Pesticide Biochemistry and Physiology 105 (2013) 224–230

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Pesticide Biochemistry and Physiology

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Butachlor induces some physiological and biochemical changes in a rice field biofertilizer cyanobacterium ⇑ Hongzhi He a, Yongjun Li b, Tianfeng Chen c, , Xiaolong Huang a, Qiu Guo a, Shufeng Li a, ⇑ Tianhong Yu a, Huashou Li a, a Key Laboratory of Agro-Environment in the Tropics, Ministry of Agriculture of the People’s Republic of China, South China Agricultural University, Guangzhou 510642, China b Zhongshan Quality Supervision & Inspection Institute of Agricultural Products, Zhongshan 528403, China c Department of Chemistry, Jinan University, Guangzhou 510632, China article info abstract

Article history: Butachlor has been widely applied in rice field in China. However, concerns are also raised about its Received 18 September 2012 potential adverse impacts on non-target organisms. In the present study, butachlor was found be able Accepted 25 February 2013 to induce toxic effects on a rice field biofertilizer cyanobacterium Nostoc sp. When treated with 80 mg L1 Available online 7 March 2013 butachlor, significant decline in the growth rate, concentrations of chlorophyll a (Chla), carotenoids

(Cars), phycobiliproteins (PBPs) and; the minimal fluorescence yield (F0), fluorescence intensity at the Keywords: J-step of OJIP (Fj), the maximum fluorescence yield (Fm), the potential quantum yield (Qy), the quantum Nostoc sp. yield of electron transport (UE ), the maximum quantum yield of primary photochemistry (UP ), and the Butachlor 0 0 performance index on absorption basis (PI ) (14.2%, 39.5%, 55.5%, 34.8%, 38.5%, 19.8%, 18.7%, 20.4%, Toxicity ABS Antioxidant system 10.1%, 10.3%, and 26.4%, respectively) was observed in Nostoc sp. In contrast, significant increase in Fluorescence Cars/Chla, PBPs/Chla, the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and, glutathione reductase (GR), content of malonaldehyde (MDA), the absorption flux per reaction center

(ABS/RC) and the effective dissipation per reaction center (DI0/RC) for 0.45, 0.65, 2.36, 2.47, 1.08, 1.16, 0.87, 0.122 and 0.205 fold was also detected by comparing with the control group. These results demon- strated that high concentration of butachlor could inhibit the growth, synthesis of pigments, and photo- system II (PSII) activities of Nostoc sp., and trigger dramatic intracellular antioxidant response in the cells. Taken together, this study may provide important information on the understanding of the changes induced by butachlor stress in nitrogen-fixing cyanobacteria and the adaptive strategy of the alga. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction acted by inhibiting the elongase responsible for the elongation of very long-chain fatty acids and geranylgeranyl pyrophosphate cyc- Chloroacetanilide herbicides (e.g. acetochlor, metolachlor and lisation enzymes [3]. As a consequence of the use in large quantity, butachlor) are important chemicals used in agriculture all over contamination of butachlor is now very common in groundwater the world. They were commonly used to control annual grasses and surface-water in China [4], and result in disruption of the envi- and certain broad-leaved weeds in both seeded and transplanted ronment and ecosystems [5]. rice [1]. In China, N-(butoxymethyl)-2-chloro-N-(2,6-diethylphe- Nitrogen is one of the most important factors that limit the rice nyl) acetamide (butachlor) is now one of the three most widely production. Nowadays, chemical N fertilizers are widely applied in used herbicides, with an annual yield of 1 104 t [2]. Butachlor rice field to promote rice yield in China [6]. However, because of

Abbreviations: Chla, chlorophyll a; Cars, carotenoids; OD, optical density; DW, dry weight; PBPs, phycobiliproteins; PC, phycocyanin; APC, allophycocyanin; PE, phycoerythrin; SOD, superoxide dismutase; CAT, catalase; POD, peroxidase; GR, glutathione reductase; MDA, malonaldehyde; ANOVA, analysis of variance; DCMU, 3-(3,4- dichlorophenyl)- 1,1-dimethylurea; 2,4-D, 2,4-dichlorophynoxyacetic acid; ROS, reactive oxygen species; GSH, glutathione; PSII, photosystem II; Ft, the instantaneous Chla

fluorescence; Fj, Fluorescence intensity at the J-step of OJIP; Qy, the potential quantum yield; M0, approximated initial slope of the fluorescence transient; VJ, relative variable

fluorescence at the J-step; UE0, the quantum yield of electron transport; UP0, the maximum quantum yield of primary photochemistry; W0, the probability that a trapped exciton moves an electron further than QA ; ABS/RC, the absorption flux per reaction center; TR0/RC, the trapping flux per reaction center; ET0/RC, the electron transport flux per reaction center; DI0/RC, the effective dissipation per reaction center; PIABS, the performance index on absorption basis. ⇑ Corresponding authors. Address: Department of Chemistry, Jinan University, Guangzhou 510632, China, Fax: +86-020-85220223 (T. Chen), College of Agriculture, South China Agricultural University, Guangzhou 510642, China, Fax: +86-020-85280211 (H. Li). E-mail addresses: [email protected] (T. Chen), [email protected] (H. Li).

