Improvement of cyanobacterial-killing biologically derived substances (BDSs) using an ecologically safe and cost-effective naphthoquinone derivative

Ecotoxicology and Environmental Safety 141 (2017) 188–198

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Improvement of cyanobacterial-killing biologically derived substances (BDSs) using an ecologically safe and cost-effective naphthoquinone derivative

MARK

Jae-Hyoung Jooa,1, Pengbin Wanga,1,2, Bum Soo Parka,3, Jeong-Hwan Byuna, Hye Jeong Choia, ⁎ ⁎⁎ Seong Hun Kimc, , Myung-Soo Hana,b, a b c

Department of Life Science, Hanyang University, Seoul 04763, South Korea Research Institute for Natural Sciences, Hanyang University, Seoul 04763, South Korea Department of Organic and Nano Engineering, Hanyang University, Seoul 04763, South Korea

A R T I C L E I N F O

A B S T R A C T

Keywords: Algicide Naphthoquinone derivatives Harmful cyanobacteria blooms Eco-friendly mitigation

In previous studies, naphthoquinone (NQ) compounds have been shown to be effective, selective, and ecologically safe algicides for controlling harmful algal blooming species (HABs) or winter bloom species, such as Stephanodiscus hantzschii. However, there are no reports on NQ-based algicides for use with cyanobacterial blooming species. In this study, we developed 31 NQ compounds to investigate algicides for mitigating cyanobacterial blooms. In addition, to better apply these compounds in the field, we reduced the number of production steps to develop a cost-effective algicide. In preliminary testing, we screened NQ compounds that showed the best algicidal activity on target cyanobacteria, including Aphanizomenon, Dolichospermum, Microcystis, Oscillatoria, and Nostoc species. The compound NQ 2-0 showed the highest algicidal activity (90%) at a low concentration (≥1 μM) on target algae. These were very limiting algicidal effects of 1 µM NQ 2-0 observed against non-target algae, such as diatoms (Stephanodiscus hantzschii, Cyclotella meneghiniana, Synedra acus, and Aulacoseira granulata) or green algae (Cosmarium bioculatum and Scenedesmus quadricauda), and the effect did not exceed 15–25% (except against S. quadricauda). NQ 2-0 (1 μM) showed no eco-toxicity, as represented by the survival rates of Pseudokirchneriella subcapitata (100%), Daphnia magna (100%), and Danio rerio (100%). Additionally, a chronic eco-toxicity assessment showed no toxicity toward the survival, growth or reproduction of D. magna. Moreover, NQ 2-0 quickly dissipated from field water samples and had a half-life of approximately 3.2 days. These results suggest that NQ 2-0 could be a selective and ecologically safe algicide to mitigate harmful cyanobacterial blooms.

1. Introduction Algicidal chemicals, such as copper sulfate (CuSO4) and herbicides, are frequently used to mitigate harmful algal blooms (HABs) in the field due to their rapid algicidal effects (Anderson, 1997). However, these chemicals can result in the secondary pollution of the aquatic environment (Liu et al., 2004; Li and Hu, 2005; Wagner et al., 2013; Closson and Paul, 2014). Thus, they are rarely applied to fields at present. Over the past few years, biologically derived substances (BDSs) such as polyphenols (Nakai et al., 2000), fatty acids (Nakai et al., 2005), amino acids (Yamamoto et al., 1998), and sesquiterpenes (Shao et al., 2011; ⁎

Wu et al., 2011) have been studied for the control of harmful cyanobacterial blooms. As these substances originate from natural sources, most of them are easily biodegraded in aquatic environments. However, most BDSs are rarely used to mitigate harmful cyanobacteria due to difficulties in applying them in the field. Unfortunately, nonanoic acid (a fatty acid) has occasionally shown unstable algicidal activity due to adaptations of the target species (Shao et al., 2009). In addition, some BDSs do not specifically target cyanobacteria in the aquatic environment and may therefore cause destruction of aquatic ecological systems due to inhibitory or lethal effects on non-target organisms. Furthermore, BDSs such as lysine, rice hulls, and wheat bran

Corresponding author. Corresponding author at: Department of Life Science, Hanyang University, Seoul 04763, South Korea. E-mail addresses: [email protected] (S.H. Kim), [email protected] (M.-S. Han). 1 These authors contributed equally as co-first authors. 2 Current address: Key Laboratory of Marine Ecosystem and Biogeochemistry, The Second Institute of Oceanography, State Oceanic Administration (SOA), Hangzhou, 310012, China. 3 Current address: Marine Science Institute, University of Texas at Austin, Port Aransas, TX 78373, USA. ⁎⁎

http://dx.doi.org/10.1016/j.ecoenv.2017.02.006 Received 25 July 2016; Received in revised form 6 January 2017; Accepted 6 February 2017 0147-6513/ © 2017 Published by Elsevier Inc.

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leachate include N and/or P, which can increase the amount of bioavailable N and/or P in the water where they are applied, thereby exacerbating eutrophication (Takamura et al., 2004; Yang et al., 2008). Therefore, there is a continuing need for the development of improved algicides based on BDSs. Naphthoquinone (NQ) derivatives, which are BDSs, are thought to be one alternative (Joo et al., 2016a). In our previous studies, NQ derivatives showed low toxicity and high algicidal activity and selectivity for the cyanobacteria Microcystis aeruginosa and the diatom Stephanodiscus hantzschii (Joo et al., 2016a, 2016b). Moreover, they did not increase nutrient concentrations or affect the microbial community structure after treatment in mesocosm studies (Joo et al., submitted). It is widely known that various cyanobacterial species, such as A. flosaquae, D. smithii, M. aeruginosa, N. paludosum, and Oscillatoria sp., are responsible for HABs in freshwater (Paerl and Otten, 2013; Zhang et al., 2016). Unfortunately, previously developed NQ derivatives (Joo et al., 2016b) have limited applicability to cyanobacterial blooms due to the narrow range of cyanobacterial species (M. aeruginosa and D. flos-aquae) they can kill. Furthermore, there are economic issues due to difficulties in the mass synthesis of these compounds. In this study, we developed an improved NQ derivative that is effective, selective, cost-saving, and can act as a common algicide against multiple target cyanobacteria species (Dolichospermum, Microcystis, Oscillatoria, Nostoc, and Aphanizomenon). In addition, the ecological risks posed by the NQ compound developed were tested using standard aquatic toxicity model species. In addition, to elucidate the effects of exposure to NQ compounds on aquatic ecosystems, we investigated the persistence and degradation characteristics of a naphthoquinone derivative in field water samples.

