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Chemosphere 193 (2018) 385e393

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Chemosphere

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Responses of Hydrilla verticillata (L.f.) Royle and Vallisneria natans(Lour.) Hara to glyphosate exposure

Guidi Zhong, Zhonghua Wu*, Jun Yin, Lulu ChaiNational Field Station of Freshwater Ecosystem of Liangzi Lake, College of Life Sciences, Wuhan University, Wuhan, PR China

h i g h l i g h t s

� Glyphosate can induce oxidative stress in H. verticillate.� Glyphosate decreased CAT activities without causing oxidative stress in V. natans.� The sensitivity of H. verticillata to glyphosate is higher than that of V. natans.

a r t i c l e i n f o

Article history:Received 26 July 2017Received in revised form27 October 2017Accepted 31 October 2017Available online 2 November 2017

Handling Editor: Jim Lazorchak

Keywords:GlyphosatePhysiological responsesH. verticillataV. natans.

* Corresponding author. Present address: College oversity, 430072 Wuhan, PR China.

E-mail address: wuzhonghua@whu.edu.cn (Z. Wu

https://doi.org/10.1016/j.chemosphere.2017.10.1730045-6535/© 2017 Published by Elsevier Ltd.

a b s t r a c t

Glyphosate is a broad-spectrum herbicide that is frequently detected in water bodies and is harmful toaquatic systems. We conducted an experiment to explore the ecological sensitivity of Hydrilla verticillata(L.f.) Royle and Vallisneria natans (Lour.) Hara to glyphosate. Our research focused on the physiologicalresponses of H. verticillata and V. natans after exposure to various concentrations of glyphosate (0, 1, 10,20, 30, 40, 50 and 80 mg/L) in hydroponic culture after one day (1D) and seven days (7D). The resultsshow that after 1D, the soluble protein content of H. verticillata was significantly stimulated under lowherbicide concentrations. Other indices for H. verticillata and V. natans had no remarkable changes at 1D.After 7D of treatment, the soluble protein content of H. verticillata showed no significant differences,while the malondialdehyde (MDA), pigment contents and catalase (CAT) activity significantly increasedat low glyphosate concentrations. Guaiacol peroxidase (POD) activity in H. verticillata significantlyincreased with increasing herbicide concentrations. The chlorophyll a/b ratio of H. verticillata sharplydecreased above 10 mg/L. For V. natans, soluble protein, chlorophyll a, and carotenoid content; and CATactivity declined significantly after glyphosate application, while other indicators showed no significantchanges. Our results indicate that glyphosate concentrations from 0 to 80 mg/L can induce oxidativestress in H. verticillate and may impede metabolism processes for protein and pigments without causingoxidative stress in V. natans. Taken together, our results suggest that the sensitivity of H. verticillata toglyphosate exposure is higher than that of V. natans.

© 2017 Published by Elsevier Ltd.

1. Introduction

Glyphosate, or N-(phosphonomethyl) glycine, is a broad-spectrum and post-emergent herbicide initially synthesized byMonsanto in 1970. It inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase, a key enzyme in the shikimate pathway thatleads to overproduction and accumulation of shikimic acid. It ac-cumulates in aboveground meristematic tissue through plant

f Life Sciences, Wuhan Uni-

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cuticles by absorption and symplastic transmission, restraining thebiosynthesis of aromatic amino acids (phenylalanine, tryptophanand tyrosine) and impeding the synthesis of proteins, shikimic acidand secondary products (Steinrücken and Amrhein, 1980; Franzet al., 1997; Sch€onbrunn et al., 2001). As a non-selective herbi-cide, it can kill grasses or broadleaf weeds and both mono-cotyledonous and dicotyledonous weeds. Glyphosate is widelyused for the control of various weeds in farmland and can also beused for the control of non-farmland weeds in locations such as ingardens, nurseries, railways, highways, forests, and lakes (Zhouet al., 2013).

Glyphosate has been used in more than 100 countries and re-gions since 1974 and is registered in the United States (Huang and

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Liu, 2015). Global glyphosate consumption has risen steadily inrecent years, and active compound consumption was more than700 thousand tons in 2014. With the development of transgeniccrops, the global planting area for transgenic crops continues torise, with a growth rate of 5% in 2014. In China, cultivation oftransgenic crops, specifically insect-resistant cotton, began in 1997.Currently the total area of transgenic cropland in China has reached4,200,000 ha (China industry information, 2016). Glyphosate-tolerant genetically modified crops have an absolute advantageover other transgenic crops. Among the 108 gene-modified cropsapproved for global registration in 2013, there are 44 glyphosate-resistant forms, accounting for 40.7%. Now, more than 20 kinds ofcrops have been successfully developed for glyphosate-tolerantcrops, such as rice, tobacco, peanuts, potatoes, wheat, tomatoes,carrots, ryegrass and oak, providing a broad market and increasingdevelopmental prospects for glyphosate (Hu, 2015).