0048-3575/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pestbp.2013.02.009 H. He et al. / Pesticide Biochemistry and Physiology 105 (2013) 224–230 225 the large requirement of fossil-fuel energy for the production of 2.3. Dry weight (DW), growth curves and specific growth rate these fertilizers, the prices of them become higher and higher. At measurement the same time, chemical N fertilizers could post many adverse ef- fects on the environment. Therefore, it is very important to survey For DW determination, 15 mL algal culture were filtered and develop alternate low-cost, assimilable nitrogen sources [7]. through a pre-dried Whatman GF/C paper, washed three times The natural nitrogen resources exhibit unique properties of biolog- with culture media, and the filters containing cells were dried at ical nitrogen fixation for agricultural use. Nitrogen-fixing cyano- 80 °C until constant weight and then weighted. A significant posi- bacteria are abundantly present in the rice field of south China tive relationship between DW and the optical density at 680 nm and are important microbes in maintenance of rice field fertility (OD680) detected by a Shimadzu UV-250IPC spectrophotometer 1 through carbon and nitrogen fixation [8]. Nowadays, microalgal showed [DW (mg L )=1.94 + 231 ⁄ OD680, r = 0.9998, biofertilizers have been proved to be a useful tool for increasing p < 0.0001] according to the results of preliminary experiment. the productivity of rice production and improving physical and Therefore, the growth was measured at 680 nm at 48-h interval chemical properties of the soil in agricultural system [9]. However, using Shimadzu UV-250IPC spectrophotometer for 12 d. During the large scale use of herbicides in modern rice agriculture has af- the exponential growth phase, the specific growth rate (l,d1) fected cyanobacteria adversely in various ways [10]. could be calculated using the equation: Till now, many studies have been conducted to assess the tox-  1 N icity of butachlor on the growth, photosynthesis and physiology l ¼ ln j ð1Þ j i N of nitrogen-fixing cyanobacteria [11–17]. However, little attention i 1 has been paid to the antioxidant system and Chla fluorescence where Ni and Nj represented DW (mg L ) at day i and day j, changes in nitrogen-fixing cyanobacteria under butachlor stress respectively. conditions. Therefore, the aim of this study was to investigate the toxic effects of butachlor on the growth, PSII activities, bio- 2.4. Determination of Chla and Cars chemical composition and antioxidant system of a nitrogen-fixing cyanobacterium Nostoc sp. FACHB-85 isolated from paddy fields in Twenty mL algal culture were filtered through 0.45 lm filters South China. and the filter membrane was transported to a 10 mL plastic tube and extracted with 6 mL ice-chilled 80% acetone for 24 h in dark at 2. Materials and methods 4 °C. The solution was then centrifuged at 5000 rpm for 10 min to re- move the cell debris, and the supernatant was collected for determi- 2.1. Chemical, organism and instrument nation of Chla and Cars according to the formulas of Lichtenthaler and Wellburn [19]. The formulas were showed below:

The tested herbicide N-(butoxymethyl)-2-chloro-N-(2,6-dieth- CChla ¼ 12:21 OD663 2:81 OD646 ð2Þ ylphenyl) acetamide (butachlor, 99.7%) was purchased from