Table 1 Comparison of the algicidal activity of naphthoquinone 2-0 on five different cyanobacterial species at the effective concentrations. Species

Effective concentration (µM)

Dolichospermum smithii Microcystis aeruginosa Oscillatoria sp. Nostoc paludosum Aphanizomenon flos-acua

EC90

EC50

0.29 ± 0.009 0.41 ± 0.063 0.99 ± 0.175 0.99 ± 0.025 0.57 ± 0.055

0.20 ± 0.002 0.27 ± 0.028 0.37 ± 0.020 0.33 ± 0.031 0.38 ± 0.011

The data show the mean ± SD. Table 2 Summary of the ecotoxicological data for the effects of naphthoquinone 2-0 from laboratory tests of acute toxicity. Species

Classification

Duration/ end point

EC50, LC50 (µM) (90% confidence intervals)

NOEC (µM)

LOEC (µM)

P. subcapitata

Green algae

0.41

Water flea

1.27

1.80

D. rerio

Fish

8.11 (7.97–8.26) 9.98 (9.89–10.06) 20.6 (18.17–22.02)

0.16

D. magna

72 h/cell density 48 h/ survival 96 h/ survival

12.97

13.60

EC50: 50% effective concentration, NOEC: No observed effective concentration, LOEC: Lowest observed effective concentration

1988) at 15 °C.

2. Materials and methods 2.1. Synthesis of naphthoquinone derivatives

2.3. Screening for the most effective algicidal NQ derivative

The naphthoquinone derivatives listed in Supplementary Table 1 were prepared starting with the basic structures shown in Supplementary Fig. 1. Naphthoquinones were reacted with various substituents to produce different naphthoquinone derivatives. The identity and purity of the NQ derivatives were determined via 1H NMR (Varian Mercury 400 MHz, Varian Inc., Palo Alto, CA, USA) and high performance liquid chromatography (HPLC). The molecular weights of the compound were determined using gas chromatography–mass spectrometry (GC-MS). A total of 31 newly synthesized NQ derivatives were provided by professor Cheon-Gyu Cho from Hanyang University.

For the primary screening of algicidal agents, 31 NQ compounds were selected as candidates (Tables 1 and 2). First, each NQ derivative was dissolved in dimethyl sulfoxide (DMSO) to prepare a 50-mM stock solution and was then serially diluted with fresh, distilled water (DW) to initial concentrations of 500, 200, 100, 50, 20, and 10 µM. Each aliquot (0.1 mL) was inoculated into 48-well plates containing 1.5 mL of mid-exponential phase M. aeruginosa (1×105 cells mL−1), D. smithii (1×105 cells mL−1), Oscillatoria sp. (about 1×105 cells mL−1), N. paludosum (about 1×103 cells mL−1), and A. flos-aquae (about 1×105 cells mL−1). The co-cultures were incubated under algal and cyanobacterial culture conditions for 7 days as described above. All experiments were performed in triplicate. The wells were examined for survival of the host cells at 7 days using a hemocytometer (Superior, Germany) or a Sedgwick-Rafter Counting Chamber (PhycoTech Inc., USA) under microscopic observation at a magnification of 200X (Olympus Co., Japan). Usually, only ‘live’ cells (an integrity form cells and cell with cell contents such as chloroplast) are counted, if without the cell content or organelles (e.g. chloroplast) in the cell, we considered this as dead cell. At this point, the algicidal activity (%) of each compound against each algal species was calculated using the following equation: Algicidal activity (%)=(1-Tt/Ct)×100, where T (treatment) and C (control) are the algal or cyanobacterial cell densities with and without each NQ compound, respectively, and t is the inoculation day. Based on algicidal activity, a four-parameter logistic curve equation (Findlay and Dillard, 2007) was used to derive a 50% inhibition concentration (IC50−240 h) after the inoculation of each NQ compound against each algal species. This function is described as follows:

2.2. Culture of algae and cyanobacteria Cyanobacteria Microcystis aeruginosa (HYK0906-B3), Dolichospermum smithii (HYUA201307-AN25), Oscillatoria sp. (HYHC1409-OS01), and Aphanizomenon flos-aquae (HYL1112-AP1) were isolated from water collected from Gyeongan Stream in South Korea. Nostoc paludosum (KMMCC-1844) specimens were obtained from the Korea Marine Microalgae Culture Center (KMMCC), Korea. The diatoms Stephanodiscus hantzschii (HYHC1405-SH01), Cyclotella meneghiniana (HYND1404CMZ3), Synedra acus (HYND1404SAZ1), and Aulacoseira granulata (HYND1404AGZ2) were isolated from water collected from Nak Dong River in South Korea. The green algae Cosmarium bioculatum (CCAP 612/17) and Scenedesmus quadricauda (CCAP 278/4) were supplied by the Culture Collection of Algae and Protozoa (CCAP), United Kingdom. Cyanobacteria and green algae were cultured in 300 mL conical flasks containing 100 mL of CB medium (Kasai et al., 2004) adjusted to pH 7 and pH 9, respectively, at 25 °C under 50 μmol photons m−2 s−1 on a 12:12 h (light:dark) cycle. Diatoms were maintained at pH 7 under 50 μmol photons m−2 s−1 with a 12:12 h (light:dark) cycle in diatom medium (DM) (Beakes et al.,

