Ecotoxicology (2014) 23:1638–1647 DOI 10.1007/s10646-014-1303-x

Aquatic environmental safety assessment and inhibition mechanism of chemicals for targeting Microcystis aeruginosa Xiao-Bo Yu • Kai Hao • Fei Ling • Gao-Xue Wang

Accepted: 6 August 2014 / Published online: 20 August 2014 Ó Springer Science+Business Media New York 2014

Abstract Cyanobacteria are a diverse group of Gramnegative bacteria that produce an array of secondary compounds with selective bioactivity against vertebrates, invertebrates, fungi, bacteria and cell lines. Recently the main methods of controlling cyanobacteria are using chemicals, medicinal plants and microorganism but fewer involved the safety research in hydrophytic ecosystems. In search of an environmentally safe compound, 53 chemicals were screened against the developed heavy cyanobacteria bloom Microcystis aeruginosa using coexistence culture system assay. The results of the coexistence assay showed that 9 chemicals inhibited M. aeruginosa effectively at 20 mg L-1 after 7 days of exposure. Among them dimethomorph, propineb, and paraquat were identified that they are safe for Chlorella vulgaris, Scenedesmus obliquus, Carassius auratus (Goldfish) and Bacillus subtilis within half maximal effective concentration (EC50) values 5.2, 4.2 and 0.06 mg L-1 after 7 days, respectively. Paraquat as the positive control observed to be more efficient than the other compounds with the inhibitory rate (IR) of 92 % at 0.5 mg L-1. For the potential inhibition mechanism, the chemicals could destroy the cell ultrastructure in different speed. The safety assay proved dimethomorph, propineb and paraquat as harmless formulations or products having potential value in M. aeruginosa controlling, with the advantage of its cell morphology degrading ability. Keywords Microcystis aeruginosa  Acute toxicity  Aquatic ecosystem  Scanning electron microscope

X.-B. Yu  K. Hao  F. Ling  G.-X. Wang (&) Northwest A&F University, Xinong Road 22nd, Yangling 712100, Shaanxi, China e-mail: [email protected]

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Introduction The global problem of harmful blooms (HABs) has expanded both in extent and public perception over the last several decades (Anderson et al. 2002). Microcystis aeruginosa Ku¨tzing is one of the main cosmopolitan and frequently outbreak blooms in freshwater systems, especially in eutrophication water, which could cause deterioration of water quality, mass mortalities of fish and shellfish, human intoxication through inhalation or water contact (Anderson et al. 2002; Azevedo et al. 2002; Zhao et al. 2006). Currently, application of chemicals is a conventional method for inhibiting M. aeruginosa in freshwater systems. There has been some researches into the use of traditional chemicals to control bloom-forming cyanobacterium M. aeruginosa (Ma et al. 2006; 2010), however, due to their potential inhibition for beneficial algae communities that are necessary to maintain the aquatic food web, copper sulfate (Ni et al. 2012) and others effective chemicals (Drabkova et al. 2007; Han et al. 2001; Jeong et al. 2000) have been no longer recommended for the control of HABs. The aquatic environment is not a simple combination of all the biota, but there is a complex competition, predation and interdependent relationship among biological communities. Therefore, it is important to develop a new generation inhibitors that only target the M. aeruginosa communities, yet that will be effective, and economically and environmentally benign. Considering the popularity of chemicals and potential effects on environment, this study mainly focuses on assessing the selected chemicals security of non-targeted organisms. In the past years, some investigators had reported that medicinal plant and microorganism inhibited cyanobacteria in hydrophytic ecosystems (Imamura et al. 2001;

