Anthropogenic inputs of pollutants, such as heavy metals (HM), into the marine environment have significantly increased within the past few years (Doney, 2010). Rivers contained highest concentrations of arsenic, cadmium, copper, mercury, and zinc. Mean concentrations of Cu, Zn, Cr, Pb, and Cd were higher than background concentrations determined for the areas (Xu et al., 2014). Lead (Pb) is one of the most common and dangerous environmental contaminants (Needleman, 2004), it in the environment can be hazardous to the health and well-being of most living species,so,estimating the toxic-effect of Pb(II) and removed it from waters and the environment is important. Acute and chronic bioassays were conducted to determine the effects of copper, lead, and zinc mixtures on Ceriodaphnia dubia and Daphnia carinata (Cooper et al., 2009). On five marine microalgae with the same biovolume quantity (Tetraselmis chuii, Rhodomonas salina, Chaetoceros sp., Isochrysis galbana (T-iso) and Nannochloropsis gaditana) 72-h exposure toxicity tests with copper and lead were performed. For both metals, 72-h EC50s showed T. chuii as the most tolerant and R. salina as one of the most sensitive n

Corresponding author. Tel: +86 15922409406. E-mail address: [email protected] (D.-j. Cao).

http://dx.doi.org/10.1016/j.ecoenv.2014.11.007 0147-6513/& 2014 Elsevier Inc. All rights reserved.

(Debelius et al., 2009). Antioxidant enzymes, such as SODs, CAT, POD, and MDA participate in antioxidant protection processes. (Alvarez et al., 2012). Trinchella et al. (2013) investigated Cd, Pb, and metallothionein (MT) contents in cultivated mussels (Mytilus galloprovincialis) in the Gulf of Naples, Southern Italy to evaluate MT content as a powerful index of the HM exposure of examined tissues. Bioremediation is an effective and low-cost interesting technology for HM (Chehregani et al., 2009). Biosorption onto living or non-living biomass, such as fungi, bacteria, yeast, moss, aquatic plants, and algae (Tien, 2002; Akar and Tunali, 2006; Sari and Tuzen, 2008; Wang and Chen, 2009), can be a feasible method for Pb(II) removal. Juknys et al. (2012) examined the effects of HM on the oxidative stress and growth of spring barley. Major green algae can be particularly useful (Hamdy, 2000; Pavasant et al., 2006) because they are fairly abundant in many regions of the world, as well as efficiently minimize secondary wastes and can be utilized as low-cost materials (Montazer-Rahmati et al., 2011; Bulgariu and Bulgariu, 2012). Bulgariu and Bulgariu (2013) reported the sorption of Pb(II) onto a mixture of algae waste biomass and anion exchanger resin in a packed-bed column. Sarada et al. (2014) analyzed the biosorption trend of biosorbent Caulerpa fastigiata (macroalgae) biomass to remove toxic HM ion Pb (II) from a solution; the resulting sorption data were consistent with various isotherm models, such as the Freundlich model.The metal-binding

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capacity of these algae can be attributed to the presence of polysaccharides, proteins, and lipids on the cell wall surface (Uncun et al., 2003). Cladophora is one of the most important components of freshwater biota and serves an important function in aquatic ecosystems. With the use of Cladophora as the biosorbent material, and aims to determine the basis for the physiological response to Pb exposure, but there are few study about these. This study focuses on bioaccumulation and the tolerance, and antioxidative defense system of Cladophora under Pb2 þ stress. The first is to assess the toxicity of lead, and use a certain antioxidant enzymes, MT of Cladophora as biomarkers for detecting toxic lead in water. The second objective is to research the potential bioaccumulation and the tolerance of Cladophora to Pb(II). The advantages of using naturally growing aquatic algae for metal removal and biological monitoring that is an effective and low-cost interesting technology.

2. Materials and methods 2.1. Algae cultivation and preparation The Cladophora was collected from rivers in Hefei in Anhui Province, China. The rivers where the species were isolated are not contaminated by metals. After isolation and purification, the plants were enlarged cultivated in a sterilized natural water, then selected Cladophora plants with superior uniform traits for the experiments. The samples were grown in a sterilized medium at 25 °C and kept under an illumination intensity ranging from 3000 lx to 4000 lx (with a light:dark photoperiod of 12:12 h). The cultures were kept at a pH of 7.070.5 and grown without antibiotics.