There are two main routes of biodegradation for glyphosate:CeN breaks to form aminomethylphosphonic acid (AMPA), andCeP breaks to produce sarcosine. Glyphosate is easily absorbed byaluminum oxide and iron oxide in the soil and can be degraded bymicrobes depending on degradation temperature and soil humid-ity. Glyphosate residues in plants degrade more slowly in soils, andglyphosate in pure water metabolizes faster than in water sedi-ment. Glyphosate in soils may come into contact with waterthrough large pores or rainfall, and the half-life of glyphosate inwater bodies is 7-14D (Borggaard and Gimsing, 2008; Mamy et al.,2016; Wang et al., 2016; Bento.et al., 2016). When glyphosate isapplied to plants, portions enter the soil and water either directly(by direct spraying) or indirectly (due to penetration and surfacerunoff). Glyphosate pesticide pollution in waters has been reportedboth domestically and overseas. In China, glyphosate pollution inTaihu is a serious problem, with the highest reported residualconcentrations at 15.21 mg/L (Wei et al., 2016). Moreover, glypho-sate has been found in Hongfeng Lake in Guiyang and in drinkingwater in Zhejiang and the Pearl River, but the content is far lowerthan the national standard limit of 700 mg/l (Wei et al., 2016).Glyphosate reached the maximum exposure concentration of1.956 mg/L in a Canadian forest pond and United States wetlands(Currie et al., 2015), which exceeds the standard limit of Canada andthe United States (65 mg/L and 700 mg/L, respectively). Argentina,Holland and other countries have also published similar relevantreports (Byer et al., 2008; Struger et al., 2008; Peruzzo et al., 2008;Glozier et al., 2012; Desmet et al., 2016).

The toxicity of glyphosate is relatively low, currently listed inToxicity Category III (low toxicity) the United States EnvironmentalProtection Agency (EPA). However, cases of glyphosate poisoningare occasionally reported (Sribanditmongkol et al., 2012; Han et al.,2016). (delete the sentence) The widespread use of glyphosate hasalso brought about other problems. First, someweeds have becomeresistance. So far, a total of 34 kinds of anti-glyphosate grasses havebeen found in different countries and regions by different cultiva-tion methods (Chen et al., 2017). Studies have found that differentweeds have different resistance mechanisms. Weeds can becomeresistant to glyphosate poisoning by reducing the absorption andtranslocation of glyphosate and enhancing EPSPS activity andtarget site mutations (Alarc�on-Reverte et al., 2015; Alc�antara-de laCruz et al., 2016). Second, impact to non-target plant growth hasbeen reported. During the glyphosate spraying process, thechemical may directly reach soils or water bodies, leading to directexposure in non-target plants and affecting their growth andmetabolism. Glyphosate has been shown to affect crop yield,chlorophyll content, chlorophyll fluorescence, C metabolism, Nfixation, gas exchange, photosynthesis, antioxidant system, andtarget site gene expression (De María et al., 2006; Davis et al, 2013;Luis Orcaray et al., 2012; Armendariz et al., 2016; Basantani et al.,

2011; Zobiole et al., 2010; Jiang et al., 2013). Glyphosate can alsoinhibit seed germination (Gomes et al., 2017).

Glyphosate enters the water through irrigation, surface runoff,and direct discharge (glyphosate is also used for control of weeds inpaddy fields), among other routes, where it can affect the growth ofaquatic plants. Previous studies on the effects of glyphosate onaquatic plants are fewer than those on terrestrial plants and mainlyfocus on effects on growth and chlorophyll content. For example,Cruz et al. (2015) found that certain doses of glyphosate inhibit thegrowth of floating aquatic plants such as Eichhornia crassipes, Pistiastratiotes and Salvinia molesta. Glyphosate at 1 mg/L and 10 mg/Lsignificantly reduced chlorophyll a and b content in Halophila ovalis(Ralph, 2000). Concentrations higher than 2.5 mg/l inhibited thetotal length, weight, root length and number of nodes in Myr-iophyllum spicatum L (S�anchez et al., 2007). Glyphosate concen-trations of 80 mg/L significantly reduced the growth rate,chlorophyll a content, and chlorophyll a fluorescence parameters inLemna minor L. (Dosnon-Olette et al., 2011), and a concentration of1mg/L significantly affected the growth and shape of Lemna gibba L.(Sobrero et al., 2007). However, the effects of glyphosate exposureon physiological indicators such as soluble protein content, shiki-mic acid content, reactive oxygen species-ROS (e.g., hydrogenperoxide, O2�), membrane lipid peroxidation, antioxidant enzymes(e.g., CAT, POD), and antioxidants (e.g., ascorbic acid, glutathione)are less studied. Among the existing studies, Kielak et al. (2011)found that exposure to 56.88, 113.76 and1137.6 mg/L glyphosateisopropylamine salts reduced chlorophyll content, biomass, accu-mulation of putrescine, spermidine and polyamines, CAT andascorbate peroxidase (APX) activity in duckweed (L. minor). Gomesand Juneau (2016) found that glyphosate reduced duckweed(L. minor) photosynthesis (100 mg/L) and total chlorophyll contentand increasedMDA, hydrogenperoxide, pheophytin a/chlorophyll aratio, APX (10 mg/L) and CAT (5 mg/L). They also found thathydrogen peroxide products from the mitochondrial transmissionchain upon glyphosate inhibition complex III activity. The effects ofglyphosate on submerged macrophytes in H. verticillata andV. natans have not been reported.

