J Appl Phycol (2008) 20:349–358 DOI 10.1007/s10811-007-9262-8

The Inhibitory Effects of Garlic (Allium sativum) and Diallyl Trisulfide on Alexandrium tamarense and other Harmful Algal Species

L. H. Zhou & T. L. Zheng & X. H. Chen & X. Wang & S. B. Chen & Y. Tian & H. S. Hong

Received: 30 March 2007 /Revised and Accepted: 21 September 2007 /Published online: 10 January 2008 # Springer Science + Business Media B.V. 2007

Abstract Using cell suspension ability as an indicator, we effective was a concentration of 0.04% on A. tamarense and studied the inhibitory effect of garlic (Allium sativum) and S. trochoidea. Moreover, the higher the concentration, the diallyl trisulfide on six species of red tide causing algae. stronger was the inhibition, and a high inhibitory rate (IR) This included: the inhibition by 0.08% garlic solution of could be maintained for at least three days when the garlic five algal species — Alexandrium tamarense, Scrippsiella concentration was above 0.04%. For A. tamarense, it was trochoidea, Alexandrium catenella, Alexandrium minutum also found that the longer the inhibitory time and the higher and Alexandrium satoanum; the effects of garlic concen- the concentration, the lower was the rate of resumed cell tration on the inhibition of A. tamarense, S. trochoidea and activity. On the contrary, garlic solution could not inhibit A. Chaetoceros sp.; the effects of inhibitory time on the minutum or Chaetoceros sp.; 2) The IR to A. tamarense was rejuvenation of algal cells; and the effects of heating and reduced slightly as the heating time of the garlic solution preservation time on algal inhibition by garlic solution. In was lengthened, but the average IR was still above 80%. addition, whether or not the ingredients of garlic solution There was no significant difference between the IR of the had a possible algicidal effect was studied by comparing supernatant and sediment of the garlic solution. Further- inhibition of A. tamarense by garlic solution and man-made more, no change of algal inhibition was found when the diallyl trisulfide. The results showed that 1) inhibition by garlic solution was preserved at 20°C for several days; 3) garlic solution was significant on A. tamarense, A. As with garlic solution, diallyl trisulfide inhibited A. satoanum, A. catenella and S. trochoidea, and the least tamarense strongly; the IR was above 93% and was maintained for at least three days, as long as the concentration was 3.2–10.0 mg L−1. Thus, diallyl trisulfide : : : L. H. Zhou T. L. Zheng (*) X. Wang Y. Tian may have been the major ingredient in garlic solution School of Life Sciences, which inhibited the algae but, in addition, more than one MOE Key Lab. for Cell Biology and Tumor Cell Engineering, ingredient may have been inhibiting the algae. In conclu- Xiamen University, Xiamen 361005, China sion, garlic was a good algal inhibitor with many e-mail: [email protected] advantages, such as being common, cheap, non toxic and : : with high efficiency, and diallyl trisulfide, one of the L. H. Zhou T. L. Zheng H. S. Hong components of garlic, was similarly effective in algal State Key Lab. of Marine Environmental Science, Xiamen University, inhibition. It would be useful, therefore, to further study Xiamen 361005, China garlic as an environmentally friendly algal inhibitor. : : L. H. Zhou X. H. Chen S. B. Chen Keywords Red tide causing algae . Inhibitory time . Fisheries College, Institute of aquaculture biotechnology, Jimei University, Inhibitory rate . Allium sativum . Diallyl trisulfide . Xiamen 361021, China Environmentally friendly algal inhibitor