Y = Min +

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(Max − Min ) ⎛ ⎛ ⎞−Hillslope ⎞ ⎟ ⎜1+⎜ X ⎟ ⎟ ⎜ ⎝ IC50 ⎠ ⎠ ⎝

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with P. subcapitata and Chlorella vulgaris. The culture water was changed three times per week. The culture and experimental solutions were maintained at 20 ± 2 °C under a 12:12 h (light:dark) cycle, and D. magna were cultured in-house in moderately hard water prepared according to the US EPA guidelines (2002). Acute toxicity tests were performed to determine the 48 h EC50 for D. magna. Four replicates of five juveniles (< 24 h old) were exposed to 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5, 10, and 20 μM of the NQ compound. Medium without NQ was used as the control. The test volume was set at 75 mL for five neonates with four replicates so that the loading density did not exceed 15 mL of medium per neonate. The temperature was kept at 20 ± 1 °C with a 12:12 h (light:dark) cycle. The organisms were not fed during the acute toxicity tests. For the duration of the experiment, death or immobility after 24–48 h of exposure was interpreted as an adverse response. Immobilization was viewed as an endpoint and was noted if no movement was detected for 15 s after gently shaking the test vessel. The acute toxicity tests using D. magna were in accordance with US EPA guidelines (U.S. EPA, 2002). The median effective or lethal concentration (EC/LC50) and the associated confidence interval was calculated using the US EPA probit analysis and the Spearman-Karber method using the computer program TOXSTAT. D. rerio were purchased from a commercial fish supplier (Sejin Aquarium Co., Seoul, South Korea). For two weeks before the start of the experiments, the fish were acclimated to laboratory conditions in 300-L glass tanks following OECD guidelines (1992). During the acclimation period, the fish were fed twice a day with a semi-synthetic diet of fish food (Tetramin). All of the animals were healthy, with a lower than 3% mortality rate observed in the stock during the acclimation period. Four-day static acute toxicity tests were performed in our laboratory to determine the LC50 values of the NQ compound for the D. rerio (OECD guidelines, 1992). On the day of the experiment, seven adult fish were placed in separate 3-L glass aquaria containing a test or control solution (rearing water) that was aerated to maintain the concentration of dissolved oxygen at least 70% of its air saturation value. NQ compound concentrations of 0.625, 1.25, 2.5, 5, 10, 20, and 40 μM were used. Medium without NQ was used as the control. After acclimation to the laboratory conditions, seven adult fish were randomLy distributed into each of the test aquariums. The control D. rerio groups were kept in untreated water. During the acute toxicity test, which lasted 96 h, the animals were not fed. The number of dead fish was recorded at 24, 48, 72, and 96 h. The dead fish were removed from the tanks. The test was conducted at 25 ± 1 °C under a 12:12 h (light:dark) cycle. At the end of the tests, the overall mortality of the fish was recorded (OECD, 1992).

where Y is the observed algicidal effect (%), Max is the highest observed value of the algicidal effect, Min is the lowest observed value of the algicidal effect, x is the NQ concentration, IC50 (the halfmaximum effective NQ concentration) is the inflection point on the calibration curve, and the –Hill slope is a slope factor indicating the largest absolute value of the slope of the curve. 2.4. The optimal concentration of the algicidal compound NQ 2-0 The algicidal activity of the NQ compound against Microcystis aeruginosa (HYK0906-B3), Dolichospermum smithii (HYUA201307AN25), Oscillatoria sp. (HYHC1409-OS01), Nostoc paludosum (KMMCC-1844), and Aphanizomenon flos-aquae (HYL1112-AP1) was examined at various concentrations (0.1, 0.2, 0.5, and 1 µM). Each experiment was carried out in 50-mL culture flasks (SPL) with a total volume of 30 mL per flask. Various concentrations of the test compound were introduced to the cultures during their exponential growth phase. All target cyanobacteria were exposed to the compounds at final concentrations of 0.1, 0.2, 0.5, and 1 µM. The control cultures were not treated with the NQ compound. The cell density of the target cyanobacteria was counted 0, 1, 2, 4, and 7 days after inoculation with the compound. The target cyanobacterial cells were counted using a Sedgwick-Rafter counting chamber under a light microscopic with 100X and 200X magnification (Zeiss Co., Tokyo, Japan). 3. Time-dependent elimination of cyanobacteria by the NQ compound Each experiment was performed in 6-well tissue culture test plates with a total volume of approximately 5 mL per well. NQ compounds were applied to all microalgae at a concentration of 1 µM. All microalgae were treated at their exponential stage of growth. The morphology was examined using a time lapse system (Olympus Co., Tokyo, Japan). 4. Eco-toxicity tests 4.1. Acute toxicity test We used the three ecotoxicological indicator species: Pseudokirchneriella subcapitata (green alga), Daphnia magna (zooplanktonic crustacean), and Danio rerio (zebrafish) to represent three levels of the food chain. The NQ compound toxicity tests were analyzed using the methods recommended by the US EPA (U.S. EPA, 1996a, 1996b, 1996c) and the guidelines from the Organization for Economic Cooperation and Development (OECD 201, 2006; OECD 202, 2004; OECD 203, 1992). The tests were conducted on P. subcapitata (CCAP 278/4) obtained from a culture collection of algae and protozoa (CCAP, United Kingdom) in Argyll, Scotland. The green alga P. subcapitata (CCAP 278/ 4) was pre-cultivated in an Erlenmeyer flask at 20 °C and an irradiance of 50 μmol m−2 s−1 with a 12:12 h (light:dark) cycle for 3–5 days to all them to reach an exponential growth phase. The growth medium was comprised of a 1:1 mixture of EG medium and JM medium (Tompkins et al., 1995). P. subcapitata were cultured in axenic conditions, and only the cultures in a logarithmic growth phase were used for the inoculations. The initial algal density of all the flasks was 1×104 cells mL−1 in a final volume of 25 mL. Triplicates samples from the exponential phases of P. subcapitata were exposed to 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5, 10, and 20 μM of the NQ compound. The medium without NQ was used as the control. The cell density of each of the replicates was measured after 24, 48, and 72 h using a hemocytometer. Among freshwater zooplankton, the cladocerans and Daphnia magna (D. magna) in particular are universally used in routine bioassays of water for safety standards. The D. magna culture was maintained at 20 ± 2 °C in a 1 L polyester jar in the Water Environmental Ecology and Restoration Laboratory at Hanyang University. D. magna was fed daily

