Toxic effects of environment-friendly antifoulant nonivamide on Phaeodactylum tricornutum

Authors


Abstract

Nonivamide, a synthetic derivate of natural capsaicin, has an effective antifouling activity. However, the poor understanding of the toxicity mechanism limits the application of nonivamide in antifouling paints. The present study investigated the inhibitory effects and toxicity mechanism of nonivamide on Phaeodactylum tricornutum. Under a 1.5 × 105 cells/ml of initial algal density (IAD), the effective concentration causing 50% inhibition at 4- d (4 d-EC50) value of nonivamide was 5.1 mg/L. Reactive oxygen species (ROS) level was significantly increased in nonivamide-treated algae. Algal antioxidants, including catalases (CAT), peroxidases (POD), superoxide dismutases (SOD), and glutathione (GSH), were all stimulated by the ROS burst. The excessive ROS substances led to the loss of algal photosynthetic pigments and also damage to the integrity of the lipid membrane. Furthermore, ROS-related genes, including psbA, psbD, psaB, rbcL, nad1, and cob, were found to be suppressed in the chloroplasts and mitochondria of nonivamide-treated algae, and the concentration of cytoplasmic Ca2+, an important regulator of chloroplast and mitochondrion, was elevated. The present study demonstrates that nonivamide could cause peroxidative damages to P. tricornutum by inducing ROS overproduction, which may be initiated by the suppression of ROS-related genes in algal chloroplasts and mitochondria. Environ. Toxicol. Chem. 2013;32:802–809. © 2013 SETAC

INTRODUCTION

Marine biofouling refers to the undesirable settling of marine organisms on the submerged surfaces. The settlement of fouling organisms brings a series of problems to ocean-going vessels, such as extra fuel consumption and poorer ship maneuverability 1. At the global level, 40% extra fuel is consumed because of marine biofouling 2. According to statistics, over 1 billion U.S. dollars are annually spent to prevent zebra mussel biofouling in the United States 1. Various strategies have been developed to prevent this problem, and one effective method is to coat antifouling paints on the surfaces. Such paints are usually used in combination with toxic chemicals called antifoulants, which can effectively protect the coated surfaces from severe bio-adhesion for three to five years 3.

However, the application of these antifoulants has also caused some environmental problems 1. Organotins, as a highly active antifoulant, have been widely used in marine antifouling paints since the 1960s. Recently, studies have revealed that organotins are difficult to biodegrade and can induce malformation and imposex of nontarget organisms 4. Organotins were prohibited for use in marine antifouling paints by the International Maritime Organization (IMO) in 2008. Some biocides, such as Cu(I)-based biocidal pigments, Irgarol 1051 and Sea Nine 211, are used as alternative antifoulants 5. However, some of these alternative chemicals are not environment friendly. For example, Irgarol 1051 was detected in the surface water column of a marina on the Côte d'Azur, France, at concentrations of up to 1700 ng/L 6. It was also reported that Irgarol 1051 can suppress algal photosynthetic functions by binding with the D1 protein in algal chloroplasts 7. In the monitoring of Diuron and DCOIT (Sea Nine 211), these chemicals were found to persistently exist in the sea and disturb the ecological balance of the marine environment 5. Thus, the development of certificated environment-friendly antifoulants is urgently required.

Natural product antifoulants are regarded as future stars in the antifouling paint industry, for the reason that many of them have shown selective antifouling activities against various marine fouling species. This advantage mainly derives from their activities of interfering with the neural signaling pathways in the target fouling organisms 1. For example, most capsaicinoids and cannabinoids have been reported to effectively prevent the byssal attachment of zebra mussels (Dreissena polymorpha) 8. These two kinds of chemical compounds are presumptive ligands of VR1 and CB receptors in zebra mussels 9. Another example is the antifouling activity studies of adrenaline and imidazole, in which both substances have exhibited efficiently inhibitory activities on the adhesion of barnacle cyprids, and the treated larva could still develop into the juvenile phase as usual 10, 11. These results have demonstrated the huge potential of natural products to be used as environment-friendly antifoulants. Yet, because the extraction of natural products from raw materials is too expensive, the application of natural products in antifouling paint formulations is actually not feasible. In marine antifouling paints, some artificial derivates of natural antifoulants are usually used 1.