DO09262; No of Pages 9 350 J Appl Phycol (2008) 20:349–358

Introduction Materials and methods

The control of harmful algae is one of the most important Algal culture parts of red tide research since, if algal blooms can be effectively mitigated as they arise, the damage by red tide The experimental algae, except Chaetoceros sp., were can be reduced. One method involves the use of algal provided by the Third Institute of Oceanography, State inhibitors, and many researchers have been trying to find Oceanic Administration, China. They were A. tamarense effective algicides. For example, bluestone (a mineral form strain DH01 (ATDH01), S. trochoidea strain XM01 of blue hydrated copper sulphate) has been proved to kill (STXM01), A. catenella strain DH01 (ACDH01), A. algae quickly and has been applied to deal with algal blooms minutum strain TW01 (AMTW01), and A. satoanum strain for a long time (Elder and Horne 1978; Murray-Gulde et al. DH01 (ASDH01). Chaetoceros sp. was provided by the 2002; Melville et al. 2002). In recent years, more and more Fisheries College of Jimei University. Algal cultures were chemicals having algicidal activity have been found, such cultivated at 20±0.5°C and day / night cycle (12 h:12 h, as amino acid (Hehmann et al. 2002), biquaternary with a light intensity of 52 μmol photons m−2 s−1 as used ammonium salt (Liu et al. 2004), and chlorine dioxide during the experiments), in F/2 media prepared with sterile- (Zhang et al. 2003). However, the obvious shortcoming of filtered seawater. Cells in the exponential growth phase using chemicals as effective algicides is that they them- were used in the experiments. selves may become pollutants in the marine environment (Lam et al. 1995; Peterson et al. 1997; Pierce et al. 2004), Preparation of garlic solution and diallyl trisulfide and serious consideration should be given before applying them to control red tide. For this reason, natural allelo- Garlic (peeled) was mixed with distilled water, in a ratio of chemicals from different organisms have been considered 100 grams garlic per 100 mL water; mashed completely as the source for potential algicides (Wu et al. 1998; using an MSL-218 blender; filtered through a screen in a Lovejoy et al. 1998; Ridge et al. 1999; Xu et al. 2002; press; the volume readjusted; and a 100% garlic concen- Brownlee et al. 2003; Etchegaray et al. 2004). Furusawa tration (100% w/v, garlic weight / garlic solution volume) et al. (2003) found that a marine bacterium, Saprospira was kept at 20°C as the experimental garlic solution. The sp. SS98-5, which was isolated from Kagoshima Bay, Japan, content of allicin (organosulfur components) in the solu- was able to kill and lyse the cells of the diatom Chaetoceros tion, measured using the fixed sulfur method (Chen et al. ceratosporum. Yang et al. (2005) reported that wood meals 2004a), was 2.43‰. The diallyl trisulfide, with a molecular from china fir can inhibit and sink the cells of Alexandrium formula of C6H10S3, was obtained in the form of an allitride tamarense in cultures. Alamsjah et al. (2005) and Wang et al. injection (product number 5E38003), at a concentration of (2006) found that the green algae Ulva fasciata and Ulva 15 mg mL−1, produced by Hefeng Medicine Corporation of pertusa showed strong algicidal activity. In the last few Shanghai. years, we found that red tide inhibitors made with Chinese herbs, such as golden thread (Rhizoma coptidis Huang Lian) Inhibitory effects of garlic solution on different algae and areca seed (Semen arecae Bing Lang), had the merits of low concentration, fast reaction (Zhou et al. 2007). Although Experiments were conducted in 125 mL clean and ster- some algicides have been applied in the control of red tide, ilized flasks. To determine the algicidal activities of garlic most of them are still undergoing laboratory research. In solution against the five red tide phytoplankton ATDH01, addition, most research has focused on fresh water, and not ACDH01, AMTW01, ASDH01 and STXM01, the garlic enough has concentrated on marine water. Thus, it is still a solution (100%, kept at 20°C for 15 days) was added into long-term task to research and develop effective algicides 100 mL algal cultures at a concentration of 0.08% (w/v), with less toxicity for use against marine algae. Garlic and a 100 mL algal culture without garlic was used as the (Allium sativum L), being a common plant, is applied control. Four duplicate treatments were cultivated under extensively in medicine, aquaculture, stock raising as well the same conditions as described