2.2. Metal solutions Cladophora was grown in a Bold Basal Medium (BBM) broth that contained various concentrations of Pb(NO3)2. All chemicals used were of analytical reagent grade and were used without further purification. All solutions and algal suspensions were prepared using Milli-Q water. Pb(II) stock solutions amounting to 1000 mg/L were prepared by dissolving Pb(NO3)2. All solutions used in the experiments were obtained by means of stock solution dilution.

a

2.3. Experiment and measurements 2.3.1. Determining the relative growth rate of Cladophora After 10 d, the exact biomass of the Cladophora was determined using the formula for relative growth rate (RGR):

RGR (%) = ln ((Mh−Mq)/Mq)/n × 100% where Mq and Mh are the biomass of Cladophora before and after the experiment, respectively; and n is the number of experiment days. 2.3.2. Determining the biological composition Chlorophyll content was determined using the method described by Arnon (1949). The total soluble sugar was estimated following the method of McCready et al. (1950). The digestion of algae materials were performed according to the method of Almela et al. (2002). 2.3.3. Determining MDA and enzymes, such as POD, CAT, and SOD, of protective systems The MDA content in the algae was determined following the method of Yajie et al. (2009) and POD, CAT, and SOD content were detected using the appropriate detection kits, which were purchased from JianCheng Institute of Biological Engineering in Nanjing, China. 2.3.4. Pb2 þ content analysis The quantitative determination of Pb2 þ content in the medium and of the cells was conducted by using an atomic absorption spectrometer. Pb2 þ concentration in the algae was determined as follows. The algae were pre-processed. A porcelain crucible was used to weigh 0.25 g Cladophora. The sample was ashed in a microwave ashing instrument at 550 °C for 4 h, dissolved in 5 mL of 2 mol/LHCl, and dilute with water to 50 mL. The samples were analyzed using flame atomic absorption spectroscopy to detect Pb concentration. A hollow cathode lamp was used, operating at 283.3 nm lamp current. 2.3.5. Metallothionein determination The Cladophora protein samples treated with different concentrations of lead were isolated. The MT was extracted from Cladophora following the method of Scarano and Morelli (2002). MT production was evaluated by means of simple spectrophotometry; the rough extract of MT was identified by means of ultraviolet spectra within 190 nm to 400 nm.

b

Fig. 1. Effect of Pb2 þ on the relative growth rate and the chlorophyll content of Cladophora.

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2.4. Data analysis The correlations between Pb2 þ and the growth and physiological characteristics of Cladophora were analyzed using was carried out using the Pearson Matrix and used the ANOVA analysis conducted with SPSS18.0. OriginPro 8.6 was used for chart drawing and data processing. Kinetic modeling was performed using Freundlich as a fitting model:

ln qe = ln K F +

1 ln Ce n

where KF ((mg/g)(L/mg)1/n) and n are the Freundlich constants, KF can be defined as the distribution coefficient and indicative of the relative adsorption capacity of the adsorbent,the value of n is indicative of the intensity of the adsorption, the values of KF and 1/n were calculated from the intercept and slope of the plot between ln qe versus ln Ce. qe is the amount of Pb2 þ adsorbed per unit mass of Cladophora at equilibrium (mg/g), and Ce is the Pb2 þ concentration of the solution (mg/L) at equilibrium. Linear regression analysis was used for isotherm data treatment.