For capsaicin and its synthetic derivatives, their antifouling activities have been evidenced in some studies. Natural capsaicin, nonivamide, and N-benzoylmonoethanolamine benzoate exhibited the best antifouling activities against zebra mussel adhesion among 19 capsaicin-like compounds 8. In a field coating test conducted by Shi and Wang, the average fouled area on the trial panels coated with capsaicin were decreased by approximately 80%, after a seven-month immersion in the sea 12. In addition, capsaicin exhibited 4d-EC50 values of 5.5 ± 0.5 mg/L, 23.0 ± 2.0 mg/L, 6.9 ± 0.2 mg/L, and 15.6 ± 0.4 mg/L in static toxicity tests conducted using Pseudomonas putida, Lake Erie bacteria, Vibrio natriegens, and Vibrio parahaemolyticus, respectively 13. Nonivamide, as a synthesized derivate of natural capsaicin, is more competitive than the latter in price. However, the poor understanding of its toxicity mechanism has hindered its application on a large scale.

Microalgae are generally used for toxicity mechanism research owing to their short life cycles, simple cultural requirements, and rich genome information 14. As a large and representative group of microalgae, diatoms are widely distributed in the world and contribute 20% of the global photosynthetic production 15. To date, more than 200 genera, approximately 100,000 species of diatoms, have been discovered 16. In recent years, the whole genome sequencing work of Phaeodactylum tricornutum, a marine diatom species with simple cultural requirements, has been finished 17. This advantage makes P. tricornutum an ideal model for scientific researching. Actually, this species of diatom has been already used as an indicator reflecting the changes of atmospheric CO2 and O2 conditions 18.

In the present study, P. tricornutum was selected as the model and an algal toxicity assay was conducted to evaluate the toxic effects of nonivamide. To understand the algal toxicity mechanism of nonivamide, the level of reactive oxidative species (ROS) and the responses of the algal antioxidants, including catalases (CAT), peroxidases (POD), superoxide dismutases (SOD), and glutathione (GSH) content, were analyzed. The possible disadvantageous effects of ROS substances on the algae were also investigated by measuring the activity of alkaline phosphatases (ALP) and the contents of malonaldehyde (MDA) and photosynthetic pigments. Furthermore, to understand the mechanism of ROS overproduction, six ROS-related genes in nonivamide-treated algae and the concentration of algal cytoplasmic Ca2+ were investigated.

MATERIALS AND METHODS

Reagents and materials

Nonivamide (CAS 618-92-8) was ordered from Haida Chemical Company. Ca2+ probe Fura-2/AM was obtained from Beyotime Institute of Biotechnology. Reactive oxidative species probe 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) was obtained from Sigma Chemical. The reagent kits used for GSH and MDA measurements and all enzyme assays were obtained from Jiancheng Institute of Biotechnology. The RNAiso plus reagent kit, diethyl-pyrocarbonate-treated water (DEPC-water), M-MLV reverse transcriptase kit, and SYBR Premix Ex Taq reagent kit were ordered from TAKARA. The primers used for the real-time polymerase chain reaction (PCR) were synthesized by Sangon Biotech. The other chemicals were analytical or higher grades, purchased from Sinopharm Chemical Reagent.

Phaeodactylum tricornutum was provided by the Institute of Oceanology, Chinese Academy of Sciences. Algae were cultured in axenic condition, in f/2 medium that was prepared based on autoclaved natural seawater (filtered through a 0.22-µm filtering membrane). The culture conditions of the illuminating incubator were as follows: fixed temperature, 20°C; photon light intensity, 48 µmol/m2/s; light cycle, 12 h (light):12 h (dark). Algae were cultured to the exponential phase before further experiments. All cultures were shaken twice a day.

The sampling and preparation of algae

Cellular inclusions contents, enzyme activities, and concentration of cytoplasmic Ca2+ were measured after 24, 48, and 72 h of treatment. To measure the concentration of chlorophyl and carotenoid, an extended exposure time of 96 h was carried out for more significant results. For RNA extraction, algal cells were collected after 24 and 48 h of nonivamide treatment. The sampling volumes of algae in these assays were all 10 ml. The initial algal density (IAD) of P. tricornutum for treatment incubation was 1.5 × 106 cells/ml.

Algal toxicity assay

Glass flasks of 100 ml were previously sterilized at 121°C for 40 min. Each of these flasks was filled with 40-ml f/2 algal culture medium (IAD = 1.5 × 105 cells/ml). Nonivamide concentrations were designed as follows: 0, 1, 2, 4, 5, 6, 8, 10, 20, and 30 mg/L. The algal densities were counted daily with a hemacytometer. Algal growth rates were obtained by normalizing the algal densities of the treatment groups with the control.