above. Samples (1 mL) as human food because of its antibacterial, antiviral and were taken 0.5, 1 and 2 hours after the addition of the nutritional functions. However, no attempt has been made to garlic, and the numbers of algal cells counted using a use garlic as an algicide. Since garlic can be used as both a 0.1 mL counting chamber under an anatomical microscope medicine and food, it should be safer than other chemicals (×40). Samples were taken 1 cm below the water surface, for use as an algal inhibitor. The algicidal effects of garlic with movement, to reduce erroneous results. However, and diallyl trisulfide (one of its components) are reported measures were taken to avoid the re-suspension of sunken here, and the purpose of this study is to discover environ- algae cells. Thus, immobility / sinking of cells to the mentally friendly algicides. bottom was assessed. Each sampling was replicated 3 J Appl Phycol (2008) 20:349–358 351 times, averaged, and the decrease of algae in suspension volumes of supernatant and sediment readjusted. This was used as a cell mobility indicator, namely inhibitory provided a supernatant of garlic solution heated for rate (IR): 20 min (abbrev. 20'S), a sediment of garlic solution heated for 20 min (abbrev. 20′D), similarly, supernatant heated for N0 NS IR ¼ 100% 40 min (40′S), sediment heated for 40 min (40′D), N 0 supernatant heated for 60 min (60′S), sediment heated for Where IR = algal inhibitory rate of garlic; N0 = control algal 60 min (60′D), respectively; and, again, 10 mL fresh garlic −1 −1 density (cell mL ); Ns = treatment algal density (cell mL ). solution (100%), which was centrifugated and its volume readjusted, served as the unheated supernatant (0′S) and the Effects of garlic concentration on three species of algae unheated sediment (0′D). Each of the above liquids (0.08 mL) was placed in a flask and then 100 mL of Three species of algae, ATDH01, STXM01 and Chaetoceros ATDH01 culture was added. Again, the algal culture sp., were tested in this experiment and various concen- without garlic was the control. trations of fresh garlic solution were added into 100 mL algal cultures to test the effect of garlic concentration. Effects of storage time on the algicidal activity of garlic Triplicate treatments were cultivated under the same solution conditions as described above, and the following garlic concentrations (w/v) were used: 0.02, 0.04, 0.08, 0.16 and To determine the effects of storage time on algicidal 0.32%; with an algal culture without garlic as the control. activity, fresh garlic solution (kept at 20°C for 0, 4 and Samples were taken 1, 3, 5 and 7 hours, and 1, 2 and 8 days) was added to the ATDH01 cultures. The final 3 days after the addition of the garlic, and the cell number concentration of garlic was 0.08%, and an algal culture of Chaetoceros sp. was counted using a blood count slide, without garlic was the control. Triplicates were used in each and that of the other two algae using a 0.1 mL counting treatment. chamber. Inhibitory effects of diallyl trisulfide on ATDH01 Effects of inhibitory time on the resumption of ATDH01 cell activity Inhibitory experiments were performed in triplicate, using 300 mL of ATDH01 culture, with the addition of different The fresh garlic solution (100%) was diluted 10-fold with diallyl trisulfide concentrations. The concentrations used filtered sea water as the experimental liquid, and various were 1.0, 1.8, 3.2, 5.6 and 10.0 mg L−1, and an algal culture volumes of experimental liquid were added into each tested without diallyl trisulfide was the control. All the flasks algal culture (10 mL) in the well of a 6-well plate. The final containing algae were shaken well after the diallyl trisulfide concentrations of garlic used were 0.08, 0.16, 0.32, 0.64 was added, and then cultivated as described above. and 1.28%, and, again, an algal culture without garlic was used as the control. After 0.5 and 1 hour inhibition, the Statistical analyses algal cells were filtered through a screen; resuspended in filtered sea water (100 mL); and cultivated under the same The difference in the inhibitory rate or resumption rate conditions as described above. Samples were taken 1 and between the treated and control groups was analyzed using 2 days later, and duplication, sampling and counting were an analysis of variance procedure (SAS) and the data the same as described above. Resumption rate (R) of algal compared using Duncan's Multiple Range Test for varia- cells was: bles. A P-value of<0.05 was considered significant. N R ¼ S 100% N 0 Results