Shanghai Pesticides Research Institute (China). Heterocystous cya- CChlb ¼ 20:13 OD646 5:03 OD663 ð3Þ nobacterium Nostoc sp. (named as FACHB-85) was originally iso- lated from rice field in Hubei Province and obtained from the 1000 OD 3:27 C 104 C C ¼ 470 Chla Chlb ð4Þ Freshwater Algae Culture Collection of the Institute of Hydrobiol- Cars 229 ogy, the Chinese Academy of Sciences. Stock culture of the alga Where OD ,OD , and OD represented absorbance at 470, 464, was maintained at 27 °C in sterilized N-free mineral medium 470 646 663 and 663 nm, respectively. BG11 [18] at pH 7.5 with 3000 lx light intensity provided by cool- white fluorescent lamps and a 16 h/8 h light/dark cycle. A Shima- dzu UV-250IPC spectrophotometer was employed for absorbance 2.5. Determination of PBPs determination. For determination of PBPs, samples obtained by filtering 20 mL algal culture were repeatedly frozen at 40 °C and thawed at 4 °C 2.2. Butachlor treatment for 6 times in 0.05 mol L1 phosphate buffer (mixing equal vol- 1 1 umes of 0.1 mol L KH2PO4 and 0.1 mol L K2HPO4 solutions, Stock solution of butachlor was prepared with analytical grade pH 7.1). And then the solution was ultrasounded for 120 s on a acetone. Firstly, preliminary experiments were conducted to exam- sonicator (YJ92-II, Yong-Jie Experimental Laboratory Apparatus ine the effects of the solvent acetone alone on the cyanobacterium Corporation, Ning Bo, China) equipped with a probe operating at and the results showed that 0.6% (v/v) acetone had no significant 80% in 5-s pulses) in ice-water bath for 10 min. The homogenized (p > 0.05) effects on the growth and Chla content of Nostoc sp. solutions were centrifuged at 18,000g at 4 °C for 30 min, and then The experiments were conducted in 500 mL Erlenmeyer flasks con- the absorbance of the supernatant was determined using Shima- taining 200 mL BG110 medium. All flasks containing medium were dzu UV-250IPC spectrophotometer. The contents of three PBPs autoclaved at 121 °C for 20 min and cooled down to room temper- including PC, APC and PE were calculated according to Bennet ature before use. Twenty mL stock algal culture (OD = 0.943, and Bogorad [20]. The formulas were showed below: DW = 4.32 mg) from mid-exponential growth phase was inocu- OD 0:474 OD lated aseptically into the flasks. All controls and concentrations ½PC¼ 615 652 ð5Þ were in three replicates. Based on the results of preliminary exper- 5:34 iments, the appropriate amounts of the freshly prepared herbicide OD 0:208 OD stock solutions were added to each culture to obtain final concen- ½APC¼ 652 615 ð6Þ trations of 0 (control), 20, 40 and 80 mg L1 and appropriate 5:09 amounts of acetone were also added to obtain a final concentration OD 2:41 ½PC0:849 ½APC of 0.6% (v/v). The culture was incubated under the same irradiance ½PE¼ 562 ð7Þ and temperature conditions as described above. All flasks were 9:62 shaken manually six times a day. The duration of the experiments Where OD562,OD615, and OD652 represented absorbance at 562 nm, was 12 days. 615 nm, and 652 nm, respectively. 226 H. He et al. / Pesticide Biochemistry and Physiology 105 (2013) 224–230

2.6. Enzyme assays measured from 50 ls to 1 s. Each transient was analyzed using

the data including F50 ls,F300 ls,Fj,Fi and Fm. To visualize the effect The activities of SOD (EC 1.15.1.1) and CAT (EC 1.11.1.6) were of butachlor on each step of the fluorescence transient, the curves determined according to Kumar et al. [21] with slight modification. were plotted as the variable fluorescence at any time on a logarith- Forty mL algal culture was filtered through 0.45 lm filter and the mic time scale. From these data, a suite of variables including the