Aquatic environmental safety assessment and inhibition mechanism

Jancˇula et al. 2008; Yi et al. 2012). However, using chemicals as the common method for controlling harmful algae is of low cost and easy operation (Kong et al. 2005). Moreover, acute toxicity tests with fish and other aquatic organisms have long played a major role in aquatic risk assessments, especially at a ‘‘screening’’ level of evaluation (Toussaint et al. 1995). Macrophytes and beneficial algae are the primary producers of water environment, keeping balance and stability of ecological system, and directly affecting the structure and function of aquatic ecosystems (Brix and Schierup 1989; Kargioglu et al. 2012; Ricardo et al. 2013). Chlorella vulgaris Beijernick and Scenedesmus obliquus Ku¨tzing selected as the toxicity test materials are two kinds of beneficial algae strains with abundant nutrients which can be used by omnivorous or herbivorous aquatic animals (Zhang et al. 2013). Moreover, fish as a consumer of aquatic ecosystems and an important source of food, has the closest relationship with human beings. So the fish is also often used as subjects of aquatic biological toxicity test (Embry et al. 2010; Folmar et al. 1979). Carassius auratus Linnaeus (Goldfish) as toxicity test material has been applied in the field of chemical safety assessment (Altinok 2004). Additionally, microbial community as an important part in freshwater ecosystems is sensitive to environmental change. So microorganism can be used for assessing the safety of chemicals. Bacillus subtilis Cohn as a beneficial microorganism has the ability to purify water by produce protease, lipase, and amylase which promotes the degradation of nutrients in feed (Fisher 1999). Therefore, B. subtilis as a popular toxicity test material has been used to measure antimicrobial activity (Bonvehı´ and Coll 1994; Singh et al. 2005). Prokaryotic cyanobacterium is classified into bacteria by scientists and has the similar ingredient with higher plant such as cell wall and chlorophyll a (Papageorgiou 1996). Accordingly, bactericide, algistat and herbicide are brought into the water environment and have developmental potential for controlling cyanobacteria (Backhaus et al. 2004; Masojı´dek et al. 2011; Schrader et al. 2010). For example, Morin et al. (2010) reported the bactericide triclosan caused a decrease in diatom growth rates and a significant delay in the exponential phase of growth. Sjollema et al. (2014) found the dissimilar modes of action on the green alga C. vulgaris of 40 herbicides in 19 different chemical structure classes. Toxic effects of chemicals on aquatic organisms are generally evaluated by toxicity tests. The aim of present study was to exploit the activity of chemicals, by screening 54 bactericide, algistat and herbicide inhibit M. aeruginosa and assess the safety of these selected chemicals. C. vulgaris, S. obliquus, goldfish and B. subtilis as representative species in the aquatic environment were evaluated in this study.

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Materials and methods Phytoplankton culture and chemicals collection M. aeruginosa (toxic strain FACHB-905), C. vulgaris (beneficial strain FACHB-9) and S. obliquus (beneficial strain FACHB-416) were obtained from the Fresh water Algae Culture of Hydrobiology Collection, Chinese Academy of Sciences. Three algae cultivated at 25 °C under a 14:10-h photoperiod at 90 l mol of photon (PAR) m-2s-1 in sterile BG11 medium (Li and Hu 2005). Algae species were cultivated in 500-mL conical flasks containing 200 mL culture media. Fifty three chemicals were tested and the detailed list was given in Table 1. Flutriafol and diniconazole were purchased from Jiangsu Rudongzhongyi Chemicals Co., Ltd (China). Thiabendazole and cyprodinil were purchased from Wuhan Jinluo Chemicals Co., Ltd (China). Fomesafen, paraquat, quizalofop-p-ethyl, fluoroglycofen and nicosulfuron were purchased from Jinan Kesaijinong Chemicals Co., Ltd (China). Mesosulfuron was purchased from bayer crop science company (Germany). Other 45 chemicals were purchased from Hubei Kangbaotai FineChemicals Co., Ltd (China). Low water-soluble chemicals (ethirimol, uniconazole, bitertanol, trifloxystrobin, epoxiconazole, cyprodinil, diniconazole, benomyl, carbendazim, dimethachlon, mancozeb, chlorothalonil, propineb, procymidone, dimethomorph and difenoconazole) were dissolved in DMSO. In this study, that in preliminary work DMSO was found to have no effect on cell division rates of the developed heavy M. aeruginosa, C. vulgaris and S. obliquus (Fig. 1). Other chemicals were dissolved in deionized water to obtain 10.0 mg mL-1 (sample/solvent) of stock solutions, which were used for the preparations of the desired concentrations for antialgae efficacy assay. All of the chemicals including fungicides and herbicides were dissolved in deionized water or DMSO to obtain stock solutions, which were used for the preparations of the desired concentrations for antialgae efficacy assay. Chemical screening and EC50 test inhibiting M. aeruginosa The cyanobacteria concentration was detected by spectrophotometry and microscope counting. This study preferred the spectrophotometry but some colored chemicals assay could not use it and was detected by microscope counting. For one, inhibition assay of developed heavy phytoplankton was monitored by spectrophotometrically at 450 nm (OD450; i.e., optical density at 450 nm) (Kim et al. 2010). Each absorbance tested in 96-well plates containing 200 lL culture media. For another, the alga cells were counted under a microscope using a

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1640

X.-B. Yu et al.