Where R =ATDH01resumptionrate;N0 = control algal −1 −1 density (cell mL ); Ns = treatment algal density (cell mL ). Inhibitory effects of garlic solution on different algae

Effects of heating time on the algicidal activity of garlic At a concentration of 0.08%, the inhibitory effects of garlic solution solution were different in the five algal species tested. Two hours after the treatment, the inhibitory rates were very high Ten mL fresh garlic solution (100%) was added to each test (over 96%) except in the case of AMTW01 (0.8%, P< tube (15 mL); heated in boiling water for 20, 40 or 60 min; 0.001). There was no significant difference between the cooled down; centrifugated for 5 min at 4000 rpm; and the other four species (IR: 96.3–99.9%) (Fig. 1). Generally, the 352 J Appl Phycol (2008) 20:349–358

Fig. 1 Inhibitory rate for five red tide causing algal species cultured in 0.08% garlic solution

longer the treatment, the higher was the IR (P<0.001), but treatment, the IR value increased sharply when the garlic the exception was still AMTW01. Furthermore, a rapid concentration increased from 0.02 to 0.04%, while, with increase of IR was found during the first hour for ATDH01, further increase in garlic concentration, IR values showed a ACDH01 and STXM01; the most rapid increase of IR slightly declining trend. When the concentration was value was from 19.7% to 94.3% from 0.5 to 1 hour in between 0.04–0.16%, the IR values were high (96.6– ACDH01 (Fig. 1). Under the microscope, acute inhibition 99.5% at 5 hours). Both higher (0.32%) and lower of algal cell mobility was observed since the algae sank to (0.02%) concentrations showed lower IR values, and the bottom of the beakers, and either remained motionless significant difference was found among the various con- or were slow swimming. With 1 hour treatment, some cells centrations (P<0.001). After 7 hours, IR increased slightly lost their walls, some were broken and the contents had with increase in concentration, but the IRs of the higher leaked out (Fig. 2). concentrations (0.08–0.32%) were close to 100% and no significant difference was found among the various con- Inhibitory effect of garlic solution on ATDH01 centrations. As for the 0.02% concentration, the IR values were generally lower and even negative at one time, but Cells of ATDH01 were inhibited significantly at concen- with some fluctuation. Based on SAS analysis during the trations above 0.02% (P<0.001) (Fig. 3a). Five hours after whole experiment there was no significant difference in IR

Fig. 2 Variation of algae cell structure after the garlic solution was added to A. tamarense culture. a: the normal cell un- treated by garlic solution; b–e: the cell losing its wall; f: the broken cell J Appl Phycol (2008) 20:349–358 353

Fig. 3 Inhibitory rate of three algal species cultured in differ- ent concentrations of garlic solution. a: A. tamarense, b: Chaetoceros sp. and c: S. trochoidea 354 J Appl Phycol (2008) 20:349–358 among the 0.04, 0.08, 0.16 and 0.32% concentrations (P> 0.05), but a significant difference was found between these concentrations and the 0.02% (P<0.001) results. It was observed also that pigments were bleached with the increase of concentration.