filter membrane was stored at 40 °C until use. The filter mem- quantum yield of electron transport (UE0), the maximum quantum brane was ground to powder with quartz sand in liquid nitrogen yield of primary photochemistry (UP0), relative variable fluores- 1 on ice and was homogenized in 0.5 mol L phosphate buffer (pH cence at the J-step (VJ), the probability that a trapped exciton 7.8, containing 1% polyvinyl pyrrolidone). The homogenate were moves an electron further than QA (W0), the absorption flux per centrifuged at 12,000g for 20 min at 4 °C, and the supernatants reaction center (ABS/RC), the trapping flux per reaction center were used for enzyme activity assays. The supernatant was used (TR0/RC), the electron transport flux per reaction center (ET0/RC), for the enzyme assays. SOD activity was assayed using a reaction the effective dissipation per reaction center per reaction center 1 1 mixture consisting of 1 mol L Na2CO3, 200 mmol L methionine, (DI0/RC), and the performance index on absorption basis (PIABS) 2.25 mmol L1 nitroblue tetrazolium chloride, 3 mmol L1 ethyl- were calculated [26]. All results were expressed as a percentage enediaminetetraacetic acid disodium, 60 lmol L1 riboflavin and of controls (%). 0.1 mol L1 phosphate buffer (pH 7.8). Absorbance was read at 560 nm. For CAT activity assay, 100 lL of enzyme extract, 1.6 mL 2.9. Statistical analysis 1 phosphate buffer, 0.2 mL 0.3% H2O2 and 3 mmol L EDTA was added in a test tube. The reaction was allowed to run for 3 min. En- The mean and standard error of the mean were calculated for zyme activity was calculated by using extinction coefficient 0.036 each treatment from three independent replicate cultures. To and was expressed in enzyme (unit/mg wet weight). One unit of determine the significant differences between the butachlor trea- enzyme was the amount necessary to decompose 1 lLofH2O2 ted groups and control groups, the data were statistically analyzed per minute at 25 °C. The absorbance of the supernatant was de- with one-way ANOVA using SPSS-13 statistical package (SPSS Inc., tected at 240 nm against blank. Chicago, IL, USA). When the probability (p) was less than 0.01 or POD (EC 1.11.1.7) activity was determined according to Wang 0.05, the values were considered very significantly or significantly et al. [22] by measuring the rate of increase in absorbance at different, respectively. And the results of one-way ANOVA tests 470 nm of a mixture containing 1 mL of 50 mM sodium phosphate indicated ⁄: P < 0.05 and ⁄⁄: P < 0.01. buffer (pH 7.0), 0.95 mL of 0.2% 2-methoxyphenol, 1 mL of 0.2% hydrogen peroxide and 0.05 mL of enzyme extract or distilled water as negative control (total reaction volume 3 mL). GR (EC 3. Results 1.6.4.2) activity was determined according to Carlberg and Man- nervik [23] by measuring the NADPH oxidation via the decrease 3.1. Effects of butachlor on the growth of Nostoc sp. in its absorbance at 340 nm of a mixture containing 50 mM K- phosphate buffer (pH 7.8), 2 mM EDTA, 0.1 mM NADPH, and As shown in Fig. 1, the growth of the alga was significantly 1 0.4 mM GSSG at 25 °C. GR activity was expressed as lM oxidized inhibited when treated with 40 and 80 mg L butachlor for 12 d. NADPH min1 mg1 protein by using its extinction coefficient The growth rates decreased dramatically with increase of buta- (e = 6.2 mM1 cm1). chlor concentrations. For instance, the growth rate of the alga trea- ted with 80 mg L1 butachlor was 0.189 d1 (n =5, r2 = 0.941), 1 2.7. MDA determination which was about 72.7% of the control group (0.260 d , n =5, r2 = 0.992). The lipid peroxidation level was determined in terms of MDA content using the method of Heath and Packer [24] with slight 3.2. Effects of butachlor on the photosynthetic pigments of Nostoc sp. modification. Forty mL culture suspension was harvested by filter- ing through a 0.45 lm filter and washed twice with 50 mM phos- Fig. 2 demonstrates the effects of butachlor on the contents of phate buffer (pH 7.0). Cells collected were homogenized in 5% (w/ Chla, Cars and the ratio of Cars/Chla in FACHB-85. With the in- v) TCA. The resulting mixture was centrifuged at 10,000g for crease of butachlor concentration, the contents of Chla and Cars 10 min. To 0.5 ml the supernatant, 2 ml of 20% TCA containing declined gradually, while Cars/Chla ratios ascended. When treated 0.5% (w/v) TBA was added. The mixture was heated at 90 °C for with 40 and 80 mg L1 butachlor, the Chla contents of FACHB-85 20 min and then quickly cooled in ice bath followed by centrifuga- tion. Absorbance of the supernatant was read at 532 and 600 nm. The value for non-specific absorption of each sample at 600 nm CK was subtracted from absorption recorded at 532 nm. The MDA 300 20 mg L-1 concentration was calculated using the extinction coefficient -1 1 1 40 mg L 155 mM cm . ) -1 80 mg L-1 200 mg L

2.8. Fast Chla fluorescence induction and JIP-test (

The instantaneous Chla fluorescence (Ft), potential quantum yield (Qy, in a dark-adapted sample it is equivalent to Fv/Fm)of 100 photosystem II (PSII) and rapid and polyphasic Chla fluorescence Dry weight emission (JIP-test) were measured using a hand-held fluorometer FluorPen-C AP-C 100 (Photo Systems Instruments, Czech Republic) 0 according to Strasser et al. [25]. The OJIP curves enable observing 024681012 major changes that occur during exposure of a sample to high irra- Time (days) diance. All samples were dark-adapted for 15 min before measure- ment. At room temperature, the fast fluorescence kinetics was Fig. 1. Effects of butachlor on the growth of Nostoc sp. H. He et al. / Pesticide Biochemistry and Physiology 105 (2013) 224–230 227

16 ** 0.08 25 2.5 Chla SOD ** ** Cars CAT DW) Cars/Chla ratios 20 POD 2.0 protein) ** protein) -1 -1 12 * 0.06 GR ** -1 mg

MDA -1 (mg g protein) 15 1.5 a -1 ratios mg 8 * 0.04 a * -1 10 1.0 ** * Cars/Chl 4 0.02 (unit min 5 * * ** 0.5 * * Activity of SOD or CAT POD *

* * or content of MDA (mM mg Activity of GR (mM min Contents of Cars or Chl 0 0.0 0 0.00 0204080 0204080 Concentration of butachlor (mg L-1) Concentration of butachlor (mg L-1) Fig. 4. Effects of butachlor on the activities of dismutase (SOD), catalase (CAT) Fig. 2. Effects of butachlor on the contents of Chlorophyll a (Chla) and carotenoids peroxidase (POD) and glutathione reductase (GR), and the content of malonalde- (Cars), and the ratios of Cars/Chla in Nostoc sp. Significant differences (p < 0.05) and hyde (MDA) in Nostoc sp. very significant differences (p < 0.01) between the test groups and the control group were indicated by ⁄ and ⁄⁄ respectively and the same below.