Algal inclusion and enzyme assays

The algal ROS level was determined by using DCFH-DA as probe 19. Sampled algal cells were suspended in 3 ml phosphate buffer solution (PBS) solution (0.1 M, pH 7.8). Then, DCFH-DA was added to make a final concentration of 5 µM in the PBS solution. After preparation, the mixture was incubated in 20°C for 1 h. Then, algal cells were resuspended in 3 ml PBS solution. The fluorescent intensity of this solution was measured by using a spectrofluorometer (LS55), with an excitation wavelength at 485 nm and an emission wavelength at 525 nm. The obtained result was used to compare the difference of the ROS levels in different treatment groups.

Protein content was measured using the Coomassie brilliant blue G-250 dye-binding method, standardized by bovine serum albumin 20. To measure the contents of algal photosynthetic pigments, algae were suspended in 5-ml 95% ethanol and then kept at 4°C for 24 h. After a centrifugation at 1250 g for 5 min, the supernatant of the algae-suspended solution was obtained and the optical density (OD) OD665, OD649, OD470 values of the supernatant were measured. Concentrations of chlorophyll a and carotenoid were calculated using the formulas from Jespersen et al. 21.

For enzyme assays and the measurement of MDA and GSH contents, sampled algae were washed twice using PBS solution (0.1 M, pH 7.8) and then were suspended in 1.5-ml 10% (v/v) trichloroacetic acid (for MDA measurement) or 1.5-ml PBS solution (0.1 M, pH 7.8, for GSH measurement and enzyme assays). The suspended algal cells were disrupted by using an ultrasonic disintegrator. After a centrifugation at 4°C, 8000 rpm for 10 min, the supernatants were drawn out and used for further operations according to the kit protocols listed as given below.

MDA measurement

A basic premixture solution was prepared by mixing solution 2 and solution 3 at a ratio of 3:1 (v/v). Sample supernatant of 0.5 ml was mixed with 0.5-ml solution 1 and 4 ml basic premixture solution. For the control, 0.5-ml distilled water was mixed with 0.5-ml solution 1 and 4 ml basic premixture solution. After preparation, both mixtures were incubated in 95°C for 40 min and the OD532 values were measured. The results were calculated by using the following formula: Relative MDA Content = (ODSAMPLEODCONTROL)/(ODSTANDARDODCONTROL).

GSH measurement

A sample supernatant of 1 ml was mixed with 1.25-ml solution 2, 0.25-ml solution 3, and 0.05-ml solution 4. For the control, 1-ml solution 1 was mixed with 1.25-ml solution 2, 0.25-ml solution 3, and 0.05-ml solution 4. For the standard, 1-ml standard solution was mixed with 1.25-ml solution 2, 0.25-ml solution 3, and 0.05-ml solution 4. After preparation, these mixtures were placed in ice for 5 min. Then, the OD420 values of these mixtures were measured. The results were calculated by using the following formula: Relative GSH Content = (ODSAMPLEODCONTROL)/(ODSTANDARDODCONTROL).

SOD enzyme assay

A sample supernatant of 0.1 ml was mixed with 0.4-ml solution 1, 0.04-ml solution 2, 0.04-ml solution 3, and 0.04-ml solution 4. For the control, 0.1-ml distilled water was mixed with 0.4-ml solution 1, 0.04-ml solution 2, 0.04-ml solution 3, and 0.04-ml solution 4. After preparation, both mixtures were incubated in 37°C for 40 min. Then 0.6-ml chromogenic reagent was added into them. After 10-min reaction, the OD550 values were recorded. The results were calculated by using the formula: SOD activity = (ODCONTROLODSAMPLE)/ODCONTROL.

POD enzyme assay

A sample supernatant of 0.1 ml was mixed with 2.4-ml solution 1, 0.3-ml solution 2, and 0.2-ml solution 3. For the control, solution 3 was replaced by distilled water of the same volume. After preparation, both the sample and the control were bathed in 37°C for 30 min. Then 0.1-ml solution 4 was separately added into two mixtures. Supernatants of the sample and control were obtained by centrifugation and the OD420 values were recorded. The results were calculated by using the formula: POD activity = ODSAMPLEODCONTROL.

CAT enzyme assay

The reaction solution supplied by the reagent kit was incubated in 28°C for 10 min. Then, 3 ml of this reaction solution was mixed with 0.5-ml sample supernatant. After the preparation, the OD240 value of the mixture was measured for two times with an interval time of 1 min recorded as OD1 and OD2, respectively. The results were calculated using the formula: CAT activity = 2.303 × log (OD1/OD2)/60.