Table 1 Percentages of growth inhibition of Microcystis aeruginosa for each treatment (20 mg L-1) after 3 and 7 days Medicine tecloftalam ethirimol fluconazole

3 days (%) Mean ± SD

7 days (%) Mean ± SD

Medicine

3 days (%) mean ± SD

7 days (%) mean ± SD

4.70 ± 1.76

27.87 ± 2.94

dimethomorph

36.34 ± 6.12

67.18 ± 0.79

11.70 ± 1.18

46.30 ± 13.74

SDIC

72.09 ± 1.65

76.39 ± 1.43

4.35 ± 7.53

–

cymoxanil

– –

triadimenol

–

41.85 ± 9.93

difenoconazole

cyproconazole

–

21.76 ± 4.61

triazolone

uniconazole

–

5.64 ± 13.10

pyrimethanil

bitertanol tebuconazole

– –

0.63 ± 3.65 5.16 ± 5.64

triflumizole nitrothal-isopropyl

validamycin trifloxystrobin

2.82 ± 2.71

3.81 ± 0.79

–

bromothalonil

34.81 ± 11.64

epoxiconazole

2.70 ± 2.82

mancozeb

–

ammonium glyphosate

44.79 ± 8.26

4.53 ± 0.24

0.12 ± 3.76

12.31 ± 1.83

29.87 ± 2.00

60.43 ± 2.14

3.29 ± 0.12 –

6.43 ± 4.69 5.48 ± 1.03

47.98 ± 6.00

70.99 ± 0.64

6.94 ± 1.88

3.18 ± 7.31

tribenuron

0.16 ± 10.96

51.38 ± 2.46

–

MCPA-Na

–

2.00 ± 2.35

–

flusilazole

1.41 ± 4.12

14.29 ± 0.95

propamocarb

36.78 ± 3.45

53.85 ± 3.85

propineb

49.16 ± 4.59

71.47 ± 0.79

tridemorph

18.39 ± 3.45

18.27 ± 8.65

50.35 ± 5.88

propiconazole

26.44 ± 2.30

24.06 ± 4.81

kasugamycin

48.28 ± 5.75

45.19 ± 6.73

myclobutanil

5.41 ± 13.29

diniconazole

8.35 ± 1.65

thiabendazole flutriafol cyprodinil carbendazim dimethachlon paclobutrazol oxadixyl chlorothalonil benomyl procymidone mesosulfuron

–

2.00 ± 6.59

8.50 ± 14.85

89.06 ± 0.47 80.12 ± 1.88

7.32 ± 1.91 23.43 ± 9.45

–

1.03 ± 1.19

2.70 ± 15.05

32.64 ± 7.23

–

8.66 ± 8.97

1.65 ± 0.12

–

68.57 ± 1.88 –

17.24 ± 6.90

56.73 ± 6.73

62.07 ± 3.45 26.44 ± 2.30

53.85 ± 1.92 40.38 ± 5.77

nicosulfuron

13.79 ± 5.75

59.61 ± 1.92

uridine

39.08 ± 10.34

56.73 ± 4.81

paraquat

96.54 ± 1.15

94.23 ± 1.92

fomesafen

44.83 ± 4.60

57.69 ± 1.92 87.50 ± 0.96

79.41 ± 1.27

terbutryn

87.36 ± 8.05

11.36 ± 1.35

bromoxynil octanoate

34.48 ± 3.45

pendimethalin

49.43 ± 6.90

–

–

87.36 ± 1.15

aquacide quizalofop-p-ethyl fluoroglycofen

– 57.69 ± 1.92

89.42 ± 2.89

– representative no inhibitory activity

Cell Division Rates (µc) (Control )

18

Microcystis aeruginosa Chlorella Scenedesmus

15 0

1

2

3 4 Time (d)

haemocytometer. The concentrations of three motherliquor and the initial concentration in the trials are M. aeruginosa (6.4 9 107cells mL-1; 2.0 9 107cells mL-1), C. vulgaris (1.7 9 107cells mL-1; 8.1 9 106cells mL-1) and S. obliquus (2.3 9 107cells mL-1; 1.3 9 107cells mL-1), respectively. The regression equations between the