H. verticillata and V. natans are usually found in ponds, lakes andditches and are the most widely distributed submerged plants inChina. As an important component of water ecosystems, they notonly provide higher primary productivity for water bodies but alsoprovide a variety of habitat for fish, amphibians, benthic fauna andplankton. Moreover, they can also absorb and degrade nutrientsubstances, concentrate and accumulate heavy metals elements(Zhang et al., 2012) and thereby gradually reduce the concentrationof pollutants in water. Thus, they play an important role in main-taining the health and stability of ecosystems and in ecologicalrestoration of water bodies. In this study, we examined the effectsof different glyphosate concentrations on chloroplast pigment,soluble protein content, membrane lipid peroxidation and antiox-idant enzyme activity in H. verticillata and V. natans. Our primarygoals were to understand the adaptability of these two submergedmacrophytes to glyphosate exposure and to provide theoreticalguidance for the selection of water purification plants and glyph-osate safety assessment.

2. Materials and methods

2.1. Plant materials and experimental design

Whole plants of H. verticillata and V. natans were collected froma mid-trophic lake in the Wuhan Botanical Garden (30�320N;114�250E), Chinese Academy of Sciences, Wuhan, Hubei Province,PR China. Each plant was thoroughly cleaned under running tapwater to remove particles and other organisms. Prior to the

G. Zhong et al. / Chemosphere 193 (2018) 385e393 387

experiment, V. natans and H. verticillata plants were cultivated in agreenhouse pond at a density of approximately 20 plants per m2.After approximately 1 week, plants were processed as follows: forH. verticillata, we chose plants with similar growth status and cutthe tops of plants (20 cm) to use as experimental material, ensuringthe integrity of plants and absence of roots; for V. natans, 5e8 leaveswithout ramets and adventitious roots and with above-groundheights no more than 20 cm were thoroughly cleaned in runningtap water and then rinsed in distilled water. The H. verticillata andV. natans plants were moved to a climate chamber for acclimati-zation. It took 3 days to allow wound healing and acclimatization.During acclimatization and for the experiment, the plants weregrown in nutrient solution in transparent plastic tanks at an airtemperature of 26 ± 2 �C; water temperature was 28 ± 2 �C duringthe light period and 26 ± 2 �C during the dark period. Light in-tensity was 120 ± 20 mmol photons m�2s�1 and was produced byhalogen lamps. The photoperiod was 12 h light: 12 h dark.

All experimental plants were grown in 10% Hoagland's solution(Hoagland and Arnon, 1950). Experiments were performed in threereplicates, and each replicate contained 3 plants of similar size.Plants were exposed to different concentrations of glyphosatetreatments (1, 10, 20, 30, 40, 50 and 80mg/L) andmaintained in 10%Hoagland's solution under the described laboratory conditions for atotal exposure period of one week. The experiment included acontrol treatment (0 mg/L). The glyphosate (biological reagent,purity�95%) used in this experimentwas obtained from SinopharmChemical Reagent Co., Ltd., Shanghai, China. The solutions werechanged every 48 h.

The plants were harvested on the first day and seventh day. Theywere first rinsed with double distilled water to remove the adsor-bed herbicide from the surface and then used for the estimation ofvarious parameters.

2.2. Photosynthetic pigment measurement

The photosynthetic pigment was measured according toJampeetong and Brixl (2009) with some modifications. Plant leaves(0.1 g freshweighteFW)were cut into pieces, ground, and placed in10-ml flasks. Then, 95% alcohol was added until the 10-ml mark.Flasks were placed in the dark for 24 h. The chlorophyll content inplant leaves was determined with a spectrophotometer at 470, 649and 665 nm for chlorophyll a, chlorophyll b and carotenoids,respectively. Values were calculated according to Lichtenthaler andWellburn (1983). All spectrophotometric analyses were conductedin a final volume of 3 ml by using a MAPADA UV-1200 spectro-photometer (Shanghai Meipuda instrument Co. Ltd.,Shanghai,China).