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compared with the control. According to Fig. 1a, increasing Pb2 þ concentrations affect Cladophora growth compared with the control. Probit analysis or sigmoidal equation using Origen Program can help to obtain the effective concentration 50 (EC50). Fig. 1(b) shows that the Chla þChlb content increase slightly in the 0.5 mg/L Pb2 þ treatment, and then gradually decreased as the Pb2 þ concentration in the medium increased. The photosynthesis of Cladophora decreases gradually as Pb2 þ concentration increases. The Chlaþ Chlb contents in Cladophora sp. then decrease to and remain at a relatively low level when the concentration of Pb2 þ exceeded 5.0 mg/L. The fitting curve of chlorophyll content and Pb2 þ concentration was Y ¼1.16065  0.19213x þ0.03173x2  0.00168x3 (r2 ¼0.84322), indicating that chlorophyll content is negatively correlated with Pb2 þ concentration. Photosynthesis is highly sensitive to Pb, probably owing to the strong detrimental effects of ROS on the critical components of the photosynthetic machinery (Calatayud et al., 1999). Qiu and Hu (2007) showed that ChlaþChlb and Chla of Chlorococcum sp. decreased, indicating that Pb2 þ could result in the decomposition of chlorophyll or inhibit the synthesis of chlorophyll in Chlorococcum sp., thereby decreasing photosynthesis in Chlorococcum sp. under Pb2 þ stress.

3. Results and discussion

3.2. Effect of Pb2 þ on biological composition of Cladophora

3.1. Effects of Pb2 þ on Cladophora growth

3.2.1. Total soluble sugar content As shown in Fig. 2(a), the total soluble sugar content in Cladophora increases under Pb2 þ treatment below 1.0 mg/L concentration, reaching 1.58% in the 1.0 mg/L Pb2 þ treatment. Total soluble sugar content in Cladophora was twice that in the control group, and then declines as the Pb2 þ concentration increases. Total soluble sugar content in Cladophora was lower than in the control when the Pb2 þ concentration exceeds 7.5 mg/L. Pb is a toxic element. To reduce cell damage resulting from HM, cells may produce more polysaccharides to increase the thickness of the cell wall or combine with HM (Lu et al., 2010). So, this study support that Cladophora has certain tolerant ability to Pb contamination, the presence of which activates detoxification processes. No significant differences were found between the Pb-treatedCladophora cells and control cells in terms of protein content (Fig. 2b). These results are consistent with the observations of Lu et al. (2010), in which Zn2 þ had no significant influence on the protein content in Nitzschia closterium (P 40.05), but the protein content increased to a certain extent when Pb2 þ concentration reached 2.5 mg/L. Alvarez et al. (2012) demonstrated that protein decreases in Trebouxia TR1 but increases in T. TR9 in response to Pb exposure. Thus, the protein content of bodies can have different reactions to Pb. In our study, protein content in Cladophora varied

Fig. 1a shows the RGR of Cladophora after treatment with various concentrations of Pb2 þ . The chlorophyll a in Cladophora is shown in Fig. 1b. Fig. 1a shows that Pb2 þ greatly influences Cladophora growth. When Pb2 þ concentration is below 1.0 mg/L, the RGR of Cladophora is higher than the control (7.88% versus 9.48%, respectively). The results clearly show that low concentrations (o1.0 mg/L) of Pb2 þ accelerate Cladophora growth. At concentrations below r7.5 mg/L, Pb2 þ has no significant inhibitory effects on Cladophora growth. However, increased concentrations of HM in nutrient solutions gradually enhance the inhibition of Cladophora growth. When Pb2 þ concentration increases to 10 mg/L, the RGR of Cladophora drops significantly, although the values are still positive, indicating that Pb2 þ may inhibit Cladophora growth, but with no obvious toxic effects. That is to say, Cladophora resists Pb2 þ stress within in the range of trial concentrations. The RGR fitting curves and Pb2 þ concentration are quadratic curves; the equations are Y¼8.13615 þ0.40411x  0.11429x2 and r2 ¼0.88775, respectively. These results are similar to the observations of Qiu and Hu (2007), in which Pb2 þ concentrations ranging from 0.1 mg /L to 10 mg /L had no obvious effects on Chlorococcum sp. growth

a 2.5

4

2.0

Protein content (mg/L)

The content of soluble sugar(%)

y

b

1.5

1.0

3

2

1

0.5

0.0

0 CK

0.5

1

2.5

5

7.5

10

ck

0.5

1

2.5

Fig. 2. Effect of Pb2 þ on the content of soluble sugar and protein of Cladophora.