4.2. Chronic toxicity tests The effect of the naphthoquinone compound on reproductive output was assessed using a semi-static test according to the standard protocol for the D. magna Reproduction Test (OECD, 1996). The D. magna culture was maintained at 20 ± 2 °C in a 1 L polyester jar in the Water Environmental Ecology and Restoration Laboratory at Hanyang University. D. magna were fed daily with P. subcapitata and Chlorella vulgaris. The culture water was changed three times a week. The culture and experimental solutions were maintained at 20 ± 2 °C under a 12:12 h (light:dark) cycle, and D. magna were cultured in-house in moderately hard water manufactured according to the US EPA guideline (2002). D. magna aged less than 24 h at the start of the test were exposed to three different concentrations of the chemical for 21 days in a two-fold geometric concentration series. Each treatment consisted of 10 beakers of 100 mL, each containing 80 mL of the test solution and a single test species. They were fed daily with 5×106 cells per animal of the algae species P. subcapitata. Test solutions were renewed three times each week. Survival and offspring production were assessed whenever solutions were renewed, and the pH and oxygen content were mea190

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da) even though the concentration of the added compound was high (10 and 20 µM). In contrast, 5 µM NQ 2-0 inhibited the growth of S. quadricauda. However, at a low concentration (1 µM), it did not show significant algicidal activity. Hence, we selected the NQ 2-0 compound for further experiments due to its strong and selective algicidal activity against the target cyanobacteria. The toxicity of DMSO was also investigated and used as the background toxicity (Kim et al., 2011). In addition, the solvent used for NQ 2-0, DMSO at a concentration of 0.1% showed no toxicity towards various phytoplankton (Dolichospermum, Microcystis, Stephanodiscus, and Cyclotella) (Supplementary Fig. 2 and 3).

sured. Test beakers were covered with a glass lid and maintained at 21 ± 0.5 °C under a 12:12 h (light:dark) cycle in an incubator. During the 21 days, mortality, immobility, frequency of molting, cumulative molting, and reproduction were measured every day. In addition, we measured Daphnia body length at the end of the experiment. 4.3. Naphthoquinone compound residual values We conducted an additional small-scale experiment to assess the change in concentration of NQ 2-0 over time. The experiment was prepared in a 12 L transparent polystyrene plastic enclosure (width=30 cm, length=20 cm, and height=20 cm) containing 7 L of freshwater from the Jungnang Streams in South Korea collected in Seoul on August 22, 2015. The synthetic NQ 2-0 compound was dissolved in dimethyl sulfoxide (DMSO) to prepare a 50 mM stock solution and then serially diluted with fresh, distilled water (DW) and stored at 4 °C until use. A stock solution (50 mM) of NQ 2-0 was diluted to produce a working solution of 100 μM of NQ 2-0 in DW. The working solution was transferred into a 25 mL glass vial and shaken at room temperature. The concentration of NQ 2-0 was analyzed using a modification of the spectrophotometric (UV-2120 Optizen, Mecasys, Korea) method of mass analysis. We measured the NQ 2-0 concentration daily. The residual concentration and the half-life of NQ 2-0 were calculated using kinetic equations based on those of Bieglmayer et al. (2002). The decrease in the NQ 2-0 compound over time in field water was determined using an exponential decay equation: t1/2=ln2/k, where t1/2 is half-life time, and k is the rate constant of the decay slope curve.

5.2. The optimal concentration of the algicidal compound NQ 2-0 Before determining the optimal concentration, we investigated the lowest concentration that had algicidal activity on the target cyanobacteria using inoculations with various low concentrations (0.1, 0.2, 0.5, and 1 µM). As shown in Fig. 2, NQ 2-0 showed the highest algicidal activity at 1 µM, and its activities gradually decreased with reduced concentrations. There was no algicidal activity against any cyanobacterial species at a concentration less than 0.2 µM. In Oscillatoria sp. and N. paludosum, 0.5 µM NQ 2-0 showed weaker algicidal effects than 1 µM. To determine the optimal concentration of NQ 2-0, we measured the effective concentrations that showed 50% (EC50) and 90% (EC90) algicidal activity based on the preliminary and lowest concentration tests (0.1, 0.2, 0.5, 1 µM). The EC50 and EC90 values were calculated to be less than 0.38 ± 0.011 and 0.99 ± 0.175 µM, respectively (Table 1). The highest concentration (0. 99 ± 0.175 µM) of NQ 2-0 was require to eliminate Oscillatoria sp. and N. paludosum, while the lowest concentration (0.29 ± 0.009 µM) was capable of killing D. smithii (Table 1).

4.4. Data analysis Toxicity data were fitted to a sigmoidal curve, and a four-parameter logistic model was used to calculate the EC/LC50 values. Analysis was performed using SigmaPlot 12.0. SPSS (Chicago, IL, USA) for windows (version 21) was used for the analyses. One-way analysis of variance (ANOVA) and Tukey test Post Hoc were applied to calculate statistically significant difference of chronic toxicity test results. A P value < 0.05 was considered significant. Weighted analysis of variance (ANOVA) was used, followed by a one-sided Dunnett's test using a 5% significance level to obtain lowest observed effect concentration (LOEC) and no observed effect concentration (NOEC). NOEC was taken to be the test concentration immediately below LOEC (Saker and Neilan, 2001). Statistical analysis of the data was by linear regression analysis using SPSS version 21.0.