2.3. Lipid peroxidation

The level of lipid peroxidation in plant leaveswas determined byestimating the MDA content. The content of MDA in leaves wasmeasured using the thiobarbituric acid (TBA) method (mmol/g FW)(Wang and Huang, 2015).

2.4. Measurement of enzyme activity and soluble protein content

Fresh leaves weighing 0.1 g were homogenized with phosphatebuffer solution (50 mM, pH 7.8) containing NaH2PO4, Na2HPO4,PVPP (1%m/v), EDTA (1 mM) and mercaptoethanol (1 mM) at 4 �C(Eppendorf Centrifuge 5417 R, Hamburg, Germany). The homoge-nate was centrifuged at 8000 � g for 15 min at 4 �C. The super-natant was stored at 4 �C and used for enzyme activity and solubleprotein content assays.

Soluble protein content was determined following the methodof Bradford (1976) using bovine serum albumin as the standard(mg/g FW). CAT activity was measured by spectrophotometry,where one enzyme activity unit (U/g$min FW) corresponds to adecrease of 0.1 in absorbance at 240 nm in 1 min (Cang and Zhao,2013). POD activity was measured using the guaiacol method,with a per-minute absorbance change of 0.01 at 470 nm as one unitof enzyme activity (U/g$min FW) (Zhang et al., 2009).

2.5. Statistical analysis

The experiment utilized a randomized block design. All valuesare expressed as the mean ± standard deviation. Results weresubmitted to normality (KolmogoroveSmirnov) and homogeneity(Levene) tests and then statistically evaluated. ANOVAs were per-formed for the full range of herbicide concentrations for the twoplants and the significant difference between two plant speciesunder the same concentration of glyphosate and exposure time.Post hoc Duncan tests were done to separate differences betweenpairs of treatments. Nonparametric data were analyzed usingKruskaleWallis tests followed by KruskaleWallis post hoc tests.The statistical analysis was performed using SPSS 19.0 forWindows(IBM Inc., Chicago, IL, USA), and graphs were generated in Sigma-Plot 12.5 for Windows (Systat Software, Inc., USA).

3. Results

3.1. Soluble protein content

After 1D of glyphosate treatment, the soluble protein content ofH. verticillata elevated slightly under low concentrations andsignificantly increased (by 61.83%) compared to the control at20 mg/L (p � 0.05); at higher concentrations, it decreased to thecontrol level. For V. natans, there were no significant differencesamong treatments (p > 0.05) (Fig. 1a).

Fig. 1b shows the change in soluble protein content within planttissues after 7D. H. verticillata had no significant changes (p > 0.05).However, V. natans declined by up to 36.43% compared with thecontrol at 80 mg/L (p � 0.05).

3.2. MDA content

As show in Table 1, the MDA content of H. verticillata did notsignificantly change after 1D of treatment (p > 0.05). After 7D oftreatment, it increased to themaximum (0.54 ± 0.01 mmoL/g FW) at40 mg/L treatment, which was significantly higher than othertreatments and the control (p � 0.05). The content of MDA 7D aftertreatment was lower than after 1D. The MDA content in V. natansdid not change significantly by glyphosate concentration on 1D or7D (p > 0.05). The content of MDA in H. verticillatawas higher thanin V. natans. At 40 mg/L after 7D treatment, the MDA content inH. verticillata was 24.6 times greater than that in V. natans(p � 0.05).

3.3. CAT activity

CAT activity in H. verticillata and V. natans showed no significantdifferences after 1D exposure (p > 0.05) (Fig. 2a). After 7D ofexposure, CAT activity increased in H. verticillata in all treatmentscompared to control but only in the 20 mg/L and 30 mg/L treat-ments reached significantly level (p � 0.05). Maximum activity(6.75 ± 1.00 U/g$min FW) was observed in the 20 mg/L treatment,which was 1.5 times higher than the control (Fig. 2b). Activity inV. natans after 7D increased from 1 mg/L to 30 mg/L, but not

Fig. 1. Soluble protein content of H. verticillata and V. natans after different concentrations of glyphosate treatment at 1D (a) and 7D (b). All values represent the mean of threereplicates ± standard deviation. ANOVA significant at p � 0.05. Bars with different letters are significantly different among different exposure concentrations (p � 0.05, Duncan test).Bars with stars are significantly different between two plant species under the same concentration of glyphosate and exposure time (p � 0.05, One way-ANOVA).

Table 1Effects of different concentrations of glyphosate on MDA content of H. verticillata and V. natans.