Inhibitory effect of garlic solution on STXM01

The inhibitory effects of different concentrations of garlic solution on STXM01 (Fig. 3c) were similar to those on ATDH01, a high inhibitory rate was observed after 3 hours in concentrations from 0.04 to 0.16%. After 5 hours, the IR Fig. 4 Resumed activity rate of A. tamarense inhibited for different increased first and then decreased with the increase of times at different concentrations concentration. After 7 hours, the higher concentrations (0.08–0.32%) had the higher IR values (94.0–100%), and and 0% respectively. However, when the ATDH01 cells there was no significant difference among them (P>0.05), were inhibited by garlic for 1 hour, the R value only of cells but there was significant difference between the higher at 0.08 and 0.16% concentrations increased slightly with an concentrations and the lower ones (namely 0.02 and 0.04%) extended resumption time, the cells at the other concen- (P<0.001). During the experiment, the IR value of the trations showed almost no activity. 0.02% concentration was generally lower but with some fluctuation. With an extended experimental time, the IR of Effect of heating time on the algicidal activity of garlic the concentrations, except the lower ones (0.02 and 0.04%), solution increased significantly (P<0.001). The IR of various groups increased with an extended Inhibitory effect of garlic solution on Chaetoceros sp. experimental time, and each was more than 90% at 7 hours. Compared with the sediment after the first 7 hours, the IR In general, there was no significant difference of IR among values of the garlic solution supernatants heated for 20 or these concentrations (0.02–0.32%), and, during the exper- 40 min were lower (P<0.05), and those heated for 60 min iment, most IR values were lower and even negative, but were similar (P>0.05). Moreover, there was no significant with some fluctuation (Fig. 3b). The cell densities of all difference among all the groups after 3 days (P>0.05). On groups declined with extended experimental time. For the whole, the supernatant and sediment of fresh garlic example, compared with 3 hours, the cell density of the solutions (without heating) had similar IRs at the same control at day 1 dropped greatly (a decrease of 90.8%), and experimental time. As for the time of heating of garlic the decreases in the other treatment groups were similar solution, the longer the time, the lower was the IR value (89.8–91.1%), showing that there was no significant after the first 7 hours; there was no significant difference in difference among these groups (P>0.05). Furthermore, the the IR among garlic solutions heated for 0–40 min after cell densities of various groups remained low from 1 to 1 day (P>0.05); but all were higher than those heated for 3 days, and no difference was observed both among 60 min (P<0.001). In total, the IR of 0′S was the highest different concentrations at the same time, or among and that of 60′D was the lowest. The sequence of IR among different times at the same concentration (P>0.05). supernatants was 0′S>20′S>40′S≈60′S. For the sediments, the 60′D was significantly lower than the others (P<0.001), Effect of treatment time on the resumption of ATDH01 cell and there was no significant difference among the other mobility three groups (P>0.05) (Fig. 5). Table 1 shows that, when comparing the mean IR values, Figure 4 shows that the longer the treatment with garlic the heating time had a slight effect on them. The supernatant solution, the less was the number of cells that resumed of garlic solution without heating had the highest IR, and swimming (P<0.001); and also the higher the concentration there was significant difference of IR between 0 and 40 or of garlic, the lower the R (P<0.01). When the treatment 60 min (P<0.001). The IR of supernatant declined with an time was 0.5 hour, the R values increased significantly at extended heating time, but there was no significant differ- the lower concentrations (0.08–0.32%) (P<0.001), but not ence among the various heating times (P>0.05). As for the significantly at the higher concentrations (0.64–1.28%) (P> sediment, only the group with 60 min heating time had a 0.05), with an extended resumption time. The 2d R values lower IR and there was significant difference between it and of cells inhibited for 0.5 hour at 0.64 and 1.28% were 3.8 the other heating time groups (P<0.001). However, no J Appl Phycol (2008) 20:349–358 355