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18

17 16

5

Cell Division Rates (µc) (0.4% DMSO)

B

6

7

ln Cell Concentration

A ln Cell Concentration

Fig. 1 Cell division rates of M. aeruginosa, C. vulgaris and S. obliquus in the control a and 4 % DMSO treatment group b lc = (ln(C2)-ln(C1))/(T2T1) where C1 and C2 are the cell concentrations (cells mL-1) at times T1 and T2 (T2-T1 = time interval in days)

17 16

Microcystis aeruginosa Chlorella Scenedesmus

15 0

1

2

3 4 Time (d)

5

6

7

cell concentration (y [105 cells mL-1]) and OD450(x) of three algal cells were respectively established as y = 629.6x-10.92 (r2 = 0.998), y = 332.6x-5.013 2 (r = 0.998), y = 438.9x-7.365 (r2 = 0.999). In the preliminary screening section, each chemical were established 20 mg L-1 designed to M. aeruginosa

Aquatic environmental safety assessment and inhibition mechanism

inhibition test for 3rd and 7th day. In consideration the specific algae inhibiting activity with different chemicals, some high-activity chemicals of inhibition M. aeruginosa were selected and designed the concentration gradient assay. A geometric series with a common ratio of 2 was prepared covering concentration from 0.0625 to 32 mg L-1 by using deionized-distilled water. All the batch experiments were prepared in triplicate. The percentages of concentration at specific test substance concentration were calculated compared to the control group. The simulation effect of the chemicals on already developed phytoplankton was estimated by the following formula: IR ð%Þ ¼ ½1ðN/N0 Þ  100 N0 and N are the concentration of cells in the control and treatment group, respectively. Environmental toxicity assessment The safety evaluation of selected chemicals was accomplished by these aquatic organisms including green alga (C. vulgaris and S. obliquus), goldfish and B. subtilis. Both the green algae cultivation method and the concentration gradient assay of selected chemicals are identical to M. aeruginosa. The detailed process of these chemicals security assessment to others aquatic organisms are as follow. Healthy goldfish (500 fish; mean weight ± SD, 11.27 ± 1.10 g; length ± SD, 47.5 ± 5.6 mm) were obtained from Shaan’xi Fisheries Research Institute (Shaanxi, China) and maintained in a 280 L aerated glass aquarium with more than 85 % oxygen content saturation at 25 ± 1 °C temporary breeding for a month. The goldfish care and treatment were maintained in conformity with the General Recommendation of Chinese Experimental Animals Administration Legislation. All the selected chemicals (against M. aeruginosa) were undertaken acute toxicity tests on goldfish. The tests were conducted in plastic pot of 2.0 L capacity, each contained 1.5 L compound solution and three goldfish. According to the toxicity preliminary experiments, each chemical toxicity test group designed appropriate arithmetic progression concentrations. Each experimental treatment of five goldfishes prepared in duplicate. The entire chemical solution group was held for observation of survival, growth, and behavior. The death of fish were recorded when the tail beat stopped and the fish no longer responded to mechanical stimulus. The dead fish was removed timely from the water to avoid the deterioration of the water quality. Fish mortalities in the treatment and control groups were recorded after 48 h. The chemicals bacteriostasis was determined by zone of inhibition (Su et al. 2007). In this study, B. subtilis was cultured in bacteria basic medium (beef extract: 0.5 %,