ALP enzyme assay

A sample supernatant of 0.03 ml was mixed with 0.5-ml buffer solution and 0.5-ml substrate solution. For the standard, 0.03 ml was mixed with a 0.5-ml buffer solution and 0.5-ml substrate solution, which was supplied by the reagent kit. For the standard, 0.03-ml distilled water was mixed with 0.5-ml buffer solution and 0.5-ml substrate solution. After preparation, the mixtures were water-bathed in 37°C for 15 min. Then, 1.5-ml chromogenic reagent was added into each mixture and the OD520 value was measured. The results were calculated using the formula: ALP activity = ODSAMPLE/ODSTANDARD.

Cytoplasmic Ca2+ concentration measurement

The cytoplasmic Ca2+ was detected using Fura-2/AM probe. Algal cells were washed twice by PBS solution (0.1 M, pH 7.8) and were suspended in 800 µl of the same solution. Then, 0.5-µl Fura-2/AM (dissolved in DMSO, 2 µM) was added into the algae-suspended solution. After preparation, the mixture was ice-bathed for 20 min and then water-bathed at 37°C for 2 h. After centrifugation, algal cells were resuspended in 4-ml PBS solution. The fluorescent signal of the sample was measured at an emission wavelength of 510 nm, with excitation wavelengths of 340 nm and 380 nm, separately recorded as F340 and F380. The relative amount of [Ca2+]cyt was determined by calculating the ratio of F340/F380 22.

RNA extraction, reverse transcription, and real-time PCR analysis

Real-time PCR was used to analyze the transcriptional level of the selected genes. Gene-specific primer pairs of psbA, psbD, psaB, rbcL, cob, nad1, and 18S ribosomal RNA are listed in Table 1.

Table 1. Primers of photosynthesis and respiratory genes used for real-time polymerase chain reaction
Gene namePrimers (sense and antisense)Transcription product
psbD5′-aggtggtgctttactttgtgctattc-3′Photosystem II protein D2
5′-ttcttctgattgtgttggcgtgaac-3′
psbA5′-caggtgtattcggtggttctttattct-3′Photosystem II protein D1
5′-cgagagttgttaaatgaagcgtattgg-3′
psaB5′-caggtcgtggtggtacttgtgatat-3′Photosystem I P700 apoprotein A2
5′-cgtagccagcccatgatgtaatttg-3′
rbcL5′-cagttgtaggtaaattagaaggcgatcc-3′Ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit
5′-taccaccagaagcaacaggcatac-3′
cob5′-ttgaaactggacaaatagccacactc-3′Cytochrome b
5′-tgcccaacagtaggaattaagataagg-3′
nad15′-cagaagcagaagcagaattggtatcg-3′NADH dehydrogenase subunit 1
5′-gaaacaaccaccctcccagaaaga-3′
18S5′-ggtaattccagctccaatagc-3′Internal reference
5′-gatggcggacccaaaacag-3′

Total RNA was extracted from the fresh algal cells by RNAiso according to the manufacturer's instructions. Briefly, the algae cells were broken in liquid nitrogen, and the RNA was obtained by centrifugation at 5000 g for 5 min. The RNA concentration and purity were determined by measuring the values of OD260/OD280, and a ratio between 1.8 and 2.2 was considered acceptable for purity.

To synthesize the template cDNA, 5.5-µl total-RNA solution was gently mixed with 0.5-µl Oligo (dT)18 primer. After 5 min incubation of 70°C, the mixture was chilled in ice for 30 s and then mixed with 2-µl buffer reagent, 0.5-µl RNase inhibitor, and 1-l dNTP Mix. The obtained solution was slowly stirred for homogenization and kept at 37°C for 5 min. The cDNA elongation was performed by adding 0.3-µl M-MLV reverse transcriptase into the solution to start a 120-min incubation at 37°C. This procedure was stopped by a 10-min water-bath of 70°C.

The real-time PCR system included 10-µl SYBR Premix Ex Taq, 0.4-µl Forward PCR primer, 0.4-µl Reverse PCR primer, 0.4-µl ROX reference dye, and 6-µl template cDNA. The 18S rRNA was used as the housekeeping standard to normalize the deviations of the mRNA and cDNA. The real-time PCR reaction was performed in a 7500 fast real-time PCR system (Applied Biosystems) using a standard two-step amplification method according to the manufacturer's instructions for SYBR Premix Ex Taq. The relative abundances of gene expressions were analyzed using the 2-ΔΔCt method, where Ct is the cycle number at which the fluorescent signal rises statistically above the background 23.