5.3. Changes in algal cell morphology due to the algicidal effects To investigate the cellular response and algicidal mechanisms of NQ 2-0, each species of cyanobacteria was observed after treatment with NQ 2-0 (1 µM) using a time-lapse system. The chain-forming cells of D. smithii began to separate within 11 h 57 min after the addition of 1 µM NQ 2-0, and cells were almost completely degraded after 12 h 17 min (Fig. 3a). The cell color of M. aeruginosa disappeared within 16 h 30 min, and cells were completely disrupted after 22 h (Fig. 3b). The chain formation of Oscillatoria sp. and N. paludosum began to break up within 24 h and cells were completely lysed after 72 h (Fig. 3c). Within 24 h of the addition of 1 μM NQ 2-0, A. flos-aquae started to degrade, with cells entirely lysed after 72 h (Fig. 3e). Based on these results, there was a time difference for cell lysis in D. smithii (12 h), M. aeruginosa (24 h), A. flos-aquae (72 h), Oscillatoria sp. (72 h), and N. paludosum (72 h), even though they are all cyanobacteria (Fig. 3).

5. Results 5.1. Screening for the most effective algicidal NQ derivate

5.4. Eco-toxicity test In a preliminary test, we investigated the algicidal activities of various concentrations (1, 5, 10, and 20 µM) of 31 types of NQ derivatives against the target cyanobacteria M. aeruginosa, D. smithii, Oscillatoria sp., N. paludosum, and A. flos-aquae (Fig. 1). Most NQ derivatives showed algicidal activities (≥80%) against D. smithii at 10 and 20 µM but (except for NQ 2-0) were barely effective against M. aeruginosa, Oscillatoria sp., N. paludosum, and A. flos-aquae. NQ 2-0 not only showed algicidal activity (≥80%) against all cyanobacteria species but also eliminated the target species at a low concentration (1 µM) (Fig. 1). To investigate the algicidal selectivity of NQ derivatives, non-target microalgae (Stephanodiscus hantzschii, Cyclotella meneghiniana, Synedra acus, Aulacoseira granulata, Cosmarium bioculatum, Scenedesmus quadricauda) were incubated in the presence of these compounds. The NQ derivatives generally showed growth inhibiting effects 60–80%) on diatoms (S. hantzschii, C. meneghiniana, S. acus, A. granulata) at a concentration of 5 µM (Fig. 1). There were few growth inhibiting effects observed in green algae (C. bioculatum, S. quadricau-

To determine whether NQ 2-0 had toxic effects beyond the target species, we investigated its eco-toxicity using the indicator species Pseudokirchneriella subcapitata, Daphnia magna, and Danio rerio, following EPA protocols (Table 2). Microalgal growth rates in the presence of NQ 2-0 were determined as the ratio of the cell density in each sample compared to the density of the control. The growth of P. subcapitata was not affected by low NQ 2-0 concentrations (0.078, 0.156, 0.312, 0.625, 1.25, and 2.5 μM), but it was significantly affected by high NQ 2-0 concentrations (5, 10, and 20 µM). The growth inhibiting effects also increased with an increase in the concentration of NQ 2-0 after a 24 h exposure. The 72 h EC50 for P. subcapitata was 8.11 µM (Table 2). D. magna mortality was calculated for each NQ 2-0 concentration after 48 h. D. magna survival was not affected by low NQ 2-0 concentrations (0.078, 0.156, 0.312, 0.625, and 1.25 μM), but it was affected by high NQ 2-0 concentrations (2.5 5, 10 and 20 µM). Based on the acute mortality test using the US EPA probit analysis, the LC50 was estimated 191

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Fig. 1. Algicidal activity of 31 compounds (from 1–1 to 2–14) at different initial concentrations (1, 5, 10 and 20 µM) against harmful cyanobacterial species (a) Dolichospermum smithii, (b) Microcystis aeruginosa, (c) Oscillatoria sp., (d) Nostoc paludosum, (e) Aphanizomenon flos-aquae and non-cyanobacterial species (f) Stephanodiscus hantzschii, (g) Cyclotella meneghiniana, (h) Synedra acus, (i) Aulacoseira granulata, (j) Cosmarium bioculatum, (k) Scenedesmus quadricauda.

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Fig. 2. Algicidal effects of various naphthoquinone 2-0 concentrations on (a) Dolichospermum smithii, (b) Microcystis aeruginosa, (c) Oscillatoria sp., (d) Nostoc paludosum, and (e) Aphanizomenon flos-aquae. Error bars indicate SD (standard deviation).

over 10 days and had a half-life of approximately 3.2 days (Fig. 4). However, in distilled water, NQ 2-0 showed little degradation over 20 days (0-day concentration: 4.28 µM, 20-day concentration: 3.52 µM) (Fig. 4).