5

7.5

10

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a

b

25

16

CAT SOD POD

14

Activity of enzyme (U/mgport)

MDA concent (µmol/g)

20

15

10

5

12 10 8 6 4 2

-1

0

1

2

3

4

5

6

7

8

9

10

2+

Concentration of Pb (mg/L)

11

0

1

2

3

4

5

6

7

8

9

10

2+

Concentration of Pb (mg/L)

Fig. 3. The malondialdehyde content and cell-protecting enzymes system of Cladophora exposed to Pb2 þ .

significantly at low concentrations of Pb, but varied slightly at high concentrations of Pb (2.5–7.5 mg/L). 3.2.2. Effect of Pb2 þ on MDA and the cell-protecting enzyme system of Cladophora MDA and enzymes of the protective system, such as POD, CAT, and SOD, were induced using different Pb2 þ concentrations, as shown in Fig. 3. Even the lowest Pb2 þ concentrations resulted in a statistically significant (P o0.05) increase in the MDA concentration of the experimental groups compared with that of the control group. The concentration-dependent increase in MDA content was observed under the different Pb2 þ concentrations. Mroczek-Zdyrska and Wójcik (2012) found that the level of MDA increased in the 50 μM Pb-treated roots by 39% compared with the control. The chain reaction can be terminated after two radicals are combined into a non-radical compound; MDA is usually a final product of the process (Valko et al. 2005; Blokhina et al., 2003). An increase in MDA concentration is therefore considered as a main biomarker of the intensity of oxidative stress (Sharma and Dietz, 2008; Benavides et al., 2005). Thus, exposure to Pb2 þ induces MDA in Cladophora. As shown in Fig. 3b, a lack of significant change in the CAT content of Cladophora was observed after treatment with Pb2 þ between 0.5 and 10 mg/L concentrations. However, Pb2 þ , like other non-redox-active HM, can indirectly initiate oxidative stress. Pb2 þ affected the CAT activity only slightly. Pb did not affect the CAT activity in the roots of Vicia faba (Mroczek-Zdyrska and Wójcik, 2012). However, the activity of catalase in Chlorococcum sp. increased at the beginning and then decreased with the increase of Pb2 þ concentrations (Qiu and Hu, 2007). Numerous authors have shown that HM can induce antioxidative enzymes (Qiu and Hu, 2007; Ekmeciy et al., 2009; Malecka et al., 2012). SOD, ascorbic acid peroxidase (APX), POD, and glucocorticoid receptor(GR) were induced in Maize leaves exposed to Pb (Ekmeciy et al., 2009). Fig. 3b shows the response of the target antioxidant enzymes in Cladophora treated with Pb2 þ . POD was clearly induced by Pb2 þ . SOD activities were reduced. SOD is considered as the cell's first line of defense against ROS (Hassan and Scandalios, 1990) because this superoxide radical is a precursor to several other highly reactive species; thus, control over the steady state of superoxide concentration using SOD constitutes an important protective mechanism (Fridovich, 1997). Interestingly, SOD is induced by its own substrate, a superoxide radical

(Allen and Tresini, 2000). Thus, the activation of cellular SOD may be an indication that the cell is experiencing pollutant-induced superoxide radical stress (Fatima and Ahmad, 2005 ). Pb and other HM ions often reduce cellular activities by generating oxidative stress and inhibiting enzyme reactions.We hypothesized increased Pb tolerance in Cladophora because Pb toxicity is associated with increased ROS formation. 3.2.3. MTs in Cladophora treated with different Pb concentrations MTs are cysteine-richmetal-binding proteins found in a wide variety of organisms, including bacteria, fungi, as well as all eukaryotic plants and animals. MTs bind essential and nonessential HM. Wei and Ru (1999) reported that the peak of ultraviolet absorption spectrum of Pb–MT is 254 nm under nearly neutral pH conditions. Thus, the amounts of MTs in the samples were estimated using the absorbance at 254 nm. The MT concentration in the control was 0.484, whereas 0.821, 1.042, 1.586, 2.094, and 2.613 in the 1.0, 2.5, 5.0, 7.5, and 10.0 mg/L Pb supplemented samples, respectively. This study demonstrated that the MT concentration in Cladophora treated with Pb increased as the Pb concentration increased, and exposure to Pb increased the MT content (Fig. 4) with respect to the control. A significant correlation was established between the Pb concentration in the samples and their MT content. This result is consistent with that of the study by Murthy et al. (2011), in which the MT concentrations in Bacillus cereus were affected by Pb supplementation. A significant increase in tissue metallothionein level was recorded in Lampito mauritii exposed to Pb and Zn contaminated soil. Thus it may be surmised that metallothioneins are directly involved in metal ion detoxification and helps L. mauritii to survive in metal contaminated soil. (Maity et al., 2011). Pb intracellular uptake induces synthesis of phytochelatins and some of their des-Gly derivatives in the green alga Stichococcus bacillaris (Pawlik-Skowron′ska, 2002).This study further revealed that MT production was induced by Pb, such that this parameter can be recommended as a biomarker of Pb exposure. The increase in HM concentration in the cells stimulates the de novo synthesis of apothioneins that can bind metal cations in a non-toxic form, thus reducing their deleterious effects (Klaassen et al., 2009; Simoniello et al., 2010). This study inferred that MT can help Cladophora survive in a Pbcontaminated environment. Therefore, the sensitivity of this biochemical parameter can be used as a reliable biomarker in monitoring metal pollution.