1 were found at 7.81 and 5.48 mg g1 DW, respectively, which were the quantum yield (Qy). When treated with 20, 40 and 80 mg L 63.5% and 44.6% of the control (12.3 mg g1 DW), while the con- butachlor, significant decline (p < 0.05) of Qy (8%, 13.5%, and tents of Cars were 69.2% and 65.2% of the control group. Moreover, 20.4% respectively) was detected by compared with the control the Cars/Chla ratios in the cells exposed to 40 and 80 mg L1 buta- group. Fluorescence emission kinetics was affected in the alga ex- chlor conditions were 0.227 and 0.306, which were much higher posed to butachlor as shown in Fig. 5. Chla fluorescence transient than that of control groups (0.209) (Fig. 2). of all samples exhibited a characteristic O–J–I–P shape composed As shown in Fig. 3, the PBPs contents of FACHB-85 were also re- of four clear transients. And visible changes in the fluorescence 1 duced with the increase of butachlor concentrations. When treated curves occurred when exposed to 20, 40 and 80 mg L of buta- with 80 mg L1 butachlor, the contents of PBPs were significantly chlor comparing to the control groups (Fig. 5). There was a contin- (p < 0.05) lower than that of control groups, and the PBPs/Chla ratio uous decrease in Fj and Fm with increase in the butachlor was 1.65-fold of the control groups. The treatment of butachlor concentration (Table 1 and Fig. 5). The decrease in Fm suggested also resulted in decrease of PC, APC, PE and total PBPs for 21.3%, an increase of closed PSII reaction centers, which do not participate 31.7%, 37.4% and 26.7%, respectively. in electron transport. It indicated that butachlor caused inhibition of the photochemical activity in FACHB-85. Values of some other fluorescence parameters resulting from 3.3. Antioxidant response of Nostoc sp. to butachlor the shape changes of the O–J–I–P curves have been analysed and the results obtained are presented in Table 1. Different parameters Exposure of the alga to 80 mg L1 butachlor significantly in- bring information about the conditions of the structure, the confor- creased the endogenous activities of SOD, CAT, POD, and GR for mation, and the function of the overall PSII. In order to further dem- 2.36-, 2.47-, 1.08- and 1.16-fold, by comparing with the control onstrate the effect of butachlor on PSII, some functional parameters group (Fig. 4). Moreover, the MDA content of FACHB-85 also in- were used to quantify the PSII behavior and its activity. As shown in creased from 0.257 mM mg1 protein (control group) to 0.385 Table 1 and Fig. 5, the yield of electron transport (UE ), which is the and 0.479 mM mg1 protein, respectively (Fig. 4). 0 product of the yield of primary electron transport (UP0) and the yield of electron transport per trapped exciton (W0), was decreased by 3.4. Effects of butachlor on the fast Chla fluorescence of Nostoc sp. 10.1%. As the value of W0 have changed little, change of UE0 were due to a decrease of UP0 (10.3%). These results indicate that buta- As shown in Table 1, treatment of the algal cells with butachlor chlor does not inhibit the redox reaction after Q but the primary 1 A (20, 40 and 80 mg L ) resulted in a dose-dependent decrease in light reaction. Butachlor at 80 mg L1 also induced increase in 80 16 ABS/RC and DI0/RC compared to the control whereas TR0/RC and PC ET0/RC showed no significant change. These changes result in a de- APC crease in the performance indexes P which is the overall expres- PE IABS sion of all the processes in the energy cascade from energy 60 PBPs/Chla * 12

DW) absorption to the reduction of the intersystem electron transport -1 * chain and the PSI end electron acceptors [25]. It showed that the

ratios activity of PSII of FACHB-85 was suppressed by high-level butachlor. 40 * * 8 a

* * PBPs/Chl 4. Discussion 20 4 * Content of PBPs (mg g 4.1. Effects of butachlor on the growth of Nostoc sp.