to be 9.98 µM (Table 2). D. rerio mortality was calculated for each NQ 20 concentration from 24 to 96 h. No mortality was observed in D. rerio after 24, 48, 72, and 96 h of exposure to 0.625, 1.25, 2.5, 5, or 10 µM NQ 2-0. However, significant mortality occurred when the zebrafish were exposed to high NQ 2-0 concentrations (20 and 40 µM). Based on these results, the LC50 value was calculated to be 20.6 µM at 96 h (Table 2). To evaluate the chronic toxicity of NQ 2-0, daily observations on survival, reproduction and growth were made over 21 days. NQ 2-0 had no significant effects on D. magna life cycle traits (Table 3). D. magna survival was calculated for each of NQ 2-0 concentration over 21 days. No mortality was observed in D. magna during 21 days of exposure to 0, 0.5, and 1 μM NQ 2-0 (Table 3). Generally, unexposed D. magna reached sexual maturity, i.e., depositing eggs in the brood pouch for the first time, with the completion of the fourth molt. During the experiment, there were 72, 68, and 77 cumulative molts of Daphnia in the 0, 0.5, and 1 µM treatments, respectively. The day of first reproduction was at 9 days in the 0, 0.5, and 1 µM treatments, and the total number of the reproductive neonates did not differ between the control, 0.5, and 1 µM treatments (Table 3). The total number of Daphnia neonates produced ranged from 221 to 293 depending on the NQ 2-0 concentration. The adult D. magna had average lengths of 3.48 ± 0.50 mm (0 μM), 3.60 ± 0.13 mm (0.5 μM), and 3.37 ± 0.27 mm (1 μM) when exposed to the different NQ 2-0 concentrations (Table 3). These results show that NQ 2-0 did not affect the survival, reproduction, or growth of D. magna.

7. Discussion The naphthoquinone (NQ) family which is the one of BDSs is widespread in nature as a secondary metabolite of micro-organisms, fungi, and plants (O’Brien, 1991; Monks et al., 1992; Tangmouo et al., 2006; Rahmoun et al., 2012), and is widely used in both home remedies and cosmetics (Rostkowska et al., 1998). Interestingly, NQs act as inhibitors at the Q site of the photosynthetic system of phytoplankton (Oettmeier et al., 1986; Biggins, 1990; Jewess et al., 2002). In a previous study, we developed a bio-derived algicidal substance (naphthoquinone) and reduced the drawbacks and side effects of its use (Joo et al., 2016a). However, the previously developed algicidal substance was not economically practical due to difficulties in the mass synthesis of the compound. Therefore, there is a continuing need for the development of new algicides with improved economic efficiency. In this study, we demonstrated efficient and selective algicidal effects and improved economic efficiency in the use of NQ 2-0 to control harmful cyanobacteria. To evaluate the algicidal activity of 31 NQ derivatives at several concentrations on several isolates of D. smithii, M. aeruginosa, A. flosaquae, Oscillatoria sp., and N. paludosum, we used a 96-well microplate bioassay that was previously developed by Schrader et al. (1997). The results of the algicidal tests revealed that at a lower initial inoculation concentration the algicidal activity of each of the compounds was within an effective species-specific range for at least one of the species tested. At a higher initial inoculation concentration, there was a wide range of activity that did not relate to general taxonomic relationships or morphological characteristics. In particular, NQ 2-0 showed the highest algicidal activity toward the target cyanobacteria (Fig. 1). As

6. Residual evaluation of NQ 2-0 To evaluate the persistence of NQ 2-0, its concentration in field water was measured daily for 20 days. As an absorption wavelength of the NQ 2-0 compound results, the maximum absorption wavelength of the NQ 2-0 compound was 265 nm. In order to eliminate the interference, the determination of NQ 2-0 compound was fixed at 265 nm against the reagent blank. NQ 2-0 was sharply degraded in field water 193

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Fig. 3. Light microscopic images of (a) Dolichospermum smithii, (b) Microcystis aeruginosa, (c) Oscillatoria sp., (d) Nostoc paludosum, and (e) Aphanizomenon flos-aquae treated with NQ 2-0 (200X). Scale bar=10 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

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Table 3 Survival, cumulative molting, body length, and cumulative neonates of Daphnia magna after 21 days of exposure to specific concentrations of naphthoquinone. Naphthoquinone concentration (µM)

Survival (%)

Molting/daphnid

Accumulative molting

Body length (mm)

Juveniles/daphnid

Accumulative neonates

0 0.5 1

100 100 100

7.2 ± 0.7a 6.8 ± 1.0a 7.7 ± 0.9a

72 68 77

3.48 ± 0.50a 3.60 ± 0.13a 3.37 ± 0.27a

22.1 ± 4.2a 22.3 ± 5.5a 29.3 ± 5.0b

221 223 293

The data show the mean ± SD. The results were analyzed by one-way ANOVA test. The letters (a, b and c) represent significant differences (n=10; *P < 0.05)

addition, in comparison with copper-based products and diuron, NQ 2-0 offers greater selective algicidal activity toward cyanobacteria relative to effects on other phytoplankton. The treatment of algal blooms using yellow clay has limitations in mitigating bloom effects because of ongoing survival cell and relatively little algal cell disruption following treatment. In contrast, NQ 2-0 showed selective algicidal activity and a high level of cell disruption (> 90%) regardless of cellular condition. Therefore, NQ 2-0 may have a selective algicidal activity against harmful cyanobacterial blooms that is better than that of yellow clay. The chemical structure of NQs may be the reason for their selective and effective of algicidal activity. However, further study is required to identify their effective concentrations, potency, and growth inhibition ratios, as well as to determine the selective patterns that correspond to the functional groups of each derivative. The high activities of particular compounds that are specific against only harmful algal species might be based on specific functional groups and their positions. The biological activity of NQ is due mainly to the presence of two carbonyl groups. From a toxicological perspective, quinones possess two principal chemical properties that confer their reactivity in biological systems: they are electrophiles and oxidants. Quinones are Michael acceptors, and cellular damage can occur through the alkylation of crucial cellular proteins and/or DNA. Alternatively, quinones are also strong redox-active molecules, which can undergo a redox cycle involving their semiquinone radicals, leading to the formation of reactive oxygen species, including superoxide, hydrogen peroxide, and ultimately a hydroxyl radical (O’Brien, 1991). This suggests that the biological activity of NQ is dependent on the electrophilicity of the quinone moiety. The reasons for the cyanobacterial selectivity of NQ 20 are not yet known. It is likely that the target cyanobacteria species lack a protective mechanisms against NQ 2-0 that may be present in non-target species, or that target cyanobacteria species possess more vulnerable sites that can be targeted by NQ 2-0. Toxicity on non-target organisms is one of the key factors that influence the use of an algicide. The special attention should be paid to ecological safety while using the toxicants (Gross et al., 1996). The toxicity of an algicidal substance to aquatic organisms, particularly those common in aquaculture, should be studied first to evaluate any potential negative effects. Most newly synthesized compounds have been described and studied as toxins due to their potential health hazards. In this study, we investigated NQ 2-0 toxicity on the freshwater unicellular green alga (P. subcapitata), zooplankton (D. magna), and zebrafish (D. rerio) using the US EPA standard and OECD protocols. Table 2 shows the results of the acute toxicity tests for all of the tested organisms. All of the eco-toxicity tests were based on the US EPA guidelines (U.S. EPA, 2002). Various concentrations (0.078–40 µM) were tested. NQ toxicity was not observed at the concentration that provided the optimal algicidal activity (1 µM). In the acute toxicity tests, we found no differences between the NQ 2-0 treatment group at a concentration of 1 µM and the control group for any of the three organisms. In addition, we compared the acute toxicity level of NQ 2-0 with that of the reported algicidal substances copper sulfate (CuSO4) and triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol]. The EC50 values of CuSO4 for P. subcapitata and D. similis were 0.344 and 0.032 mg/L, respectively, and the LC50 value for D. rerio was 0.094 mg/L (De Oliveira-Filho et al., 2004). The EC50 values of