D. Cao et al. / Ecotoxicology and Environmental Safety 112 (2015) 231–237

235

CK

Pb-1.0

Abs=0.484 2

2

Pb-MT

Abs

Abs

0

-2

Abs=0.821

0

-2

-4

254

264

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-4

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254

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wavelength(nm)

wavelength(nm) Pb-2.5

Pb-5.0 Abs=1.586

Abs=1.042

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0

Abs

Abs

2

0

-2

-2

-4

-4 254

264

274

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254

294

264

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294

wavelength(nm)

wavelength(nm) Pb-7.5

Pb-10.0

Abs=2.613

2

2

0

0

Abs

Abs

Abs=2.094

-2

-2

-4

-4 254

264

274

284

294

wavelength(nm)

254

264

274

284

294

wavelength(nm)

Fig. 4. UV scanning spectrograms of metallothionein inner Cladophora exposed to Pb2 þ .

3.3. Biosorption effects of Pb2 þ on Cladophora Pb2 þ concentrations were measured in Pb-treatedCladophora cells over a period of 10 d. The sorption data were fitted using the Freundlich model. The results depicted in Fig. 5 clearly indicate that Cladophora had strong accumulation characteristics with Pb2 þ (Fig. 5a). The cumulative Pb2 þ capacity of Cladophora ranged from 1724 mg/kg to 14,502 mg/kg. The cumulative Pb2 þ capacity of Cladophora increased gradually with the increase in the Pb2 þ concentrations in the medium. In particular, Pb2 þ accumulation improved significantly when the Pb2 þ concentrations exceeded 2.5 mg/L. The cumulative Pb2 þ capacity of Cladophora reached 1.45% when the

Pb2 þ concentration was 10 mg/L. Brooks et al. (1977) proposed the novel concept of a hyperaccumulator plant, and pointed out that the overground parts an hyperaccumulator plant can be enriched with 100 mg kg  1 Cd, 1000 mg kg  1 (Cu, Pb, Ni), or 10,000 mg kg  1 (Zn). Liu et al. (2002) showed the hypertolerance to HM Pb of Vicia amoena, Melilotus suaveolens, Rumex acetosa, Elymus dahuricus, Medicago sativa, and Bidens maximowicziana, as well as the cumulative Pb2 þ capacity of six plants. Thus, Cladophora can be considered as a hyperaccumulator plant with regard to Pb2 þ . The results of the adsorption isotherm indicated that the correlation coefficient (r2) value of the Freundlich isotherm of Pb2 þ was 0.00528. The equation used in the Freundlich model was

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Fig. 5. The cumulative capacity of Pb2 þ in Cladophora and Freundlich fit. Table 1 Correlation analysis of the effect of Pb2 þ on the growth and physiological characteristics of Cladophora. r