0 0 In China, there are growing concerns on the ecotoxicological ef- 0204080 -1 fects of herbicides. As important primary producers in aquatic and Concentration of butachlor (mg L ) agricultural ecosystems, nitrogen-fixing cyanobacteria are Fig. 3. Effects of butachlor on the contents of phycocyanin (PC), allophycocyanin vulnerable non-target species. Many previous studies have (APC) and phycoerythrin (PE), and the ratios of PBPs/Chla in Nostoc sp. evaluated the toxic effects of various herbicides on nitrogen-fixing 228 H. He et al. / Pesticide Biochemistry and Physiology 105 (2013) 224–230

Table 1 Some Chla fluorescence parameters of Nostoc sp. cells treated with different concentration of butachlor.

a b c d e f g h i j k l m n o Butachlor F0 Fj Fm Area Qy M0 VJ UE0 UP0 W0 ABS/RC TR0/RC ET0/RC DI0/RC PIABS concentration (mg L1) 0 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 20 100.8 95.8⁄ 92.7⁄ 106.7 92.0⁄ 104.3 103.5 82.9⁄ 87.6⁄ 94.2 115.5⁄ 101.0 95.3 126.0⁄ 62.7⁄ 40 91.9 88.3⁄ 86.8⁄ 87.0⁄ 86.5⁄ 103.5 101.4 91.1⁄ 93.1 97.6 109.8⁄ 102.1 99.7 115.3⁄ 77.3⁄ 80 85.8⁄ 81.7⁄ 80.2⁄ 72.2⁄ 79.6⁄ 100.8 100.2 89.9⁄ 89.7⁄ 99.7 112.2⁄ 100.7 100.5 120.5⁄ 73.6⁄ a, initial Chla fluorescence; b, fluorescence intensity at the J-step (2 ms) of OJIP; c, maximal fluorescence intensity; d, area between fluorescence curve and FM; e, quantum yield, in a dark-adapted sample this is equivalent to Fv/Fm; f, approximation of the slope at the origin of fluorescence rise; g, VJ =(FJ F0)/(Fm F0); h, UE0 =Fv/Fm (1 VJ); i,

UP0 =Fv/Fm;j,W0 =1 VJ; k, ABS/RC = M0 (1/VJ) (1/UP0); l, TR0/RC = M0 (1/VJ); m, ET0/RC = M0 (1/VJ) W0;n,DI0/RC = (ABS/RC) (TR0/RC); o, PIABS = (RC/

ABS) [UP0/(1 UP0)] W0/VJ. Note: all parameters were normalized with controls. Significant differences at p < 0.05 between control and treated cultures are marked by asterisks.

9 nitrogen-fixing cyanobacteria, such as molinate on A. cylindrical CK [10], molinate on A. sphaerica [27], thiobencarb on N. sphaeroides -1 20 mg L [28], oxyfluorfen on N. muscorum and Phormidium foveolarum 8 -1 40 mg L [29], and 2,4-D on A. fertilissima [32]. -1 80 mg L Significant decrease in Cars contents of some nitrogen-fixing 7 cyanobacteria induced by herbicides has also been documented [29,32]. For instance, Galhano et al. [10] addressed that Cars in A. cylindrical cells were significantly depressed by molinate in a time- 6 and dose-dependent manner. However, it was slightly but significantly stimulated by bentazon after 72-h treatment. To our knowledge, no information about the change of Cars contents in Relative variable fluorescence 5 nitrogen-fixing cyanobacteria in response to butachlor is available. In the present study, it was shown that the Cars contents declined