Fig. 4. Changes in NQ 2-0 concentration in field freshwater and in distilled water. The black dotted line illustrates the half-life of the inoculated sample.

shown in Table 1, NQ 2-0 required higher concentrations to inhibit the growth of Oscillatoria sp. and N. paludosum, although it showed high algicidal activity against D. smithii, M. aeruginosa, and A. flos-aquae at a low concentration. However, the inoculation concentrations of ≥1 µM can exert significant lysis in M. aeruginosa, D. smithii, Oscillatoria sp., N. paludosum, and A. flos-aquae. If the NQ compound concentration used under natural conditions is too high, there will be negative effects on other aquatic organisms, even if the target algae are controlled. Therefore, 1 µM was determined to be the optimal application concentration for NQ 2-0 for the control of harmful cyanobacterial blooms. Our results indicate that because of concerns regarding environmental safety, the determination of the EC value is necessary to verify speciesspecific control without resulting in undesired consequences. Traditionally, most BDSs focusing on limiting cyanobacterial growth and have been done under laboratory conditions due to complex chemical synthesis and comparative higher cost for field applications (Jančula and Maršálek, 2011; Shao et al., 2013). By contrast, in the case of naphthoquinone, it showed a high algicidal activity at a low concentration (272.3 μg L−1) compared to the other BDSs such as a nonanoic acid (1 mg/L) and L-lysine (10 mg/L) (Kaya et al., 2005; Techer et al., 2016). In addition, we compared the EC50 level of NQ 2-0 with that of the reported BDSs gallic acid, nonanoic acid and L-lysine. The EC50 values of NQ 2-0 for M. aeruginosa was 73.5 µg L−1 (0.27 µM). But, the EC50 values of gallic acid, nonanoic acid and L-lysine for M. aeruginosa was 1.0, 0.5 and 3.65 mg/L, respectively (Nakai et al., 2000, 2005; Takamura et al., 2004). Above all, NQ 2-0 compound may attain > 90% desired algicidal activity merely using once, however, even repetitive application of gallic and nonanic acid did not allow the complete elimination of cyanobacteria (Techer et al., 2016). Therefore, the NQ treatment is more safe and economical application than other BDSs (e.g. gallic, nonanic acid and Llysine) for mitigation of harmful cyanobacterial blooms in field. The feasibility of using NQ 2-0 depends on its ability to selectively target and inhibit cyanobacterial blooms. Therefore, we tested the algicidal activity of NQ 2-0 against various non-target freshwater algae. NQ 2-0 showed algicidal effects on the target cyanobacteria but had no effect on other freshwater algae, such as green algae and diatoms, implying that NQ 2-0 has selective algicidal activity (Fig. 1). In 195

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produce residual compounds. However, additional information is required for field applications of NQ 2-0, including data on its environmental persistence and the identity of its breakdown products. Considering the cost and dilution by current velocity, we think that it is more reasonable to apply algicides in enclosed waters than in an open river. In addition, it is also very important to select algicides that are cost-effective, user-friendly, and environmentally sound even when used in large quantities. Therefore, future studies should compare the economic efficiency of NQ 2-0 to diuron or copper-based products used to help manage musty off-flavor problems in various freshwater columns. A seven-step synthesis procedure is typically required for the synthesis of naphthoquinone for the control of M. aeruginosa (Joo et al., 2016b). However, NQ 2-0 was produced here in a total of four synthesis steps (Supplementary Fig. 4 and 5). Moreover, basic NQ 2-0 is highly economically efficient. Recently, yellow clay has been widely applied to control red tide in Korea (Park et al., 2013; Lee et al., 2013). In 2003, a total of 228,000 t of yellow clay was dispersed for the removal of C. polykrikoides in the southern sea of Korea, at a total cost of $10 million US (Kim, 2006; Park et al., 2013). The cost of applying NQ 2-0 (2280 kg) as an algicide over the same area is estimated to be approximately $0.9 million US (production and field application costs). The costs of yellow clay and NQ 2-0 are substantially different (Supplementary Table 2), considering the labor and transportation costs, NQ 2-0 is cost-effective substance more than yellow clay. In addition, the dispersal and loading of NQ 2-0 are easier than in the application of yellow clay. Although this study identified a novel NQ compound, NQ 2-0, with strong and selective algicidal activity toward harmful target cyanobacteria, more studies are needed to determine the specific site of damage in the target cyanobacteria strains to elucidate the mechanism by which NQ 2-0 acts. Moreover, as a results of our experiment, there is an algicidal activity at > 5 µM concentrations on S. quadricauda, and the application concentration is 1 µM, which is relatively low algicidal activity. NQ 2-0 itself was far more toxic to harmful cyanobacteria than to the S. quadricauda. In other words, NQ 2-0 showed a much widerspectrum activity against cyanobacteria, although the latter affected also the S. quadricauda. However, to safety application in the field, we need more specific experiments such as various cell density of Scenedesmus sp. and various concentration of NQ 2-0. On the other hand, NQ 2-0 proved to be quite specific towards harmful cyanobacteria at low concentrations apparently non-toxic to any of the other freshwater organisms screened. In the present study, algicidal effects were observed at a different time in each species tested. This compound also produced different morphological changes in the target cyanobacteria (M. aeruginosa, D. smithii, Oscillatoria sp., N. paludosum, A. flosaquae) (Fig. 3). Although there have been reports of disruptions to photosynthesis in phytoplankton (Oettmeier et al., 1986; Biggins, 1990; Jewess et al., 2002), this was not confirmed in our study. Therefore, a study on the algicidal mechanisms of NQ 2-0 is required to ensure selective control and ecological safety in field applications of this compound. In addition, to better understand and more safety apply NQ 2-0, future studies should be conducted to properly assess the ecological risks it may pose in the field beyond the effects seen in the laboratory experiments.

