The Toxic Effects of Lindane On Microcystis Aeruginosa And The Inuence of pH And DOM Dependent Effects of Lindane On Microcystis Aeruginosa

The toxic effects of Lindane (γ-BHC) on Microcystis aeruginosa were studied under lab culture conditions. Total protein levels, as well as malondialdehyde (MDA) levels and superoxide dismutase (SOD) enzyme activity, in algal cells, were determined after exposure to different concentrations of Lindane. The bioaccumulation of Lindane, as well as the inuence of pH and dissolved organic matter (DOM), on the toxic effects was also evaluated in algal cells. The growth of M. aeruginosa was inhibited by Lindane treatment (96 h), resulting in 50% of maximal effect (EC 50 ) concentration of 442 μg/L. In addition the lowest observed effect concentration (LOEC) was found to be 120 μg/L, the no observed effect concentration (NOEC) 60 μg/L, and the maximum acceptable toxicant concentration (MATC) 85 μg/L. With increasing concentrations of Lindane and exposure time, M. aeruginosa growth was signicantly inhibited; in addition, total protein levels and SOD activity signicantly decreased. MDA concentration, however, showed an insignicant increase after 96 h. Lindane has the potential for bioaccumulation in algal cells with a bioconcentration factor (BCF) of 340. Furthermore, the toxic effects of Lindane on M. aeruginosa were inuenced by environmental factors, such as pH and DOM. The toxic effects decreased with increasing pH and humic acid concentrations. Ultrastructure cell images were used to depict Lindane induced apoptosis.


Introduction
Lindane (γ-BHC) is an organochlorine insecticide that is classi ed as a persistent organic pollutant (POP) and serious health problems can arise after exposure to it (Saez et al., 2012). It is a highly chlorinated compound that has been used worldwide, as a broad spectrum insecticide, on a variety of crops (Bidlan et al., 2004). The compound has also been used in human health applications to treat scabicide and pediculocide in the form of lotions and shampoos (Phillips et al., 2005). Lindane is the main insecticide produced in China. The highly stable nature of Lindane leads to its ready accumulation in the environment, and thus in organisms. Today, Lindane is no longer in production, however, it persists in the environment and has been found in water, sediment, soil, plant and animal samples (Carvalho et  Algae are primary producers in aquatic ecosystems and provide a source of food and energy for zooplankton, sh, and other aquatic organisms, the levels of algae are used to monitor and evaluate water environment quality (Kobraei and White, 1996). Blue-green algae (cyanobacteria) are a common, naturally occurring component in most recreational water environments. Excessive cyanobacteria populations in surface water are indicative of a eutrophic water environment. Microcystis aeruginosa is a single-celled alga which belongs to Microcystis, Chroococcales, Cyanophyta. M. aeruginosa is the dominant species in most eutrophic lakes in China and is found in eutrophic bodies of water all over the world (Saez et al., 2012). The aim of this study is to investigate the toxic effects of Lindane on M. aeruginosa as well as some environmental factors affecting its toxicity.

Algal Cell density measurements
A series of different concentrations of M. aeruginosa were prepared. Algae concentrations were measured by cell counting with a hemacytometer and determining their absorbances at 680 nm with a spectrophotometer. A signi cant linear correlation between cell number and absorbance was observed (y = 0.0002x + 0.0116, R 2 = 0.9971). The absorbance was then used as an index to measure the growth status of M. aeruginosa in the present study.