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peptone: 1 %, NaCl: 0.5 %, dipotassium hydrogen phosphate: 0.1 %, agar: 1.5 %) at 37 °C and preserved in our laboratory. The strain was cultured in 500 mL flask containing 200 mL liquid medium which were used for the preparations of the assay. Each 0.192 mg chemicals was respectively dissolved to 3 mL sterile water and mixed with 3 mL basic medium and keep the final test concentration at 32 mg L-1. After mixing with a vortex, aliquots (6 mL) were poured into 35 mm diameter plates and then placed in sterile bench natural cooling and solidification. Uniform growth of B. subtilis was punched holes of equal size (diameter 5.0 mm) onto each media plate. Each chemicals group was repeated 3 times. After 48 h, the growth surface area of B. subtilis was measured. The growth inhibition rate was calculated from mean values as: IR ð%Þ ¼ 100ðy-zÞ=ðx-zÞ where IR = growth inhibition rate, x = bacterial growth surface area in control, y = bacterial growth surface area in sample, z = the growth surface of the hole puncher. Effect on M. aeruginosa damage Based on the bioassays, safe and efficient chemicals inhibiting M. aeruginosa were selected for further mechanism studies. To explore the effects of screened chemicals on M. aeruginosa cell damage, concentration was set to twice of the 7 days EC50 value. Samples of control and chemical treated cells were collected at 3rd day and 7th day, then fixed with 2.5 % glutaraldehyde at 4 °C overnight. After that, samples were washed with phosphate buffer solution, dehydrated in a graded series of ethanol, passaged through acetone, replaced with isoamyl acetate and fully dried with a critical point dryer. Treated samples were mounted on copper stubs and sputter-coated with gold–palladium (Zhang et al. 2013). The specimens were observed and photographed using scanning electron microscopy (SEM-6360 LV, HITACHI, JAPAN) at 10 kV. Statistical analysis The differences of phytoplankton concentration at each time were analyzed using Log phase cell specific division rate (lc) in the controls. lc ¼ðlnðC2 Þ - lnðC1 ÞÞ=ðT2 - T1 Þ where C1 and C2 are the cell concentrations (cells mL-1) at times T1 and T2 (T2-T1 = time interval in days). Besides, when phytoplankton concentrations was significantly down-regulated compared with control, the effective concentration causing a 50 % inhibitory response at 3 and 7 days (EC50) was estimated with Probit analysis. The data were analyzed by one-way ANOVA and expressed as the arithmetic mean ± SD. Differences was determined by Tukey’s test in SPSS statistical software (SPSS Inc., USA)

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1642 Table 2 Inhibition activity of ten chemicals on Microcystis aeruginosa, Chlorella vulgaris and Scenedesmus obliquus for 3 days

X.-B. Yu et al.

Chemical

SDIC mancozeb paraquat terbutryn

Data at the same medication with different letters are significantly different (a \ b \ c, p \ 0.05)

Microcystis aeruginosa EC50 (mean ± SD) (mg L-1)

Scenedesmus obliquus EC50 (mean ± SD) (mg L-1)

0.73 ± 0.07a

6.77 ± 0.37c

6.24 ± 0.05b

a

b

14.42 ± 1.75b

b

0.48 ± 0.06a

a

1.80 ± 0.20a

b

7.80 ± 1.10

a

0.30 ± 0.04

b

7.18 ± 1.71

a

11.85 ± 1.29

5.26 ± 0.66 2.79 ± 0.62

mesosulfuron

1.63 ± 0.12

3.52 ± 0.66

1.61 ± 0.25a

chlorothalonil

1.39 ± 0.09a

33.99 ± 1.14b

43.25 ± 1.80c

a

c

dimethomorph pyrimethanil

18.47 ± 2.92 [50

47.46 ± 1.81 [50

44.87 ± 0.75b 37.14 ± 0.68

propineb

11.31 ± 1.48a

47.08 ± 2.09b

45.72 ± 1.20b

with P values less than 0.05 being accepted as significant. The 50 % lethal concentration (LC50) and 95 % confidence level were calculated by Probit analysis and Regression equation (Finney 1971).

Results The chemicals preliminary screening test on M. aeruginosa The chemicals and the screening result are shown in Table 1. Of the chemicals tested, chlorothalonil, dimethomorph, SDIC, pyrimethanil, propineb, mancozeb, paraquat, terbutryn and mesosulfuron were found to have significant inhibitory activity (C60 %) at 20 mg L-1. Meanwhile, the cyanobacteria cell stopped growing and remained in an inhibited state in some groups. Fluconazole, Trifloxystrobin, diniconazole, oxadixyl, procymidone, tribenuron MCPA-Na and bromoxynil octanoate did not measured the inhibitory activity with the relative concentration greater than 100 %. Distinctively, most of the chemicals for 7 days relative concentrations are greater than or approach for 3 days except bromoxynil octanoate. Besides, the positive control paraquat had the best inhibiting effect compared with other 53 chemicals. Sensitivity analysis of phytoplankton In this study, 0.4 % (v/v) DMSO had no effect the concentration and cell division rates (lc) of M. aeruginosa, C.vulgaris and S. obliquus (Fig. 1) compared with control. Moreover, those cultures were moving into stationary phase by day 7 because lc (i.e. the slopes of the lines joining the points of the attached graph) were lower for all 3 species between day 6 and 7 than between day 0 and day 1. The EC50 value of screened chemicals (chlorothalonil, dimethomorph, SDIC, pyrimethanil, propineb, mancozeb,