decreased rapidly at the same time (P<0.001) (Fig. 7b). With an extended experimental time, the IR values remained high at the higher concentrations but lower, and with some deviation, at the lower concentrations (1.0 and 1.8 mg L−1).

Discussion

Garlic, being common as a food and natural medicine, has Fig. 5 IR value of A. tamarense inhibited for different times by garlic been used widely for thousands of years all over the world. solution which had been given different heating treatments. 0′S= For example, it is often used as an additive to fish feed or as supernatant and 0′D = sediment control; 20′S = supernatant and 20′D= a medicine in aquaculture. In general, garlic and its ′ ′ sediment heated for 20 min; 40 S = supernatant and 40 D = sediment products are believed to have many functions, such as heated for 40 min; 60′S = supernatant and 60′D = sediment heated for 60 min antibacterial, parasiticidal, improving immunity and so on (Roth et al. 1993; Madsen et al. 2000; Buchmann et al. significant difference in the IR values was observed among 2003). In particular, it has been shown to have no the sediments exposed to 0, 20 and 40 min heating time (P> significant side-effects on animals. So far, there has been 0.05). Finally, there was no significant difference between much research about garlic in many fields, but little has supernatant and sediment at the same heating time (P>0.05). been reported as yet involving the use of garlic to control algal blooms. In this study, the results showed that the Effect of storage time on the algicidal ability of garlic effect of garlic was different on various species. In the six solution species tested, the cells of A. minutum and Chaetoceros sp. showed almost no inhibition. Even though they belonged to Based on the SAS analysis, there was no significant the same genus, algae were inhibited differently, thus A. difference in the IR of ATDH01 cells, no matter how long tamarense, A. catenella and A. satoanum were strongly (0, 4 or 8 days) the garlic solution had been stored at 20°C inhibited, the 2 h-IR (0.08%) being more than 96.3%, (P>0.05) (Fig. 6). whereas A. minutum was not effected by garlic. Among the species of algae inhibited by garlic, there was no positive Inhibitory effects of diallyl trisulfide on ATDH01 correlation between the IR value and the concentration of garlic solution after the first 5 hours, indeed the IR values At an diallyl trisulfide concentration of 1.0–10.0 mg L−1, were lower at both higher and lower concentrations, while the IR of different concentrations after the first 0.5 hours they were higher at intermediate concentrations. Under the varied from 42.7 to 80.4%. No significant correlation was microscope, it was seen that the higher the concentration of observed between the concentration of diallyl trisulfide garlic solution, the more glutinous was the water. This is and the IR values, and this same situation occurred after because garlic contains many glutinous components such as the first 3 hours. SAS results showed that there was no polysaccharides and proteins. Obviously, this glutinous significant difference among various concentrations (P> characteristic of garlic solution restrains the sinking of 0.05) (Fig. 7a). The higher the concentration of diallyl inhibited algal cells. For example, at the concentration of trisulfide, the higher was the IR at 1 day. The IR values of 0.32% garlic solution, the algal cells were inhibited quickly the higher concentrations (3.2-10.0 mg L−1)weremore and completely, but immobile cells were still suspended in than 93% from 1 to 3 days, and no significant difference the water after the first 5 hours (Fig. 3a and c). Of course, of IR was observed among these concentrations (P>0.05); these inhibited cells would finally sink to the bottom of the but the IR of lower concentrations (1.0 and 1.8 mg L−1) flask 5 hours later and this is why the IR values of higher