Acute and chronic toxicity test
M. aeruginosa was cultured in 250mL BG11 medium and different initial concentrations of Lindane (0, 50, 89, 158, 281, and 500 µg/L) were added. Experiments were performed in triplicate. The initial cell density was 1.7×10 4 cells/mL. The algal optical density at 680 nm (OD 680 ) was measured every 24 hrs by spectrophotometer and the growth inhibition rate (I a ) was calculated using the following equation: Where N represents the test group and N 0 represents the control group at OD 680 . In accordance with the principle of the linear relationship between the natural logarithm of the toxicant concentration and the percentage of biological effect, we can calculate the concentration for 50% of maximal effect (EC 50 ) at the 96 hr time point through the probability method (Hoekstra, 1991). Lowest observed effect concentration (LOEC) is the lowest concentration which has a signi cant difference from the control and An algal solution (50 mL) was centrifuged for 10 minutes at 4000 rpm, the supernatant was discarded, and then the algal cells were washed using Mili-Q water (3x). Phosphate buffer solution (PBS) (0.05 M, pH = 7.8, 5 mL), a small amount of liquid nitrogen, and quartz sand were added to grind the cell material. The homogenate was then centrifuged for 10 minutes, at 4000 rpm; the resulting supernatant contained a crude enzyme solution. Total protein levels in algal cells were determined using the Coomassie Brilliant Blue G-250 staining method (Bradford, 1976). The superoxide dismutase (SOD) enzymatic activity was determined according to the nitrogen blue tetrazolium photoreduction method (Beauchamp and Fridovich, 1971). Malondialdehyde (MDA) levels were measured according to the thiobarbituric acid method (Heath and Packer, 1968) with 10% trichloroacetic acid (TCA) substituting for PBS.

Determination of Lindane concentration in algal cells
An algal solution (70 mL) was centrifuged for 10 minutes, at 4000 rpm and the supernatant was discarded. The cell pellet was washed 3x using Mili-Q water. Acetic acid (100nL), a small amount of liquid nitrogen, and quartz sand were added to lyse algal cells. The homogenate was centrifuged for 10 minutes at 4000 rpm. The supernatant was ltered through a GF/C membrane (0.45 µm) and passed through a C18 column. The column was then washed with 10 mL methanol and 10 mL H 2 O to clear away contaminates. The extract was eluted with 10 mL petroleum ether, and then concentrated to 1 mL using a vacuum rotary evaporator and gentle stream of pure nitrogen gas. Lindane concentration was determined using capillary gas chromatograph with electron capture detector (GC-ECD, Agilent 7890) tted with HP-5 column (30m×0.25mm×0.25µm, Agilent, USA). The injector port temperature was 200 ºC. The oven temperature started at 140 ºC for 2 min, and increased to 260 ºC at a rate of 10 ºC min − 1 . The temperature was then maintained at 260 ºC for 10 min. The temperature of detector was set at 300 ºC. A standard curve was prepared using Lindane solutions of various concentrations (5, 10, 20, 50, 100, 200, 500 µg/L). A linear regression equation (y = 294.27x-863.53) (R 2 = 0.9998) for the standard curve was obtained for quanti cation of Lindane concentration in M. aeruginosa cells. The recovery e ciency and precision of the process were determined in three different concentrations of Lindane in these experiments we observed 84.6-91.9% recovery and 1.98-4.62% relative standard deviation (RSD). The bioconcentration factor of Lindane, in treated M. aeruginosa cells, was determined using the Kukkonen method (Kukkonen, 1991). The Kukkonen formula is below.
Here C f represents the concentrations of Lindane (mg/kg) in the algal cells and C W represents the concentration of Lindane in the water (mg/L).

The in uence of pH and DOM
To determine the in uence of pH and DOM, M. aeruginosa growth, after exposure to different concentrations of Lindane (5, 10, 20, 50, 100, 200, 500 µg/L) for 96 hrs, was assessed. In these experiments cells were grown in various pH conditions (pH 5,7,9) and various humic acid (HA) concentrations (0, 2.5, 5.0, 7.5, 10.0 mg/L).Cell growth was monitored every 24 hrs to evaluate the in uence of pH and DOM.

2.7
The ultrastructural observations of M. aeruginosa M. aeruginosa were grown in different concentrations of Lindane (5,10,20,50,100,200, 500 µg/L) for 96 hrs and then samples were taken. Algal solutions were centrifuged for 10 minutes at 4000 rpm and the supernatant was discarded. Cells pellets were washed 3x, using 0.2M PBS (pH = 7.4). The algal cells were xed with 2.5% glutaraldehyde (Sigma) and 2% paraformaldehyde, rinsed with 0.2 M PBS (pH = 7.4), and then suspended in 2% agar in PBS. Next, the small blocks of agar-suspended algae were post-xed in 1% osmium tetroxide, dehydrated with ethanol, and embedded in Epon812 epoxy resin. Ultrathin sections were cut with a diamond knife and sequentially stained with 3% methanolic uranyl acetate followed by lead citrate (Pollio et al., 1993). The ultrastructure of M. aeruginosa was studied by transmission electron microscopy (TEM) using the JEM-200CX (JEOL, Japan).