Data analysis

The median effective concentration (EC50) value estimation was calculated using SPSS 17.0. All data shown in the present study were the means ± standard error of three independent experiments and were evaluated by using one-way analysis of variance (ANOVA) followed by the least significant difference test (LSD), p < 0.01 and p < 0.05 (Origin 8.0 for Windows). Figures were shaped using Origin 8.0 for Windows.

RESULTS

Algal toxicity assay and protein content

Algal toxicity assay was conducted to evaluate the effects of nonivamide on P. tricornutum. The results are shown in Figure 1. Dose and time responses could be clearly seen from the results of algal toxicity assay. A significant growth inhibitory effect (p < 0.01) was observed in the group treated with 1 mg/L of nonivamide for 4 d, and the EC50 value (4 d-EC50) was 5.1 mg/L. After 10 d of treatment, the cell densities of the P. tricornutum exposed to nonivamide were 89.1, 60.9, 42.6, 37.0, and 16.2% of the control, in the algae treated with 1, 4, 10, 20, and 30 mg/L nonivamide, respectively. The lowest growth rates in treatment groups, from low to high nonivamide concentration, were 0.44, 0.30, 0.28, 0.23, 0.08, 0.08, 0.14, 0.02, and 0.08. Protein content was measured to investigate the preliminary response of the algae treated with nonivamide. After 24-h exposure, the protein contents in the treatment groups were 1.20, 1.37, and 1.64 times of the control. After 48-h exposure, the protein contents in the treatment groups were 1.66, 2.16, and 1.73 times of the control. After 72-h exposure, the protein contents in the treatment groups were 2.39, 3.09, and 2.76 times of the control. Protein content was significantly increased in the nonivamide-treated algae.

Figure 1.

(A) Survival curves of Phaeodactylum tricornutum treated with nonivamide at an initial algal density (IAD) of 1.5 × 105 cells/ml. (B) Effects of nonivamide on the protein content in P. tricornutum. All error bars indicate standard error of the three replicates. The asterisk (*) indicates a statistically significant difference at p < 0.05 level when compared with the control, and (**) indicates a statistically significant difference at p < 0.01 level.

ROS burst and antioxidative responses in algae

The results shown in Figure 2 are the changes of ROS level and antioxidant enzyme activities in nonivamide-treated algae. The content of ROS substances was obviously increased in the algae exposed to the higher concentrations of nonivamide (10 and 15 mg/L) for 48 h or 72 h. The values in the treatment groups were 0.95, 1.54, and 2.32 times after 48 h exposure, and 1.42, 2.66, and 3.52 times of the control after 72 h exposure.

Figure 2.

Effects of nonivamide on (A) reactive oxygen species (ROS) level, (B) superoxide dismutase (SOD) activity, (C) peroxidase (POD) activity, (D) catalase (CAT) activity, and (E) glutathione (GSH) content in Phaeodactylum tricornutum. All error bars indicate standard error of the three replicates. The asterisk (*) indicates a statistically significant difference at p < 0.05 level when compared with the control, and (**) indicates a statistically significant difference at p < 0.01 level.

The antioxidants were stimulated in the algae treated with nonivamide. For SOD, the activities of the treatment groups and the control showed no significant difference after 24 h of exposure. The SOD activities in the treatment groups were 1.06, 1.62, and 1.98 times of the control after 48 h of exposure, and were 1.43, 2.27, and 2.70 times of the control after 72 h of exposure. Cellular POD activities were obviously increased in the experiment. Cellular POD activity values in the treatment groups were 1.08, 1.74, and 1.28 times of the control at 24 h; 1.65, 1.85, and 2.66 times of the control at 48 h; and 1.61, 2.24, and 2.92 times of the control at 72 h. Similar to POD, the activities of CAT were also significantly increased when algae were treated with nonivamide. The CAT activity values in the treatment groups were 0.95, 1.51, and 2.19 times of the control at 24 h; 2.12, 2.56, and 3.99 times of the control at 48 h; and 6.92, 9.42, and 13.19 times of the control at 72 h.

The GSH contents were increased in the algae exposed to nonivamide. After 24 h of treatment, a significant increase of GSH content was observed in the group of highest concentration (2.58 times of the control, p < 0.01, 15 mg/L). The GSH contents in the treatment groups increased to 2.46, 2.76, and 3 times versus the control after 48 h exposure, and 2.73, 2.91, and 3.78 times of the control after 72 h exposure.

ROS-induced damages

The MDA content, ALP activity, and the contents of chlorophyll a and carotenoid were measured in the algae treated with nonivamide. They were used as parameters reflecting the disadvantageous effects of ROS substances on algal lipid membrane integrity, energy balance, and photosynthetic pigments. The results of the measurements are shown in Figure 3.