Concentration Of glyphosate (mg/L) MDA content (mmol/g FW)

H. verticillate V. natans

1D 7D 1D 7D

0 0.59 ± 0.14a 0.02 ± 0.00a* 0.30 ± 0.08b 0.02 ± 0.01a*1 1.37 ± 0.45a 0.02 ± 0.01a* 0.33 ± 0.01b 0.02 ± 0.00a*10 0.86 ± 0.33a 0.02 ± 0.00a* 0.29 ± 0.05b 0.02 ± 0.00a*20 0.93 ± 0.18a 0.02 ± 0.00a* 0.36 ± 0.07b 0.02 ± 0.00a*30 0.94 ± 0.22a 0.02 ± 0.00a* 0.36 ± 0.08b 0.02 ± 0.00a*40 0.89 ± 0.16a 0.02 ± 0.00a* 0.54 ± 0.01a 0.02 ± 0.00a*50 1.05 ± 0.31a 0.02 ± 0.00a* 0.40 ± 0.09b 0.02 ± 0.00a*80 1.08 ± 0.16a 0.02 ± 0.00a* 0.34 ± 0.02b 0.02 ± 0.00a*

Means with different letters in the same columns are significantly different among different exposure concentrations (p� 0.05, Duncan test). Means with stars are significantlydifferent between two plant species under the same dose of glyphosate at the same exposure time (p � 0.05, One way-ANOVA).

Fig. 2. CAT activity of H. verticillata and V. natans after different concentrations of glyphosate treatment at 1D (a) and 7D (b). All values represent the mean of threereplicates ± standard deviation. ANOVA significant at p � 0.05. Bars with different letters are significantly different among different exposure concentrations (p � 0.05, Duncan test).Bars with stars are significantly different between two plant species under the same dose of glyphosate (p � 0.05, One way-ANOVA).

G. Zhong et al. / Chemosphere 193 (2018) 385e393388

significantly (p> 0.05), and sharply decreased (by 49.73% comparedto the control) in the 80 mg/L treatment (p � 0.05) (Fig. 2b). After 7D of exposure, H verticillata plants submitted to glyphosate con-centrations �20 mg L-1 shown greater CAT activity than V. natans.

3.4. POD activity

After 1D of glyphosate exposure, POD activity in H. verticillatadecreased, but not significantly compared to the control (p > 0.05).

G. Zhong et al. / Chemosphere 193 (2018) 385e393 389

After 7D of glyphosate exposure, there was a concentration-dependent increase up to 40 mg/L (p � 0.05), and it reached themaximum value at 80 mg/L (1686.7 þ 344.0 U/g$min FW), which is1.84 times higher than the control (p � 0.05). The POD activity ofV. natans did not significantly differ among treatments nor betweentreatments and the control after 1D or 7D (p > 0.05). POD activity inH. verticillatawas generally higher than V. natans (Table 2) (p� 0.05,except controls in 7D).

3.5. Pigment content

Chlorophyll a and b, carotenoid, and total chlorophyll (aþb)contents and chlorophyll a/b showed no significant changes after1D of treatment within the two plant tissues (p > 0.05) (Fig. 3a, b, c,d, Table 3). The values for H. verticillatawere higher for all pigmentscompared to V. natans.

As shown in Fig. 4aed and Table 3, after 7D exposure chlorophylla and carotenoid content in H. verticillata changed similarly. Theywere higher than control from 1 mg/L to 40 mg/L but only signifi-cantly changed at 10mg/L compared to the control (p� 0.05). Thesevalues under high glyphosate doses (50 mg/L and 80 mg/L) weresharply decreased compared to the 10 mg/L treatment (p � 0.05)but did not change significantly in comparison with the control(p > 0.05). The chlorophyll b content of H. verticillata significantlyincreased from 10 mg/L to 50 mg/L, while the total chlorophyll(aþb) content significantly increased at 10 mg/L, 30 mg/L and40 mg/L (p � �0.05). After reaching the maximum at 40 mg/Ltreatment (0.39 ± 0.03, 2.89 and ±0.24 mg/g FW, respectively),chlorophyll b and total chlorophyll (aþb) contents showed amarked decline compared to the 40 mg/L treatment (p � 0.05),although these changes were not significantly different from thecontrol (p > 0.05). Chlorophyll a/b in H. verticillatawas significantlylower than the control in all treatments (p� 0.05), except for a non-significant decline at 1 mg/L (p > 0.05). At the lowest value, thisrepresented a decrease of 50.39% compared to the control (40 mg/Ltreatment, 2.38 ± 0.22). Glyphosate reduced chlorophyll a andcarotenoid content in V. natans, with significant effects in the30e50 mg/L treatments and 30e80 mg/L treatments, respectively,compared to the control (p � 0.05). Minimum values for bothchlorophyll a and carotenoid (0.47 ± 0.09, 0.14 ± 0.03 mg/g FW,respectively) were observed at 50 mg/L.