Statistical analysis
The T-test was used to evaluate statistical differences among different test groups using the SPSS software (Version 16.0). All charts were generated with Origin 8. The effect of Lindane exposure on SOD enzyme activity is shown in Fig. 2B. The SOD enzyme activity decreased with increasing concentrations of Lindane (50-500 µg/L for 96 hrs). In fact, marked enzymatic activity reduction occurred at all concentrations of 50 µg/L and higher (p < 0.01). SOD enzyme activity was only 12.8% of the control sample in 500 µg/L Lindane sample. The SOD enzyme catalyzes excess O 2 − to H 2 O 2 and O 2 , in order to prevent free-radical toxicity and protect cells (Scandalios, 1993). In this study, the activity of SOD decreased with increasing concentrations of Lindane. This decrease in SOD activity resulted in a decrease in the cell membrane's osmotic adjustment ability (Mittler, 2002;Zhu, 2002).
The cellular content of MDA increased gradually from 0.386 µmol/L to 0.427 µmol/L with increasing concentrations of Lindane (Fig. 2C.). The increasing MDA levels in the treatments varied from 4.0-10.6% compared to the control (p < 0.05). MDA is an oxidized product of membrane lipids. MDA has been shown to accumulate when plants are exposed to oxidative stresses. Cellular levels of MDA are considered to be an indicator of lipid peroxidation and cellular stress (Chaoui, 1997). In this experiment, MDA levels gradually increased in correlation to Lindane concentration levels. These results suggested Lindane promotes lipid peroxides in M. aeruginosa. Overall, we observed, Lindane exposure caused a reduction in total cellular protein content and SOD activity, as well as elevated MDA levels in M. aeruginosa. Moreover these results indicate high concentrations of Lindane may destroy the anti-oxidative system of M. aeruginosa.

Bioaccumulation of Lindane in M. aeruginosa
The concentration of Lindane in M. aeruginosa was determined using GC-ECD after exposure to different concentrations of Lindane (Supplementary material 1). As expected, the cellular concentration of Lindane increased with increasing initial concentrations of Lindane in the algal solution. The BCF of Lindane in M. aeruginosa was obtained when the initial concentration of Lindane was 400 µg/L and algae cell population was 2.0 × 10 4 cells/mL (Supplementary material 2). The BCF value of 340 (log BCF, 2.53) indicated that Lindane has a relatively strong bioaccumulation potential in M. aeruginosa. Lindane has been previously shown to bioaccumulated in different aquatic organisms (Geyer et al., 1997;Thybaud and Le Bras, 1988;Vigano et al., 1992). Moreover, the BCF value in this study is similar to previously reported values (Arnot and Gobas, 2006). This review reported the BCF values of Lindane in over a 140 aquatic organism which ranged from 0.52-3.32 for logBCF and acceptable log BCF ranging from 2.16-3.32.