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Chlorella vulgaris EC50 (mean ± SD) (mg L-1)

paraquat, terbutryn and mesosulfuron) inhibiting M. aeruginosa, Chlorella and Scenedesmus after 3 and 7 days are shown in Table 2 and 3, respectively. The results indicated nine kinds of chemicals have varying degrees inhibition effect on M. aeruginosa and EC50 less than 10 mg L-1 after 7 days. The paraquat has the highest activity targeting M. aeruginosa than other chemicals. The effects of the nine screened chemicals on Chlorella and Scenedesmus are also shown in Table 2 and 3. Some algae were not sensitive to chemicals and the EC50 value greater than 50 mg L-1 and data were not appeared in the results. Among them five chemicals (SDIC, mancozeb, paraquat, terbutryn and mesosulfuron) obtained the EC50 value of inhibition both Chlorella and Scenedesmus after 3 and 7 days. From the screened results of each treatment group, there are only two groups assay of the 7 days EC50 value greater than 3 days. They are paraquat inhibition of C. vulgaris and mesosulfuron inhibition of S. obliquus. Moreover, a comparative analysis of algae EC50 showed that C. vulgaris and S. obliquus were less sensitive than M. aeruginosa. Toxicity test of goldfish Based on the results of safety tests on goldfish, the LC50 values were calculated by SPSS and the results are shown in Fig. 2. Chlorothalonil, SDIC and Mesosulfuron were discovered had higher toxicity than others. Besides, no fish were dead in control group and some treatment groups of dimethomorph, pyrimethanil, propineb, mancozeb at 32 mg L-1. Bacteriostatic test of B. subtilis The bacteriostasis for B. subtilis of the nine chemicals was assessed on the basis of bacteria growth inhibition rate. The control group of B. subtilis growth surface area (348 ± 75 mm) was deemed to 100 %. And the treatment group growth percentage was shown in Fig. 3. All the

Aquatic environmental safety assessment and inhibition mechanism Table 3 Inhibition activity of ten chemicals on Microcystis aeruginosa, Chlorella vulgaris and Scenedesmus obliquus for 7 days

Microcystis aeruginosa EC50 (mean ± SD) (mg L-1)

Chlorella vulgaris EC50 (mean ± SD) (mg L-1)

SDIC

0.55 ± 0.02a

4.01 ± 0.09c

4.61 ± 0.20b

4.10 ± 0.38

a

b

11.06 ± 0.68c

0.06 ± 0.01

a

b

0.24 ± 0.10a

0.61 ± 0.18

a

b

0.30 ± 0.01a

mesosulfuron

0.73 ± 0.01

a

a

2.02 ± 0.61b

chlorothalonil

0.62 ± 0.01a

paraquat terbutryn

6.42 ± 0.63 5.33 ± 0.77 1.86 ± 0.39 0.82 ± 0.10

4.12 ± 0.17b

24.39 ± 1.75c

dimethomorph pyrimethanil

a

5.23 ± 0.26 7.54 ± 0.24a

b

35.33 ± 1.47 14.31 ± 3.59b

45.56 ± 1.36c 34.05 ± 1.38c

propineb

4.20 ± 0.35a

44.64 ± 1.06c

38.01 ± 2.54b

100%

100%

Chlorothalonil (LC50=0.81 mg L-1)

80%

Mortality

Fig. 2 The toxicity of chemicals to goldfish. X-axis and Y-axis are chemical concentration and goldfish mortality (48 h), resipectively. The chemical names and half lethal concentration (LC50) are presented in each area

Scenedesmus obliquus EC50 (mean ± SD) (mg L-1)

Chemical

mancozeb

Data at the same medication with different letters are significantly different (a \ b \ c, p \ 0.05)