Figure 3.

Effects of nonivamide on (A) malonaldehyde (MDA) content, (B) alkaline phosphatase (ALP) activity, (C) chlorophyll a content, and (D) carotenoid content in Phaeodactylum tricornutum. All error bars indicate standard error of the three replicates. The asterisk (*) indicates a statistically significant difference at p < 0.05 level when compared with the control, and (**) indicates a statistically significant difference at p < 0.01 level.

The MDA contents in the treatment groups increased quickly and significantly in the algae treated with nonivamide. The MDA contents in the treatment groups were 1.47, 1.70, and 2.08 times of the control at 24 h; 1.67, 2, and 2.11 times of the control at 48 h; and 1.46, 2.12, and 2.80 times of the control at 72 h. The ALP activity values in the treatment groups showed no significant change until after 48 h. The ALP activity values were 1.95, 2.09, and 2.12 times of the control at 48 h, and were 1.12, 1.41, and 1.44 times of the control at 72 h. The chlorophyll a and carotenoid contents in the treatment groups were not obviously affected by nonivamide in the initial 48 h. The chlorophyll a contents in the treatment groups were 0.88, 0.70, and 0.62 of the control at 72 h, and were 1.01, 0.54, and 0.50 times of the control at 96 h. The contents of carotenoid in the treatment groups were 0.87, 0.84, and 0.72 times of the control at 72 h, and came to 0.99, 0.68, and 0.66 times of the control at 96 h.

ROS-related genes and [Ca2+]cyt concentration

The transcriptional abundances of genes, psbA, psbD, psaB, rbcL, nad1, and cob in the algae were analyzed by real-time PCR. [Ca2+]cyt was considered to be involved in the ROS burst and was also measured in nonivamide-treated algae. The results are shown in Figure 4. It can be seen from Figure 4A that the gene expression of psbA was significantly suppressed in algae treated with nonivamide. The transcriptional levels in treatment groups were 0.74, 0.11, and 0.04 of the control after 24-h exposure, and 0.83, 0.73, and 0.11 times of the control after 48-h exposure. Remarkable suppression of psbD was only observed in the groups treated with higher concentrations of nonivamide (10 and 15 mg/L). Similar to psbD, the transcriptional levels of psaB were obviously suppressed when algae were treated with 10 and 15 mg/L nonivamide. For rbcL, the transcriptional levels of the gene in the treatment groups were 0.48, 0.19, and 0.14 times, and 1.08, 0.51, and 0.08 times of the control after 24- and 48-h exposure, respectively. For nad1, at 24 h, the transcriptional levels in the treatment groups were 2.97, 0.39, and 0.35 times, and 0.98, 0.77, and 0.59 times of the control at 48 h of treatment. For cob, the transcriptional levels in the treatment groups were obviously higher than that of the control after 24-h exposure. However, the transcriptional abundance significantly decreased after 48-h exposure.

Figure 4.

Effects of nonivamide on the transcriptional levels of (A) psbA, (B) psbD, (C) psaB, (D) rbcL, (E) nad1, (F) cob, and (G) cytoplasmic Ca2+ content in Phaeodactylum tricornutum. All error bars indicate standard error of the three replicates. The asterisk (*) indicates a statistically significant difference at p < 0.05 level when compared with the control, and (**) indicates a statistically significant difference at p < 0.01 level.

Shown in Figure 4G, [Ca2+]cyt in the treatment groups were increased with the concentration of nonivamide increasing except for 24-h exposure. The values were separately 1.09, 1.14, and 1.16 times of the control after 48-h exposure, and 1.12, 1.12, and 1.39 times of the control after 72-h exposure.

DISCUSSION

The use of antifouling coatings based on organotins or biocides can lead to severe environmental pollution 1, 3, 6. To prevent great environmental disasters, it is necessary to evaluate the toxic effects of these antifoulants. Algal toxicity assay and protein measurement are commonly used for this purpose 24. In the present study, P. tricornutum was found to be sensitive to nonivamide treatment (Fig. 1A). A slight increase of the treatment concentration can lead to an obvious enhancement of the growth inhibitory effects. When treated with 4 mg/L nonivamide (close to 4-d EC50 value) the inhibition rate of the algae was approximately 40% after 10 d of treatment, which suggests that nonivamide had a chronic growth inhibition on P. tricornutum. In addition, when the treatment time was prolonged to 7 d, hormesis phenomenon was observed in the group treated with 1 mg/L nonivamide. It indicated that the algae possibly adapt to the nonivamide stress.