The in uence of pH and DOM on Lindane toxicity
The in uences of pH and DOM on Lindane toxicity in M. aeruginosa are represented in Table 1. There was little impact observed on the LOEC, NOEC and MATC of the samples. The EC 50 did increase with an increase in pH; this result implies weak alkaline conditions could reduce Lindane toxicity in M. aeruginosa. Jin et al. (2004) reported the optimal pH range is 8.5-9.5 for the growth of M. aeruginosa. Lindane is hydrolyzed in alkaline conditions. Hydrolysis is an important mechanism in the abiotic transformation of Lindane (Ngabe et al., 1993). Previous studies revealed that Lindane hydrolysis could be catalyzed by hydroxide and hydrogen ions, especially in alkaline conditions (Ngabe et al., 1993;Saleh et al., 1982). The values of LOEC, NOEC and MATC at pH 5 and 9 were slightly higher than that at pH 7 (Table 1). Liu et al. (2011) reported that the hydrolysis of Lindane was not signi cant under certain growth conditions; speci cally pH 5 or pH 7, at 25ºC. This study reported the half-life of Lindane hydrolysis was approximately 2310 d (pH 5) and 1386 d (pH 7), however the half-life of this reaction was 28.1 d at pH 9. Thus pH has a signi cant effect on the rate at which Lindane is hydrolyzed in environment. The products of Lindane hydrolysis, including 1,2,4-trichlorobenzene, 1,2,3-trichlorobenzene 1,3,5-trichlorobenzene and pentachlorocyclohexene have been identi ed and their distributions shown to be depended on pH (Liu et al., 2002). Maybe these products are less toxic and persistent than Lindane in the environment. Therefore, support previous data in that, a weak alkaline condition degrades the Lindane toxicant and favors M. aeruginosa growth. The toxic effects of Lindane on M. aeruginosa decreased dramatically with increasing concentrations of HA (Table 1). The EC 50 value of Lindane increased from 554 µg/L to 920 µg/L when the concentration of HA increased from 2.5 mg/L to 10 mg/L. DOM is regarded as natural chelator (Boggs et al., 1985).
Previous studies have shown the hydrophobic component of DOM has a high a nity to pesticides, thus DOM improves solubility in water and migration of hydrophobic organic contaminants (Edwards and Cole, 1996;Thevenot et al., 2009). DOM has been reported to decrease the bioconcentration of organic chemicals in aquatic animals. These decreases in bioconcentration have been attributed to DOM interactions with chemical compounds. Under these conditions aggregates are formed that are too large and/or too polar to be taken up by test organisms (Haitzer et al., 1998). In the present study, the increased EC 50 value maybe the result of a stable chelate formation involving HA and Lindane.

The ultrastructure of M. aeruginosa
The ultrastructure photos of M. aeruginosa, exposed to different concentrations of Lindane are shown in Fig. 3 (1-cell growth; 2-cell division). Here, the M. aeruginosa cells grown in BG-11 medium, without Lindane, demonstrate a dense cytoplasm an intact cell wall and membrane. The thylakoids were abundant and with a compact, neat arrangement in the cytoplasm. The central nuclei were obvious, ribosomes were evenly distributed throughout the cells, and there were many phosphate granules around the central nucleus ( Fig. 3 -A1, A2). Under the stress of Lindane (50 µg/L) exposure the vast majority of M. aeruginosa cells began to degrade and thylakoids gradually blurred (Fig. 3 -B1, B2). The disintegration of thylakoids became more obvious and the nucleus began to appear as an empty cavity in cells treated with 89 µg/L of Lindane ( Fig. 3 -C1, C2). In samples where the concentration of Lindane was greater than 158 µg/L, the thylakoids were further broken and empty nuclei cavities became more abundant (Fig. 3 -D1, D2). The structure of algal cells became blurred, and the nuclei gradually disappeared (Fig. 3 -E1, E2). When the concentration of Lindane was increased to 500 µg/L, cells had all but disintegrated, however the cell walls remained intact ( Fig. 3 -F1, F2).
The structural integrity of a cell's periphery plays an important role in protecting the internal structure, as well as maintaining normal viability and cellular activity. These TEM images of M. aeruginosa indicated that Lindane can destroy the structure of these cells. This results in the cells' inability to perform photosynthesis thus leading to death.

Conclusions
Here we report that M. aeruginosa growth is inhibited by Lindane. This study demonstrated Lindane exposure resulted in an EC 50 442 µg/L, LOEC 120 µg/L, NOEC 60 µg/L, and MATC 85 µg/L in M.
aeruginosa after 96 hrs of exposure. The observed decrease in protein levels, SOD activity, and the increase in MDA levels suggested the main mechanism of Lindane toxicity might result from oxidative damage of lipid and other biological macromolecules. Furthermore, the BCF value of 340 indicates Lindane has the potential to bioaccumulate in M. aeruginosa. Here we observed the toxic effects of Lindane decreased as pH increased and as concentrations of HA increased. Finally, the ultrastructure images of M. aeruginosa showed that increasing concentrations of Lindane destroyed cellular structures. This destruction resulted in the loss of photosynthesis, subsequently preventing cellular metabolism and reproduction eventually leading to cell death. Availability of data and materials

Declarations
All the data are included within the text.

Figure 1
The growth inhibition of Microcystis aeruginosa by Lindane. Each value is the mean ± S.D.