1643

60%

60%

40%

40%

20%

20%

0% 0.3

0.5

Mortality

100%

0.9

1.1

1.3

0% 1.5

80%

60%

60%

40%

40%

20%

20% 17

19

100%

21

23

25

27

29

31

40%

40%

20%

20% 0.3

0.4

0.5

Concentration (mg

chemicals had not significant difference against B. subtilis compared with control group (Fig. 3). Effects on cellular microstructure The M. aeruginosa cell microstructure changes were reflected in Fig. 4a–h. Figure 4a and b showed normal cells were round and plump and had spherical shapes with a clear edge. Cells were aggregation or dispersion in the field of vision. After 3 days of exposure to paraquat,

8

9

L-1)

0.6

0%

2.3

2.5

2.7

2.9

3.1

3.3

10

11

12

13

14

15

Dimethomorph Pyrimethanil Propineb Mancozeb (LC50 32 mg L-1)

80% 60%

0.2

2.1

Terbutryn (LC50=11.78 mg L-1)

0% 7

60%

0% 0.1

1.9

100%

Mesosulfuron (LC50=0.33 mg L-1)

80%

1.7

100%

Paraquat (LC50=23.12 mg L-1)

80%

0% 15

Mortality

0.7

SDIC (LC50=2.43 mg L-1)

80%

0

10

20

30

40

Concentration (mg L-1)

dimethomorph and propineb, some cells were obviously depressed or distorted. The result of paraquat showed obvious change at 3 days (Fig. 4c). To 7 days with exposuring,dead cells were appeared more seriously with rupturing, having holes, leaking intracellular matrix and producing debris (Fig. 4d). The speed of cells apoptosis treated by dimethomorph and propineb has been slowly than paraquat. There were not appeared mass of cells deformation and ruptured until as exposure time extending to 7 days (Fig. 4e–h).

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Percentage of growth (%)

1644 200.00 180.00 160.00 140.00 120.00 100.00 80.00 60.00 40.00 20.00 0.00

X.-B. Yu et al.

*

Fig. 3 The surface area percentage of B. subtilis with different treatments. All treatments compared with control (100 %), where ‘‘*’’ represent significant difference (p \ 0.05)

Of the chemicals tested, we found dimethomorph, propineb and paraquat have significant inhibitory activity on M. aeruginosa at low concentration and safe to aquatic environment. The unicellular cyanobacterium M. aeruginosa stopped growing and remained in an inhibited state for 30 days. Among all the chemicals screened, there was positive correlation between the concentration of the chemical and environmental safety.

Discussion The control of water blooms has become an important issue of environmental water management and aquaculture industry, that many methods cannot give assurance both the control effect and environment problem. This study was screened out 9 chemicals inhibiting M. aeruginosa effectively but among them only dimethomorph, propineb, and paraquat were identified safe to aquatic environment. Interestingly, the chemicals could destroy the cell ultrastructure in different characteristic and speed. Besides, we found that all three phytoplankton cultures were moving into stationary phase by day 7. The growth of phytoplankton may be limited by developed heavy cell concentration but not affect screening results. Considerable attention has been received about screening of biologically active chemicals, especially for environmental safety. Therefore, chemicals including fungicides, algicide and herbicide were selected and assessed for the activity inhibiting M. aeruginosa and assessment the safety of aquatic organisms. M. aeruginosa, C. vulgaris and S. obliquus are growing luxuriantly in pond, rivers, lakes and their tributaries (Nakai et al. 1999; Zhou et al. 2012). Toxic M. aeruginosa is one of the most dominant species in freshwater systems, which caused the deaths of aquatic organism due to consumption dissolved