-5 -4 -3 -2 -1 0 slightly with the increase of butachlor concentrations. Cars are 10 10 10 10 10 10 important accessory pigments in microalgal cells. In photosynthetic Time (s) organisms, they play a vital role in the photosynthetic reaction cen- Fig. 5. The fast fluorescence kinetics of Nostoc sp. treated with different concen- tre and they either participate in the energy-transfer process, or tration of butachlor. protect the reaction center from auto-oxidation [33]. Moreover, we found that the Cars/Chla ratios increased significantly (p < 0.05) when treated with 40 and 80 mg L1 butachlor by com- cyanobacteria [10,27–29] and the results demonstrated that the paring with the control group (Fig. 2). Similar changes were also in- sensitivity of cyanobacteria towards herbicides was depended on duced with some other herbicides on cyanobacteria documented by the species and kinds of the herbicides. Attention has also been Galhano et al. [10], Sheeba et al. [29], and Kumar et al. [32]. We cal- paid to the toxicity of butachlor toward nitrogen-fixing cyanobac- culated the Cars/Chla ratios according to the data in these refer- teria. The results showed that butachlor exhibited moderate or ences, whereas the contents of Chla and Cars were provided high toxicity to some cyanobacteria, such as N. muscorum [11], individually. The results showed that the Cars/Chla ratios increased Anacystis nidulans, N. muscorum and Anabaena doliolum (com- after treated with herbicides, such as bentazon and molinate on A. pletely inhibited at 2.5, 5 and 20 mg L1, respectively) [12], N. linc- cylindrical [10], oxyfluorfen on N. muscorum and P. foveolarum kia, N. calcicola, Nostoc sp., and A. doliolum (lethal doses 6– [29], and 2,4-D on A. fertilissima [32]. Taken together, these results 8mgL1) [13]. However, butachlor also showed slight toxicity to suggest that the inhibitory effects of herbicides on the Chla of nitro- some other cyanobacteria, such as Nostoc [14], Nostoc sp., N. punc- gen-fixing cyanobacteria are more serious than those on Cars. tiforme, N. calcicola, A. variabilis, Gleocapsa, Aphanocapsa and A. fer- PBPs are antenna pigments in photosynthetic system of cyano- tilissima (6 d IGC values between 9.7 and 15 mg L–1) [15], Ge– 50 bacteria cells, and they act by capturing light energy and then pass- Xian–Mi (Nostoc) (96 h-EC = 169 lM) [16], Aulosira fertilissima 50 ing it to chlorophylls during photosynthesis [34]. Changes in PBPs (16 d-EC =65lM) [17]. In the present study, butachlor also 50 contents of nitrogen-fixing cyanobacteria induced by butachlor showed slight toxicity to FACHB-85 (the lethal concentration more have been observed in some studies. For instance, Singh and Datta than 120 mg L1). The variation in the survival potential and lethal [31] registered that, the contents of PC and PE in six nitrogen-fixing dosages of herbicides on different strains suggest the presence of cyanobacteria declined significantly when treated with sublethal different degrees of inherent natural tolerance among strains from dosages of four herbicides including butachlor. Chen et al. [16] diverse ecosystems [30]. showed that the content of PC and APC significantly increased when Ge–Xian–Mi colonies were treated with 10 lM butachlor, 4.2. Effects of butachlor on the photosynthetic pigments of Nostoc sp. but declined with increasing butachlor concentration. Kumari et al. [17] also addressed a dose-dependent rise in PE, APC and Decline of photosynthetic pigments is a common response of PC of A. fertilissima cells. In the present study, our results also sup- cyanobactria to various herbicides. In the present study, we dem- port this conclusion (Fig. 3). Moreover, some other herbicides also onstrated a significant decline in Chla contents of FACHB-85 ex- induced similar changes in nitrogen-fixing cyanobacteria, such as posed to butachlor. Similar changes were also found in other molinate on A. cylindrical [10], molinate on A. sphaerica under dif- nitrogen-fixing cyanobacteria, such as Ge–Xian–Mi (Nostoc) [16], ferent light intensities after 9-d treatments [27], thiobencarb on A. fertilissima [17], Nostoc sp., N. punctiforme, N. calcicola, A. variabi- N. sphaeroides [28], oxyfluorfen on N. muscorum and P. foveolarum lis, Gleocapsa, Aphanocapsa, and N. muscorum [31] under butachlor [29], and 2,4-D on A. fertilissima [32]. PBPs are important reserves stress. Some other herbicides also induced similar response in for nitrogen in cyanobacteria. Singh and Datta [31] considered that H. He et al. / Pesticide Biochemistry and Physiology 105 (2013) 224–230 229 the decline in PBPs suggested that under herbicide stress, there by molinate in a time- and concentration-dependent manner after was a diversion to meet the nitrogen demand, possibly through 72-h treatment. The results obtained in the present study were the induction of proteolytic enzymes. However, Galhano et al. similar to Sheeba et al. [29]. [10] addressed that PBPs contents of A. cylindrical increased with the increase in bentazon concentrations after treatments for 24 h 4.4. Effects of butachlor on the PSII activity and 48 h. In addition, the PBPs/Chla ratios of cyanobacteria com- monly increased when treated with herbicides, such as dimethoate The potential quantum yield (Qy) of electron transfer through on Ge–Xian–Mi (Nostoc) [16]. In the present study, we demon- PSII is a reflection of photosynthetic efficiency. Under physiological strated similar results (Fig. 3). However, Chen et al. [16] docu- stress, this parameter in plant is typically expressed as the ratio of mented that the PBPs/Chla ratios did not show any significant Fv/Fm. In the present study, the quantum yield of electron transfer difference compared to the control group for butachlor and bensul- through photosystem II (PSII) in FACHB-85 cells declined signifi- furon-methyl. Moreover, Sheeba