C

POD

SOD

CAT

MDA

MT

AQ

RGR

chl

SS

P

C POD SOD CAT MDA MT AQ RGR chl SS P

1 0.959nn  0.227  0.655 0.961nn 0.983nn 0.958nn  0.882nn  0.853n  0.813n  0.012

1  0.191  0.701 0.918nn 0.900nn 0.905nn  0.897nn  0.706  0.852n  0.033

1 0.329  0.466  0.229  0.427  0.105 0.471  0.116  0.622

1  0.745  0.551  0.679 0.508 0.537 0.456  0.275

1 0.934nn 0.968nn  0.774n  0.877nn  0.672 0.136

1 0.957nn  0.836n  0.908nn  0.800n 0.022

1  0.745  0.934nn  0.780n 0.264

1 0.565 0.775n 0.381

1 0.618  0.359

1  0.066

– 1

Note: C – concentration of Pb2 þ ; AQ – acumulative capacity of Pb2 þ in Cladophora; SS – the content of soluble sugar of Cladophora; P – the content of protein of Cladophora; r – Pearson correlation coefficients. nn n

Correlation is significant at the 0.01 level (2-tailed). Correlation is significant at the 0.05 level (2-tailed).

ln Qe¼4.44106þ0.65365 ln Ce (Fig. 5b). The Freundlich model could not describe the adsorption isotherms in this analyzed cases. Arecoa et al. (2012) reported that the correlation coefficient (r2) value of the Freundlich isotherm of Pb2 þ was 0.942. Sarada et al. (2014) analyzed the biosorption trend of biosorbent C. fastigiata (macroalgae) biomass for removal of toxic HM ion Pb (II) from a solution, indicating that the Freundlich model is the best one with correlation coefficient of 0.999. Scanning electron microscopy energy dispersive X-ray analysis (SEM-EDS) confirmed the presence of Zn(II), Cu(II), Pb(II), and Cd(II) ions on the biomass surface of Ulva lactuca (Arecoa et al., 2012). The Freundlich isotherm is an empirical equation assuming that the adsorption process takes place on a heterogeneous surface through a multilayer adsorption mechanism (Fan et al., 2011). Freundlich model better describes the adsorption isotherms in surface adsorption. So the adsorption on Cladophora is not a simple surface adsorption and therefore has a significant relationship with its metabolic process. The cumulative characteristic of Pb2 þ in Cladophora is different from that in U. lactuca. Cladophora can be an effective biological adsorbent for the removal of Pb from synthetic solutions. The experimental results also show that Cladophora can accumulate Pb. Thus, we can use Cladophora to clean the water system. 3.4. Correlation analysis of the effect of Pb2 þ on the growth and physiological characteristics of Cladophora The results of the correlation analyzes demonstrate that Pb content in Cladophora was strictly correlated with POD, MDA, MT, and the growth of Cladophora (P o0.01) (Table 1). POD was clearly

induced by Pb as a defense to the toxicity of Pb. MDA in Cladophora was likewise induced by the exposure to Pb2 þ . These results show the relationship of Pb2 þ accumulation in Cladophora to the formation of MT, as well as to the ion exchange. Thus, MT can help Cladophora survive in a Pb-contaminated environment. The results of this study clearly indicate the capability of Cladophora to tolerate moderately polluted environments and reveal that the physiological features of Cladophora increase its Pb tolerance level. Over expression of metal-binding proteins such as MTs in Cladophora cells resulted in enhanced metal accumulation. This phenomenon presents a potential strategy for the development of microbe-based biosorbents for the removal and recovery of metals from contaminated water or soil.

4. Conclusion Low Pb2 þ concentrations accelerated the growth of Cladophora, whereas Pb2 þ concentrations increases to 10.0 mg/L inhibited its growth. The photosynthesis of Cladophora decreased under Pb2 þ stress. Pb2 þ treatment affected the content of total soluble sugar in Cladophora, but not its protein content. The experimental results also showed that Cladophora can accumulate Pb. The results of the correlation analyzes demonstrated that the Pb content in Cladophora was significantly correlated with POD, MDA, MT, and the growth of Cladophora. POD and MT can help Cladophora survive in Pb-contaminated environments.

D. Cao et al. / Ecotoxicology and Environmental Safety 112 (2015) 231–237

Acknowledgements This research is completed, under the financial aid of the National Science-Technology Support Plan Projects (2012BAD14B00). Funding for this study was provided by Nature Fund of Anhui Province of China (070411025) also.

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