4. Discussion

Proteins play important roles in maintaining cell structure andregulating physiological metabolism in organisms. Our resultsshowed changes in the soluble protein content in H. verticillata andV. natans after 1D exposed to glyphosate and that these changes in

Table 2Effects of different concentrations of glyphosate on POD activity of H. verticillata and V. n

Concentration Of glyphosate (mg/L) POD activity (U/g$min FW)

H. verticillate

1D 7

0 2268.3 ± 832.0a 61 1490.0 ± 212.1a 610 1747.5 ± 294.6a 620 1793.3 ± 280.5a 630 2044.2 ± 93.1a 640 1921.7 ± 300.0a 650 1878.8 ± 146.7a 680 1648.8 ± 58.3a 6

Means with different letters in the same columns are significantly different among differendifferent between two plant species under the same dose of glyphosate at the same exp

H. verticillata were significantly higher at a glyphosate concentra-tion of 20 mg/L compared other concentrations (except 1 mg/L).V. natans showed no significant changes, indicating that in earlystages of exposure, glyphosate did not affect soluble protein con-tent in V. natans. It may be that H. verticillata obtains nutrientsmore easily under low glyphosate concentrations, as it was re-ported that low concentration of organophosphorus pesticides maycause a dose-response relationship in algae called an “excitatoryeffect” (Yuan et al., 2016). After 7D exposure, there were no sig-nificant differences in protein content in H. verticillata, possiblybecause the plant antioxidant enzymeswere activated, maintainingorganism stability. However, with increased concentrations ofglyphosate, V. natans protein content decreased gradually and at80mg/Lwas significantly lower than that of 0e30m/L. Reduction inprotein content of V. natans leaves under high glyphosate concen-trations may be due to breakdown of soluble proteins or due toincreased activity of protease or other catabolic enzymes whichwere activated and destroyed the protein (Singh et al., 2006).Glyphosate competitively inhibits the activity of 5-enolpyruvylshikimate-3-phosphate in the shikimate pathway andinhibits the biosynthesis of aromatic amino acids such as phenyl-alanine, tryptophan and tyrosine. This leads to the blocking ofprotein synthesis and secondary products of some metabolic re-actions, thereby reducing the content of proteins, hormones, fla-vonoids and lignin (Steinrücken and Amrhein, 1980; Franz et al.,1997; Sch€onbrunn et al., 2001; Radwan and Fayez, 2016). Studieshave shown that plants can slow down the rate of protein synthesiswhen under stress, resulting in reduced content (Zhang et al., 2010;Mu et al., 2016). The sensitivity and response of different plants tothe same pollutants also differ. Romero found that the proteincontent of Chlorella kessleri significantly increased under highglyphosate concentrations (60, 70 mg/L), suggesting that glypho-sate may not inhibit C. kessleri protein synthesis (Romero et al.,2011).

Chlorophylls and carotenoids are photosynthetic pigments ingreen plants, and the latter are accessory pigments. Photosyntheticcapability depends partly on photosynthetic pigment content.Photosynthesis provides the driving force for growth in plants;thus, all factors affecting photosynthetic ability will inevitablyaffect plant growth. Glyphosate is a strong cation chelator due to itscarboxyl and phosphonate groups and can form complexes withnutrients in plant tissues, thus making them unavailable for bio-logical processes (including photosynthesis). It also inhibits thesynthesis of plant secondary metabolism products, including someof the substances involved in photosynthesis such as quinones.Glyphosate inhibits the synthesis of plastoquinone, which is acofactor of the enzymes phytoene desaturase and z-carotenedesaturase, which are important enzymes for the synthesis of

atans.

V. natans

D 1D 7D

83.3 ± 112.2a* 915.8 ± 358.0b 376.7 ± 93.6a30.8 ± 173.2a* 930.8 ± 244.0b 284.2 ± 44.5a*46.7 ± 167.5a* 1003.9 ± 283.2b 419.2 ± 43.1a*83.3 ± 143.0a* 1312.5 ± 121.9 ab 455.0 ± 80.9a*39.2 ± 147.5a* 1547.5 ± 76.1a 408.3 ± 42.0a*55.8 ± 118.8a* 1665.0 ± 172.7a 430.0 ± 23.8a*98.3 ± 162.6a* 1595.0 ± 188.6a 390.0 ± 15.6a*90.8 ± 54.3a* 1686.7 ± 344.0a 425.8 ± 143.8a*

t exposure concentrations (p� 0.05, Duncan test). Means with stars are significantlyosure time (p � 0.05, One way-ANOVA).

Fig. 3. Chlorophyll a (a), carotenoid (b), chlorophyll b (c) and total chlorophyll (aþb) content (d) of H. verticillata and V. natans after 1D of different doses of glyphosate treatment. Allvalues represent the mean of three replicates ± standard deviation. ANOVA significant at p � 0.05. Bars with different letters are significantly different among different exposureconcentrations (p � 0.05, Duncan test). Bars with stars are significantly different between two plant species under the same dose of glyphosate at the same exposure time (p � 0.05,One way-ANOVA).