et al. [29] addressed that the cantly induced by 40 and 80 mg L1 butachlor (Table 1). Measure- PBPs/Chla ratios of N. muscorum and P. foveolarum declined with ment of Chla fluorescence induction curve provides a powerful tool the increase of oxyfluorfen concentrations. for the detection of toxic factors on photosynthesis of phytoplank- ton precisely [40]. PSII, as a highly susceptible site to several types 4.3. Effects of butachlor on the intracellular antioxidant system of stresses, its status under various environmental stresses can be estimated by analyzing the fluorescence transients (O–J–I–P) Studies have showed that some cyanobacteria have developed according to the JIP-test [25,26,40,41] which has been developed an efficient antioxidant system to protect them from oxidative to characterize the fluorescence rise kinetics based on the theory stress, including antioxidants (such as carotenoids, ascorbate, of energy fluxes in the membranes in the photosynthetic apparatus GSH, praline and peroxiredoxin) and antioxidant enzymes (such [25]. The results in the present study showed that the value of W0 as SOD, CAT, POD, GR and ascorbate peroxidase) [17,21,35,36]. have changed little, decrease of UE0 were due to a decrease of UP0 The coordination of enzymatic and nonenzymatic antioxidants and it indicated that butachlor does not inhibit the redox reaction plays critical roles in scavenging intracellular ROS and maintain- after QA but the primary light reaction. The adverse effects of buta- ing the physiological redox status of the cyanobacteria [37,38]. chlor on the PSII reaction center activity was also confirmed by the However, nowadays, only a few reports about the alteration of performance index PIABS which can be used as overall measures of antioxidant system in nitrogen-fixing cyanobacteria in response the potential of the plants for energy conversion [25,26]. In the to herbicides are available [29,38,39]. For instance, Galhano present study, a significant decrease showed for PIABS in the pres- et al. [38] documented that the activities of the antioxidant en- ent study when treated butachlor. Our results suggested that buta- zymes showed significant increase upon treatment of bentazon chlor may increase ABS/RC and DI0/RC compared to the control in a time- and dose- dependent manner in N. muscorum cells. whereas TR0/RC and ET0/RC showed no significant change. These Sheeba et al. [29] showed that in N. muscorum cells, the activities changes resulted in a significant decrease in the performance in- of SOD and CAT increased when treated with 10 mg L1 of oxyflu- dexes PIABS. Similar to our results, Pan et al. [41] documented that orfen, while decrease was observed when treated with 20 mg L1 an increase of amoxicillin concentration results in decrease of UE0, of oxyfluorfen. However, in case of P. foveolarum, the activities W0, UP0,ET0/RC, PIABS, and increase in ABS/RC and DI0/RC in cyano- SOD, CAT and POD increased with oxyfluorfen. These results sug- bacterium Synechocystis sp. Jena et al. [42] also recorded that four gested that P. foveolarum was better equipped with enzymatic organophosphorus insecticides induced decrease in UE0, W0, UP0, antioxidants to counteract the adverse effects of oxyfluorfen- PIABS and increase in ABS/RC and DI0/RC in Chlorella vulgaris. It sug- induced ROS and thus had greater tolerance by comparing with gested that, in order to fulfill the energy requirement of photosyn- N. muscorum. In the present study, our results also showed that thesis under stress conditions, the photosynthetic reaction center the activities of SOD, CAT, POD and GR of FACHB-85 increased of these microalgal cells absorbed more energy to offset higher en- significantly by comparing with control group, when treated with ergy dissipation. However, Petit et al. [26] addressed that 1 mg L1 40 and 80 mg L1 butachlor (Fig. 4). In contrast, Galhano et al. of G2 poly(amidoamine) dendrimer and 2.5 mg L1 of G4 [39] demonstrated contrary results that the activities of SOD, poly(amidoamine) dendrimer induced significant increase in UE0, CAT, APX, GR, and GST in N. muscorum cells showed a dramatical W0, UP0,PIABS, and decrease in ABS/RC and DI0/RC in Chlamydo- fall exposed to 0.75–2 mM of molinate in a time and concentra- monas reinhardtii. It demonstrated that PSII changes induced by tion dependent manner. All these results mentioned above sug- different toxicant in microalgae were different. gested that antioxidant enzymes were stimulated by relatively The suggested concentration of butachlor in China is 1.0–5.0 kg low-level herbicide and high-level herbicide was adverse to anti- active ingredient hm2 (depth of water: 2–5 cm). Therefore, the fi- oxidant enzymes. The oxidative damage to cyanobacteria cells by nal concentration of butachlor applied in rice field is 2–25 mg L1. ROS could be examined by measuring the MDA content [38]. The Wang et al. [43] recorded more than two times the applied concen- results in the present study combined with some results in refer- tration of butachlor in field experiments with 2.82% organic matter ences mentioned above showed that MDA content increased sig- in the paddy field. In the present study, the results showed that 40 nificantly with decline in herbicide concentrations [29,38,39]. For and 80 mg L1 butachlor significantly inhibited growth, PSII activ- this reason, it suggested that although antioxidant enzymes are ity, and synthesis of pigments in Nostoc sp. (FACHB-85). Therefore, induced by herbicides, they are not enough to completely elimi- there is no doubt that butachlor residue in the field may do harm to nate ROS in cyanobacteria. N-fixing cyanobacteria. Under herbicide stress, some nonenzymatic compounds could also be involved in antioxidant reaction in cyanobacteria. Sheeba et al. 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