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oxygen, production toxins (Jensen and Cox 1983; Oh et al. 2000; Vasconcelos and Pereira 2001). With environmental consequences on non–target organisms that are still difficult to assess but surely significant. C. vulgaris and S. obliquus are beneficial algae having the potential to remove heavy metals from freshwater (Zhou et al. 2012) and having other function in aquatic ecosystem (Feuga 2000). In the present study, different species as test organisms have been demonstrated different sensitivity in their response to chemicals, which were consistent with the research of Ma et al. (2002). Simple organism usually has the higher sensitivity than complex organism because the low-level biotransformation and excretion of chemical (Hill 1985; Lumaret et al. 2012). Similarly, chemicals have potential toxicity in aquatic organisms. So it is necessary to assess the safety of chemicals after activity screening inhibit M. aeruginosa. Goldfish, B. subtilis C. vulgaris and S. obliquus are suitable non-target toxicology materials used for such study. This study had identified that nine chemicals had good efficacy than others for simulation of effect on already developed heavy bloom of M. aeruginosa. And among them, only three chemicals showed low toxicity on foregoing toxicology material. At present the compared standards about effective concentration and safe concentration of chemicals is controversial (Abbasi and Soni 1986; Pimentel and Bulkley 1983; Sprague 1971). Currently, the proportional relations of effective concentration and median lethal concentration are usually displayed as standards of chemicals toxicity. We deem it is safety if the acute toxicity test results (LC50 or EC50) greater than triple EC50 of inhibiting M. aeruginosa (Zou et al. 2012). dimethomorph, propineb, and paraquat are safety on Chlorella, Scenedesmus, goldfish and B. subtilis within effective concentration. Moreover, the phenomenon of dose-dependent response was appeared in the entire acute exposure test. Comparing the efficacies of the dimethomorph, propineb, and paraquat, it was observed that the paraquat showed better inhibition effect against M. aeruginosa than others, which is consistent with other literature (Wong 2000). Paraquat is widely used about half century as a weed killer in about 130 countries (Sa´enz et al. 1997) and some literatures reported its inhibition efficacies against alga (Prado et al. 2009). The National Institute of Agricultural Technology (INTA) recommended its use for aquatic weed control with an application rate of 0.1 to 2 mg L-1 of active ingredient in water (Sa´enz et al. 1997). Nevertheless, water ecological environment safety assessment of the paraquat is relatively lacking. In this study, it is demonstrated that the paraquat can be used as the EC50 is far below the concentration recommended by INTA and safe to other aquatic organisms. Moreover, dimethomorph and propineb as fungicides (Lo et al. 1996; Stein and Kirk

Aquatic environmental safety assessment and inhibition mechanism

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Fig. 4 SEM images of M. aeruginosa treated by twice of EC50. a, b Control; c, d paraquat; e, f dimethomorph and g, h propineb after (3–7 days), respectively

2004) were discovered having the inhibition effect on M. aeruginosa and being safe to the environment. Chemicals are now being used in aquaculture, but fewer can be used in freshwater system. The main factors usually considered when selecting harmful bloom control methods

are effectiveness and speed of action (Yi et al. 2012). This study confirms some chemicals can inhibit cyanobacteria within 7 days or even shorter and have no toxic side effects, also are economical and faster compared with others. Nevertheless, chemical residue is an inevitable problem attracted

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much attention for a long time and many chemicals are strictly prohibited from use in aquaculture all over the world (Gorbach 2001). Although this study obtained some potential safe chemicals inhibiting cyanobacteria, we should strictly follow control measures and standards to reduce chemical residues in practical application. It was pointed that deep understandings of the inhibition were important. Hence, we conducted additional studies to further explore the inhibition mechanisms of chemicals on M. aeruginosa. By SEM, we observed the significant difference of cell morphology between paraquat, dimethomorph and propineb, although previous studies had showed the morphology distinction of many cells at a specific time point. The cellular membrane damage and substantial release caused by chemicals would no doubt result in disintegration of cells since there was no place for the normal physiological metabolism, and therefore, may act as one of algal inhibiting mechanisms (Zhang et al. 2013). Additionally, the speed of cell apoptosis and substantial release can be affected by different chemical and dosage, which is consistent with toxicity data. Moreover, this study screened three chemicals for targeting M. aeruginosa and low toxicity to other organisms added the process and mechanism of M. aeruginosa apoptosis.

Conclusion In this research 9 chemicals were found inhibiting M. aeruginosa effectively, and among them dimethomorph, propineb, and paraquat were identified safe to aquatic environment. For the potential inhibition mechanism, the chemicals could destroy cell ultrastructure in different characteristic. The sensitivity of three chemicals to the M. aeruginosa ranked as paraquat [ propineb [ dimethomorph. In conclusion, it will provide water mangers with a wider choice of ecologically appropriate compounds to control blooms of toxic M. aeruginosa. Besides, the issue of toxicity of breakdown products is also important in aquatic environment, and thereby, a new work about the environmental toxicity of toxins is needed to fill those knowledge gaps. Acknowledgments This research was supported by the China Spark Program (2012GA651002). We would like to thanks the anonymous referees for their writing assistance on this paper. Conflict of interest of interest.

The authors declare that they have no conflict

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