When treated with nonivamide at low concentrations, the increase of algal protein in the treatment groups indicates that P. tricornutum would synthesize extra protein to cope with the nonivamide stress. However, when the nonivamide concentration was gradually increased, the protein synthesis would be suppressed. It was probable that much more metabolic energy was saved for basic survival (Fig. 1B).

Plants can survive detrimental environment through intracellular physiological adaption. In this process, cellular chemical metabolites, functional peptides, and enzymes are necessary 25. The ROS substances, mainly including superoxide anion, hydrogen peroxide, and hydroxyl radical, were reported to act as principle signaling molecules involving multiple cellular functions, such as cellular stress responses, aging, and apoptosis processes 26. Under normal conditions, excessive ROS substances could be scavenged by various antioxidative enzymes; therefore, the production and scavenging of ROS can reach a dynamic equilibrium. However, when plant cells suffer from persistent environmental stress, the metabolism of ROS substances will be in severe imbalance 24. The accumulated ROS substances would destroy the integrity of cellular lipid membrane and cause metalloenzyme deactivations and DNA damage in plant cells 27. In the present study, ROS content was obviously increased in nonivamide-treated algae (Fig. 2A), which indicated the equilibrium of ROS substances was broken. In addition, we found that when algae were treated with 5 mg/L of nonivamide, the ROS level stayed unchanged until the treatment time was prolonged to 72 h. This result indicates that the ROS-eliminating function of the algal antioxidative system was initiated and excessive ROS substances could not be accumulated in the short exposure time when the algae were treated with a low dose of nonivamide.

To find out the ROS-eliminating mechanism, the content of GSH and the enzyme activities of several antioxidants including SOD, POD, and CAT were measured. The SOD enzymes can catalyze the dismutation of superoxide into oxygen and hydrogen peroxide 28. The POD enzymes, including nicotinamide adenine dinucleotide (NADH) peroxidase, cytochrome-c peroxidase, and many other antioxidative enzymes, can transport electrons from electron donors such as NADH and GSH to the electron receptors like H2O2 and Omath image 29. The CAT enzymes, mainly located in the chloroplasts, mitochondria, and peroxisomes, are used to catalyze H2O2 into H2O and O2 26. The GSH was selected as the parameter to indicate the response of an enzyme-independent antioxidative system 29. These antioxidants were all stimulated (Fig. 2), suggesting that algal antioxidative systems can rapidly respond to a ROS burst. In fact, ROS levels in most treatment groups did not increase with the treatment time; this might have been caused by the ROS-eliminating effects of algal antioxidant systems.

To confirm whether the growth inhibition of nonivamide-treated algae was caused by ROS substances, some biochemistry parameters relating to ROS damages were measured. The MDA is the main by-product of lipid membrane peroxidation, which is correlative to the integrity of algal lipid membrane 30. The ALP enzymes are an important group of isoenzymes involved in the protein dephosphorylation process, which is needed for shutting down the unnecessary enzyme functions. The presence of excessive ROS substances in the algae can consume some additional metabolic energy, which might lead to a shortage of metabolic energy. As a result, unnecessary enzymes would be inactivated and by this means metabolic energy could be saved for the basic survival of algae 31. The ALP enzymes participate in this process by inactivating the unnecessary enzymes that require a large amount of metabolic energy. In addition, the contents of carotenoid and chlorophyll a were measured to check whether the photosynthetic efficiency was decreased by the attacks of ROS substances. In plant cells, the biosynthesis pathway of chlorophyll a and carotenoid require the formation of Mg-protoporphyrin IX from magnesium chelatase and magnesium 32; the excessive ROS substances would block this process. As a result, the algal photosynthetic efficiency would be influenced and the energy deficiency would be aggravated.

The MDA contents in the treatment groups were obviously increased (Fig. 3A), suggesting that the integrity of the algal lipid membrane was seriously damaged by the peroxidative attacks of ROS substances. It is worth noting that in the single-treatment group, no significant change of MDA content was observed from 24 to 72 h, which might be caused by the ROS-eliminating effects of algal antioxidants. Figure 3B shows that ALP activity was significantly enhanced in the treatment groups. The result suggests that enzyme dephosphorylation widely occurred in the nonivamide-treated algae, which further implied the shortage of metabolic energy caused by ROS overproduction. The contents of chlorophyll a and carotenoid were significantly decreased in the treatment groups (Fig. 3C,D). The results indicated that overproduced ROS substances might reduce the efficiency of algal photosynthesis by causing photosynthetic pigment losses. The membrane integrity, algal energy metabolism, and photosynthetic efficiency are very important for the proliferation of algae 24. Therefore, a ROS burst was considered as the direct reason for the algal growth-inhibitory effects of nonivamide.