Table 1 Mean inhibitory rate and difference of A. tamarense inhibited by garlic solution after different heating times

0′S0′D20′S20′D40′S40′D60′S60′D

Mean IR(%) 98.6 95.3 93.5 98.0 88.9 97.0 89.8 81.8 difference* A ABC ABC AB DC ABC BCD D

*there was no significant difference among the items having the same letters, but there were significant differences among the items having different letters 356 J Appl Phycol (2008) 20:349–358

high IR at the 0.04–0.32% concentration, the 0.04% concentration can be adopted in the control of algal bloom as it has good algicidal effect, and the water was not too glutinous to restrain cell sinking, further more, it is more economical. In the case of Chaetoceros sp., as a whole, there was no significant difference of inhibitory effect between the treatment and the control. Mostly, the IRs of different concentrations of garlic solution were negative and there was no significant difference among them (negative IR shown in Fig. 3b). At the end of the first hour, the cell Fig. 6 Inhibitory rate of A. tamarense inhibited by 0.08% garlic density increased as the concentration increased, but this solution stored for different times also may be because the glutinous nature of the garlic solution prevented the cells from sinking. After this time, concentration groups were low at the beginning and then the cell densities of the treatment tended to be the same as, increased with an extended experimental time. or even higher than, the control. This result showed that According to this research, the algicidal effect was not garlic solution was useless in the control of red-tide caused obvious when the concentration of garlic solution was by Chaetoceros sp. However, Chaetoceros sp. is an below 0.02% and, indeed, the cell density of the treated important feed for many zooplankton, and control is not groups was higher than that of the control group and the IR the aim in a majority of cases. To find material which is not value sometimes became negative (Fig. 3a). Generally, the harmful to beneficial algae is very important in developing algal cells treated by garlic could not multiply rapidly algicides, and thus garlic is worth further study in this enough to exceed the numbers in the control within a few regard. hours. When observed under the microscope, the cells The efficiency of an algicide relies heavily on the speed treated with a 0.02% concentration of garlic solution moved of action. If the inhibitory action begins slowly, then the faster and were distributed more uniformly in the water algicide may be diluted by currents. Generally, natural than those of the control, and so the probability of counting allelopathic substances require a longer time to control the cells was higher than in the control. According to the some algae than chemicals such as bluestone. For example, the maximum inhibition effect occurred after 2–3 days of the experiment when aqueous extracts (0.25–2.0 g L−1)of U. pertusa were tested for allelopathic activity against three species of red tide microalgae, Heterosigma akashiwo, Skeletonema costatum, and A. tamarense (Nan et al. 2004); while the inhibition of algal activity by barley straw usually occurred only after three months or even one year (Welch et al. 1990; Xu et al. 2002). Our research showed that garlic solution can inhibit algae quickly, and the 3 h-IR value was more than 90% when the concentration was 0.04% or more, and thus, garlic solution may be suitable in an emergency to control algal blooms. In terms of the recovery of inhibited algal cells, garlic is satisfactory as an effective potential algicide since, for example, cells inhibited after 0.5 hours at a concentration of 0.08% showed a resumption rate of cell activity of only 34.8% after 1 day; and after inhibition for 1 hour the rate was only 20.8%. Moreover, algal cells inhibited at a concentration of 0.64% or higher could not recover completely even after 2 days. These results implied that algal cells can be seriously inhibited after even a short contact with garlic solution, and the inhibitory effect can persist for a long time. Microscopic observation revealed Fig. 7 The inhibitory effect of diallyl trisulfide on A. tamarense. a: that the higher the concentration of garlic solution, the more acute inhibition and b: subacute inhibition cells were broken. Furthermore, garlic solution can cause J Appl Phycol (2008) 20:349–358 357 the cells to become motionless, even if they did not break, ASDH01, ACDH01 and STXM01, treated for 2 hours with and gravity will then cause the algae cells to sink, so that 0.08% garlic solution kept at 20°C for 15 days, were greater they will not grow normally because of the lack of light. than 96%. All of this meant that the algicidal activity of Thus, when a large number of cells sink or die, the red tide garlic solution was quite stable. Nan et al. (2004)reportthat will disappear or be lessened and the water might return to the algicidal effect of aqueous extracts of U. pertusa may be normal. due to various components, some of which may lose their Compared with other algal inhibitors such as chemicals activity under high temperature, so that the total inhibitory or Chinese herbs, garlic is a feedstuff additive being widely effect of the extracts decrease. Some researchers also used in aquaculture, and thus should be specially suitable consider that higher plants may inhibit algae by a mixture for application in aquaculture farms, lakes or inshore of components (Li and Hu 2004;Brownleeetal.2003;Xuet aquaculture areas as an algicide with the advantages of al. 2002;Chenetal.2004b). Compared with their work, our having no toxic side-effects, leaving no residues, being results showed that the algicidal action of garlic was economical, and causing no secondary pollution. Thus, it is accomplished by a mixture of many components, and that worthy of further investigation as an algal inhibitor. heating and storage did not seriously impact its inhibitory The major medicinal compound obtained from garlic is ability. Thus, further study is necessary to analyze the allicin, a powerful antibiotic and antifungal. Allicin does algicidal compounds in garlic solution, by separation and not occur in "ordinary" garlic, it is produced when garlic is purification of the components, in order to understand the finely chopped or crushed. And allicin starts to degrade mechanism of algicidal