Table 3Effects of different concentrations of glyphosate on chlorophyll a/b ratio of H. verticillata and V. natans.

Concentration Of glyphosate (mg/L) chlorophyll a/b ratio

H. verticillate V. natans

1D 7D 1D 7D

0 2.30 ± 0.43a 2.69 ± 0.14a 4.79 ± 1.39a 2.94 ± 0.03a*1 2.59 ± 0.18a 2.96 ± 0.31a 4.12 ± 0.35 ab 3.22 ± 0.45a10 2.31 ± 0.01a 2.91 ± 0.26a* 3.53 ± 0.34bc 3.01 ± 0.27a20 2.55 ± 0.28a 2.87 ± 0.26a 2.83 ± 0.34cd 2.93 ± 0.17a30 2.54 ± 0.22a 3.07 ± 0.25a* 2.67 ± 0.30d 2.94 ± 0.08a40 2.06 ± 0.73a 2.87 ± 0.20a 2.38 ± 0.22d 2.98 ± 0.31a50 2.42 ± 0.25a 3.05 ± 0.10a* 2.56 ± 0.20d 2.83 ± 0.14a80 2.48 ± 0.12a 2.52 ± 0.27a 2.66 ± 0.23d 2.68 ± 0.13a

Means with different letters in the same columns are significantly different among different exposure concentrations (p� 0.05, Duncan test). Means with stars are significantlydifferent between two plant species under the same dose of glyphosate at the same exposure time (p � 0.05, One way-ANOVA).

G. Zhong et al. / Chemosphere 193 (2018) 385e393390

carotenoid precursors; glyphosate can thus affect carotenoid con-tent (Gomes et al., 2016). In addition, glyphosate has been proposedto interfere with d-aminolevulinic acid (ALA) biosynthesis by con-trolling the conversion of alpha-ketoglutarate to ALA and/or thecondensation of glycine with succinyl-CoA to form ALA and CO2.ALA is the precursor of porphyrin synthesis, which is involved inthe formation of chlorophyll, CAT and POXs. Other studies have alsoshown that the effects of glyphosate on the synthesis of chlorophyll

may be due to glyphosate metabolite AMPA because AMPA reducesglycine, serine and glutamic acid contents, which are componentsof the process ALA synthesis. Amino acid reduction leads to reducedsynthesis of ALA and blocks the synthesis of chlorophyll (Gomeset al., 2014). The chlorophyll, carotenoid and total chlorophyllcontents in H. verticillata and V. natans did not change significantlyafter 1D treatment, which may be explained by a temporal effect ofglyphosate on pigments. At 7D after application, the chlorophyll a,

Fig. 4. Chlorophyll a (a), carotenoid (b), chlorophyll b (c) and total chlorophyll (aþb) content (d) of H. verticillata and V. natans after 7D of different doses of glyphosate treatment. Allvalues represent the mean of three replicates ± standard deviation. ANOVA significant at p � 0.05. Bars with different letters are significantly different among different exposureconcentrations (p � 0.05, Duncan test). Bars with stars are significantly different between two plant species under the same dose of glyphosate at the same exposure time (p � 0.05,One way-ANOVA).

G. Zhong et al. / Chemosphere 193 (2018) 385e393 391

b, carotenoid and total chlorophyll contents in H. verticillata werestimulated at low concentrations but decreased under high con-centrations. Other studies have also shown that glyphosate canpromote plant growth at low concentrations (Cedergreen et al.,2009). However, after 7D of exposure in V. natans, chlorophyll aand carotenoid contents decreased, while chlorophyll b and totalchlorophyll contents did not change. The decrease in chlorophyll acontent may be related to the decrease in ALA synthesis, while thedecrease of carotenoid content may be related to the inhibition ofplastoquinone synthesis. Chlorophyll b and total chlorophyll con-tents did not change. It may be that they are insensitive to glyph-osate compared to chlorophyll a and carotenoids. Some studieshave suggested that the decrease in chlorophyll content afterglyphosate treatment is due to the decrease in carotenoid content(Moldes et al., 2008). Chlorophyll a/b explains the sensitivity of thelight harvesting complex Ⅱ enzyme system to external factors(Romero et al., 2011). The results showed that chlorophyll a/b inV. natans after 1D and 7D and in H. verticillata after 1D did notchange significantly, while in H. verticillata, it decreased after 7D oftreatment. It has been reported that the reduction of chlorophyll bcontent will affect photosystem stability, eventually leading toinstability of the photosynthetic mechanism and decreasing planttolerance to stress. In contrast, it is speculated that chlorophyll bcontent increases under stress, perhaps due to enhancement ofplant tolerance to stress, and that the plant can adapt to challenging