In plant cells, the function-related electron flows in the chloroplasts and mitochondria are known as the original cause of ROS generation 24. Investigations were conducted to check whether the ROS overproduction might come from these two cellular organs, and the expressions of ROS-related genes in the chloroplasts and mitochondria were measured through real-time PCR.

In chloroplasts, photosystem II (PS II) is the primary functional unit in the electron transporting chain for capturing electrons from water 34. The psbD and psbA encode protein component PsbD1 and PsbD2, respectively, and the two proteins construct a dipolymer functioning reaction center in PS II. The psaB encodes a protein component in the center of the photosystem I (PS I). The rbcL encodes the large subunit of ribulose-1, 5-bisphosphate carboxylase oxygenase (Rubisco), which is the key enzyme of the rate-limiting step in the CO2 assimilation process for producing carbohydrates 35. Figure 4 shows the transcriptional suppression of these genes in the treatment groups. The transcriptional suppression of psbA, psaB, psbD, and rbcL may lead to the block of the electron transporting chain in the chloroplasts and further bring a ROS burst. In mitochondrion, nad1 encodes one of constitutional components of NADH dehydrogenase, which is also known as the complex I, whereas cob encodes complex III. Both complexes are the source of ROS substances in mitochondrion 4. Therefore, the transcriptional expression of nad1 and cob was surveyed to confirm whether ROS substances can also be generated in mitochondrion. As shown in Figures 4E and 4F, transcriptional levels of the two genes were found to be downregulated after the 48-h treatment, indicating that ROS substances may also be induced from mitochondrion.

How could nonivamide induce the gene transcriptional changes in the mitochondria and chloroplasts? Caterina et al. reported that capsaicin could couple with the vanilloid receptor (VR1) to transport Ca2+ across the cellular membrane 36. Although there were no findings about the roles of capsaicin in the Ca2+ pathway and VR1 in microalgae, algae indeed have various Ca2+ conducting channels that are similar to animals 37. Algal chloroplasts and mitochondria could be effectively regulated by cytoplasmic Ca2+. For example, Ca2+ influx from cellular plasma mediated by ionophore A23187 can induce the rotation of chloroplasts; Balsera et al. reported that a Ca2+ channel-forming protein Tic110 was located on the membrane of chloroplasts in Mougeotia cells 36, 38. Figure 4G showed that the concentration of cytoplasmic Ca2+ increased with nonivamide. Based on the results, the following hypothesis was proposed: As a derivative of natural capsaicin, nonivamide can couple with some kind of Ca2+ channel receptor of P. tricornutum and the combination will consequently raise the cytoplasmic Ca2+ concentration, which will further induce the suppression of ROS-related genes and finally lead to ROS overproduction.

Of course, there are many other causes that need to be considered. Organotin compounds and heavy metals can both destroy algal Ca2+ homeostasis by interacting with calmodulins 39. Sea-Nine 211 was reported to induce the efflux of the cytoplasmic Ca2+ from rat liver mitochondria through membrane leakage toxicity 6. Therefore, further studies needs to be worked on the origin of a ROS burst.

CONCLUSIONS

In the present study, it was shown that the concentration of cytoplasmic Ca2+ was increased in the algae treated with nonivamide, and the ROS-related genes in algal mitochondria and chloroplasts were suppressed. It is estimated that the transcriptional suppression of the ROS-related genes was caused by the increase of cytoplasmic Ca2+. The gene suppression can disturb the electron transporting chains in mitochondria and chloroplasts and ROS substances will be consistently produced. The overproduction of ROS substances can consume large amounts of metabolic energy and also lead to the loss of photosynthetic pigments and lipid membrane damages, thereby inhibiting algal growth. The algal toxicity mechanism of nonivamide was studied for the first time to promote the acceptance of nonivamide as an environment-friendly antifoulant. However, to better understand the environmental performances of nonivamide, much work still needs to be done, such as the natural degradation rate, sedimental adsorption, and so forth.

Acknowledgements

The present study was financially supported by the National Science and Technology Supporting Item (2011BAC02B04), the National High Technology Research and Development Program of China (2010AA065105), the Cooperative Program of the Chinese Academy of Sciences and Local Governments (Y12B051011), and the Project from Yantai Science and Technology Bureau (2011063). The authors give their thanks to J. Morrison from the University of Wollongong for his language-editing help.

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