triclosan for P. subcapitata and D. magna were 0.0047 and 0.39 mg/L, respectively, and the LC50 for D. rerio was 0.42 mg/L (Orvos et al., 2002; Tatarazako et al., 2003; Oliveira et al., 2009). However, the EC50 values of NQ 2-0 for P. subcapitata and D. magna were 2.20 and 2.71 mg/L, respectively, and the LC50 for D. rerio was 5.61 mg/L. These results show that the acute toxicity of NQ 2-0 was lower than that of the algicides CuSO4 and triclosan. The chronic effects of NQ 2-0 are highly important to the aquatic environment because they represent a more realistic exposure scenario than do the acute toxicity test results. When the chronic toxicity results were compared with and without NQ 2-0, we observed a similar trend in the life cycles of D. magna (Table 3). The results show that at 0.5 or 1 µM concentrations, NQ 2-0 did not affect the growth or reproduction of this species (Table 3). Based on the results of the acute and chronic toxicity assessments, it appears that NQ 2-0 would not negatively affect the freshwater ecosystem if the intact, unmodified algicide was applied to a freshwater cyanobacterial bloom. However, when applying NQ 2-0 via spraying, the actual toxic effect may be lower in an open site where various factors can reduce the toxicity, including the dilution of the algicide by wind or tidal currents, and the coexistence of various species, none of which exist in a closed laboratory system. Therefore, we assume that the toxic effect of NQ 2-0 in a natural ecological system might not be as strong as approximated by our laboratory results. Algicidal compounds produced by specific organisms can be an effective and safe way to control blooms, but determining their proper use is not always an easy task. Rice straw and barley straw, for example, inhibit algal growth in laboratory and field experiments (Everall and Lees, 1996; Park et al., 2006), but the algicidal compounds from barley are unsatisfactory because their inhibitory effect is likely to be a synergistic effect of various compounds (Ball et al., 2001; Ferrier et al., 2005). Therefore, many countries strongly regulate the use of biocides due to their potential effects on aquatic organisms (e.g., Federal Food, Drug, and Cosmetic Act and the Federal Insecticide, Fungicide and Rodenticide Act in USA). The use of aquatic herbicides has been implicated in the decline of pelagic organisms in the upper San Francisco estuary (Scholz et al., 2012). The desirable properties of any algicide include a relatively short half-life and its chemical break-down into non-toxic compounds. These are some of the primary reasons why persistent chemicals have been replaced by organic biocides that characteristically degrade in the environment or become biologically inactive. In the present study, the concentration of the algicide NQ 2-0 decreased over 20 days in field water tests (Fig. 4). The half-life of NQ 2-0 is approximately 3.2 days, which is lower than that of other US EPA-approved herbicides, which have half-lives ranging from a few days to one month (http://plants.ifas.ufl.edu/manage). For example, NQ 2-0 has a much lower persistence in field freshwater samples (halflife of 3.2 day) than diuron, which has been shown to persist for weeks in the water column after application to catfish aquaculture ponds (half-life of two weeks in pond water, Schrader et al., 2003). Environmental safety issues also persist after the use of copper-containing algicidal compound in ponds because the copper accumulates in the pond sediment, and long-term applications may adversely affect microbial activity in these pond sediments (Han et al., 2001). Although NQ 2-0 was not degraded in distilled water, it was quickly degraded in field water (Fig. 4). The reason for this phenomenon is thought to be the existence of a viable route for the removal of many of the alternative factors. This is likely due to degradation by a variety of organisms present in the field water. Additionally, persistence can depend on the physicochemical properties of a compound and the conditions of the environment, such as photolysis by ultraviolet radiation, in combination with a variety of chemicals present in the field water. NQ 2-0 is derived from natural compounds, which are found in certain plants and bacteria (Tangmouo et al., 2006). Even with this degradation, the reduction in cell abundance caused by NQ 2-0 was ≥80% of the initial population in the present experiment. These results indicate that NQ 20 would not negatively affect a freshwater ecosystem and that it did not

8. Conclusion A series of naphthoquinone compounds were synthesized for use as algicides, and their efficacies were examined against the harmful cyanobacteria A. flos-aquae, D. smithii, M. aeruginosa, Oscillatoria sp., and N. paludosum. In particular, NQ 2-0 was found to be the most active candidate, significantly decreasing the growth of the target cyanobacteria. NQ 2-0 had better selectivity for the target cyanobacteria than for the non-harmful algae, and it had a low toxicity, quickly dissipated from field freshwater samples and was found to have a half-life of approximately 3.2 days. Therefore, NQ 2-0 has potential for use as an 196

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