conditions by adjusting the chlorophyll a/b ratio (Jian et al., 2016).Environmental pollutants such as heavy metals, surfactants,

pesticides and other plants may cause oxidative stress responses,resulting in the accumulation of reactive oxygen species. ExcessiveROS can cause membrane lipid peroxidation, leading to increasedMDA content (MDA is the main product of membrane lipid per-oxidation, which is a biological indicator of the degree of damage tothe cell membrane). Plants can cope with oxidative stress by syn-thesizing antioxidant enzymes and antioxidants to remove excessROS (Wu et al., 2010; Schweikert and Burritt, 2012; Garanzini andMenone, 2015; Mu et al., 2016; Radwan and Fayez, 2016). CATand POD are important antioxidant enzymes in plants and play animportant role in the removal of hydrogen peroxide. In thisexperiment, the MDA, CAT and POD contents in H. verticillata didnot change significantly 1D after treatment of glyphosate, whichindicates that glyphosate did not cause oxidative stress over theshorter time period. After 7D of exposure, MDA content inH. verticillata in the 40 mg/L treatment was significantly higherthan that in the other treatments, indicating that H. verticillatasuffered membrane damage. CAT activity at 20 mg/L glyphosateconcentration in H. verticillatawas significantly higher than that ofthe control and 80 mg/L treatment, followed by a decrease withincreased herbicide concentration. POD activity in H. verticillatasignificantly increased at 40 mg/L and showed an upward trendoverall. The results show that the antioxidant systemwas activated,

G. Zhong et al. / Chemosphere 193 (2018) 385e393392

ROS were cleared, and MDA content reduced. This result agreeswith previous studies (Kielak et al., 2011; Zhang et al., 2015;Radwan and Fayez, 2016). After 1D and 7D of glyphosate expo-sure, the MDA content in V. natans did not change, indicating theabsence of membrane damage. CAT and POD activity in V. natansdid not change after 1D of glyphosate treatment. CAT activity wassignificantly inhibited after 7D at 80mg/L treatment; however, PODactivity did not change significantly after 7D. Moldes et al. (2008)found that glyphosate had no significant effect on MDA contentin sensitive and tolerant soybean roots and that neither of the twotypes of soybeans showed significant changes in APX, CAT, and PODactivity. However, the authors found significantly increased solubleamino acid content in sensitive and tolerant soybean, both in leavesand roots. Soluble amino acids such as antioxidants can removeROS, which may explain why MDA content did not change in soy-bean. MDA did not change, so enzyme activity was not activated;thus, enzyme activity showed no significant changes. Wang et al.(2014) found that glyphosate presence in the aquatic environ-ment slightly inhibited CAT activity in Pyramimonas delicatula andAlexandrium tamarense, significantly increasing MDA content, andthat low concentrations of glyphosate have stimulating effects onsuperoxide dismutase (SOD) activity. Gomes et al. (2016) studiedthe effects of glyphosate on Salix miyabeana and found that MDAand hydrogen peroxidewere significantly increased after treatmentwith glyphosate and that SOD, CAT and APX were significantlydecreased, possibly due to inhibition of ALA synthesis. Non-significant differences in POD activity and inhibition of CAT mayexplain why glyphosate did not generate stress in V. natans. Eitherother antioxidant enzymes or non-enzymatic antioxidants mayhave been active, or V. natans may have another defense mecha-nism to cope with stress. Moreover, the reduction in CAT activitymay due to the decrease in ALA, as ALA is a synthetic precursor ofporphyrin, which is involved in the synthesis of chlorophyll, CATand POD. The trend of chlorophyll a and carotenoid content isconsistent with CAT activity in V. natans after 7D of exposure.

The sensitivity of H. verticillata and V. natans to glyphosatediffered. After 1D of treatment, glyphosate only significant affectedsoluble protein content in H. verticillata and did not cause markedchanges in other indicators in plant tissues. However, the physio-logical response indices such as MDA content, CAT activity and PODactivity of H. verticillata were higher than those of V. natans afterglyphosate exposure. After 7D of treatment in H. verticillata, therewere significant effects on physiological indicators except for sol-uble protein content. Soluble protein content, CAT activity, andchlorophyll a and carotenoid contents in V. natans significantlychanged after exposure to glyphosate, while the other indices werenot significantly affected. Furthermore, the measured responses inH. verticillata were generally higher than those in V. natans. Takentogether our results suggest that the sensitivity of H. verticillata toglyphosate exposure is higher than that of V. natans.

Acknowledgments

This research was funded by the National Science Foundation ofChina (No. 31270410, No. 30970303 and No. 30670206).

Appendix A. Supplementary data

Supplementary data related to this article can be found athttps://doi.org/10.1016/j.chemosphere.2017.10.173.

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