activity. immediately after it is produced, so its medical effective- The results showed that various concentrations of diallyl ness decreases over time. Heating speeds up this degrada- trisulfide (1.0–10.0 mg L−1) could inhibit ATDH01 cells. tion (Chen et al. 2004a). Conversely its breakdown can be At a concentration of 1.0 mg L−1, the highest IR value was slowed by refrigeration. Our results demonstrated that the 90.7% after treatment for 1 day; and the IR values of higher inhibitory effect of garlic solution supernatant declined with concentrations (3.2–10.0 mg L−1) remained high (over increase in heating time, and that the supernatant without 93%) during the experiment. Thus it can be seen that diallyl heating had the highest IR. However, the difference trisulfide had effects on the red tide causing alga, ATDH01. between the highest and lowest IR value was only 9.9% The allicin concentration of the garlic solution during (Fig. 5). This indicated that some of the algicidal treatment can be estimated by measuring the quantity of components in garlic can be destroyed or their activity garlic, the specific gravity of the garlic solution and the decreased by heating. The rapid degradation of allicin under content of allicin in the garlic solution. Taking the lowest high temperature, and the lack of difference in IR values effective garlic concentration (0.04%, see Fig. 3a) as an among the various heating times (20–60 min), also example, the concentration of allicin was about 1.0 mg L−1 demonstrated that allicin may not be the only algicidal of water, and the average IR value was 93.7% in 3 days. component, or that the changes of its quantity and character However, when the concentration of diallyl trisulfide was may not seriously influence the inhibitory effect of garlic 1.0 mg L−1, the average IR value in 3 days was only 69.7%, solution. and at the highest concentration (10.0 mg L−1) it was only Garlic solution is composed of many natural compounds 86.3% (Fig. 7a and b). According to this comparison, the such as allicin (organosulfur components), allithiamine, inhibitory effect of diallyl trisulfide seems to be slightly protein, fat, carbohydrate, coarse fibre and all kinds of weaker than that of garlic solution having a similar microelements. After heating, the cloudy garlic solution concentration of allicin. On the one hand, this demonstrated clarified on standing, and a light brown supernatant was that other algicidal components besides diallyl trisulfide easily separated from the sediment. Not only can the allicin may exist in garlic solution, and this was consistent with in garlic solution degrade, but so can the other components the fact that heating garlic solution would not significantly (for instance, protein and fat). Thus, various complicated influence its inhibitory effect. On the other hand, allicin in changes may have occurred as the garlic solution was garlic solution is more stable than when it is purified (Xu heated. However, in general, there was no significant et al. 2001), and this may cause the difference in inhibitory difference among the various sediment IR values. This effect. showed that heating time was irrelevant to the components Allicin, being an important component of garlic solution and quantity of algicidal materials in the sediments. Another with its associated volatile and offensive smell, has been result of this study showed that no significant difference of widely applied in the medicine, food and aquaculture IR values was observed between the supernatant and the industries because of its broad-spectrum antimicrobial sediment with the same heating time; or between the fresh properties, no drug resistance, and so on. Allicin degrades garlic solution and those preserved at 20°C for 8 days. rapidly to produce various diallyl sulphides, and now, Furthermore, the IR of four species, namely ATDH01, diallyl sulphides (such as diallyl trisulfide), as its degrada- 358 J Appl Phycol (2008) 20:349–358 tion products, can be synthesized easily, conveniently and Liu JS, Zhang H, Yang WD, Gao J, Ke Q (2004) Studies on cheaply. Although the diallyl sulphides are less powerful biquaternary ammonium salt algicide for removing red tide algae. Mar Sci Bull 6:60–65 than allicin, they can still reportedly provide some benefits Lovejoy C, Bowman JP, Hallegraeff GM (1998) Algicidal effects of a to animal health. Moreover, they are also less volatile than novel marine Pseudoalteromonas isolate (class Proteobacteria, allicin, do not degrade as quickly and, importantly, the gamma subdivision) on harmful algal bloom species of the health benefits survive heating. According to our research, genera Chattonella, Gymnodinium,andHeterosigma.Appl Environ Microb 64:2806–2813 man-made diallyl trisulfide has a similar algicidal effect to Madsen HCK, Buchmann K, Mellergaard S (2000) Treatment of garlic solution, and so we believe that it could also be used trichodiniasis in eel (Anguilla anguilla) reared in recirculation to control algal blooms in aquaculture areas. systems in Denmark: alternatives to formaldehyde. Aquaculture 186:221–231 Melville PA, Benites NR, Sinhorini IL, Costa EO (2002) Susceptibil- Acknowledgements This work was supported by the National Sci- ity and features of the ultrastructure of Prototheca zopfii ence Foundation of China (No. 30370276; 40376032), Program for following exposure to copper sulphate, silver nitrate and Changjiang Scholars and Innovative Research Team in University chlorexidine. Mycopathologia 156:1–7 (No. 40521003), the Special Fund for PHD Program in University Murray-Gulde CL, Heatley JE, Schwartzman AL, Rodgers JH Jr (No. 20050384002) and the 973 project (No. 2001CB409710). We (2002) Algicidal effectiveness of clearigate, cutrine-plus, and would like to thank Professor I. J. Hodgkiss for his assistance with copper sulfate and margins of safety associated with their use. English. Thanks also to Associate Professor A. Y. Liu and Dr. W. S. Environ Contam Toxicol 43:19–27 Huang for their help in the experiments. Nan CR, Zhang HZ, Dong SL (2004) Growth inhibition of aqueous extracts of Ulva pertusa on three species of microalgae in red tide. Acta Sci Circums 24:702–706 References Peterson HG, Boutin C, Freemark KE, Martin PA (1997) Toxicity of hexazinone and diquat to green algae, diatoms, cyanobacteria and duckweed. 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