Anthracene phytotoxicity in the freshwater flagellate alga Euglena agilis Carter

Abstract

The freshwater flagellate alga Euglena agilis Carter was exposed to the polycyclic aromatic hydrocarbon (PAH) anthracene for 96 h under optimal photosynthetically active radiation (PAR), and responses of growth, photosynthetic pigment production, and photosynthetic efficiency were assessed. Anthracene reduced the growth rate (μ) and levels of chlorophyll a (Chl a), chlorophyll b (Chl b), and total carotenoids. The growth rate was more sensitive than photosynthetic parameters, with a median effective concentration (EC₅₀) of 4.28 mg L⁻¹. Between 5 and 15 mg L⁻¹, anthracene inhibited the maximum quantum yield (F_v/F_m) of photosystem II (PSII) and the maximum photosynthetic electron transport rate through PSII (rETR_max) with EC₅₀ values of 14.88 and 11.8 mg L⁻¹, respectively. At all anthracene concentrations, intracellular reactive oxygen species (ROS) were elevated, indicating increased oxidative stress. Anthracene presumably reduced the PSII efficiency of photochemical energy regulation and altered the photochemistry through intracellular ROS formation. Acute exposure to PAHs may induce severe physiological changes in phytoplankton cells, which may influence vital ecological processes within the aquatic environments. Additionally, growth and Chl a content may serve as sensitive risk assessment parameters of anthracene toxicity in water management since EC₅₀ values for both overlap with anthracene levels (8.3 mg L⁻¹) permitted by the US Environmental Protection Agency (USEPA).

Introduction

Polycyclic aromatic hydrocarbons (PAHs) comprise a diverse family of hydrocarbons, each composed of two or more fused benzene rings. They are ubiquitous in terrestrial and aquatic ecosystems and are introduced by natural and man-made processes such as volcanic eruptions, crude oil spills, fossil fuel combustion, oil refining, automobile exhausts and industrial effluents¹. PAHs are hydrophobic in nature and their persistence in the environment is a consequence of their low water solubility². Due to their toxic, carcinogenic, mutagenic and photosensitising effects, especially their ability to generate singlet oxygen and other ROS, PAHs are considered one of the most prevalent groups of aquatic contaminants of high global concern³.

Anthracene is a three-ring, low molecular weight PAH with relatively high water solubility than other toxic PAHs⁴. Anthracene adversely affects the growth and photosynthesis of natural phytoplankton communities, with a toxicity threshold value less than its aqueous solubility¹. Moreover, it is one of the most rapidly modified hydrocarbons and is assumed to be a strong photosensitizer⁵ that induces intracellular oxidative stress and blockage of the photosynthetic electron transport chain⁶ through the formation of ROS. Given the wide occurrence and high toxicity to aquatic organisms, anthracene is now on the list of priority pollutants, with a recommended water quality criterion of 8.3 mg L⁻¹ and interim water quality guidelines of 0.012 μg L⁻¹ for the protection of freshwater life set by the Canadian Council of Ministers of the Environment^7,8.

Microalgae are an important group of primary producers in aquatic habitats that play a pivotal role in aquatic ecosystems, forming the food and energy base for all organisms, and powering food webs and biogeochemical cycling. They are readily exposed to toxic waterborne contaminants and tend to reach an equilibrium with pollutants rather rapidly because they are small with a proportionally large surface area⁹. Various studies have reported the toxic effects of PAHs to freshwater algae in terms of growth, photosynthesis, and respiration, with special attention given to interactions with solar and ultraviolet (UV) radiation^10–12. However, Brack et al.¹³ suggested that anthracene toxicity is largely independent of irradiation and the intact compound itself contributes to toxicity. EC₅₀ values reported for anthracene in freshwater microalgae range between 0.024 and 5 mg L⁻¹ (Table 1). Among the various toxicity criteria studied, inhibition of photosynthesis is particularly pertinent as it inevitably results in reduced growth, biomass yield and loss of competitive ecological advantage.

Table 1.

Anthracene toxicity data for freshwater microalgae.

Tested taxa	Criterion	Test period	Effect (mg L−1)	Reference
Chlamydomonas angulosa	Photosynthesis	3 h	0.54, EC50	42
Chlorella vulgaris	Photosynthesis	3 h	0.24, EC50
Selenastrum capricornutum	Growth	96 h	>40, EC50	63
Pseudokirchneriella subcapitata	Growth	34 h	0.037, EC50	1  *
P. subcapitata	Primary production	36 h	0.024, EC50
Chlorella protothecoides	Growth	96 h	0.85, EC50	64
Natural Phytoplankton	Fv/Fm	30 min	<0.2, EC50	10
Chlorella vulgaris	Growth	96 h	1.27, EC50	10
Coenochloris pyrenoidosa	Growth	96 h	1.47, EC50	10
Scenedesmus subspicatus	Growth, area under curve	7 d	1.04, EC50	65
S. armatus	Population density	24 h	0.25, EC50	32
S. vacuolatus	Population density	24 h	0.5, EC50	11
Scenedesmus spp.	Growth	24 h	0.25, significant reduction	66
Chlamydomonas reinhardtii	Population density	24 h	0.28, EC50	38
Desmodesmus subspicatus	Growth	72 h	0.26, EC50	67
Microcystis aeruginosa	Growth	72 h	>0.06, significant reduction	68
Anabaena fertilissima	Growth	8 d	5.0, EC50	62

^*Under UV-A radiation (12.5 Wm⁻²).

Currently, most countries have legislation and regulations on accepted values for toxicity derived from bioassays that are applied to regulate agricultural and industrial chemicals, biocides, cosmetics, food additives, medicines and other substances¹⁴.

The genus Euglena contains motile, unicellular, photosynthetic eukaryotes found in many aquatic habitats, especially shallow eutrophic water bodies. These flagellates have rapid growth rates and can be easily cultured in the laboratory at low cost, ensuring year-round availability. Euglena spp. are sensitive to physicochemical changes and pollution in the surrounding environment, therefore, are potentially used as model organisms in ecotoxicological studies^15,16. Widely used endpoints in bioassays involving Euglena are, growth inhibition¹⁷, photosynthesis and respiration¹⁸, chlorophyll content¹⁹, gene expression²⁰ and motility/orientation²¹. Despite large quantities of toxicity data from the analysis of pollutants such as metals and herbicides^21–23, the effects of PAHs on Euglena remain poorly understood.

The ultimate goal of bioassay tests is to provide representative and incorporative criteria regarding exposure conditions, thereby improving risk assessment and management of water quality. In this respect, multiple, rather than single, endpoint assays may be more reliable for comprehensive risk assessment of toxicants. Such an approach may facilitate insight into the mechanisms of toxicity and provide information on the relative sensitivity of selected parameters to toxicant concentration and/or exposure duration, thereby establishing methods for detecting changes caused by particular phytotoxicants²⁴. In the present study, we investigated the ecotoxicological effects of anthracene on three endpoints of Euglena agilis Carter, including growth, pigmentation, and Chl a fluorescence which were then compared with permitted levels of anthracene in aquatic environments set by the US Environmental Protection Agency (USEPA). Phytoplankton is the main biomass producers in aquatic ecosystems, contributing ca. 50% of the atmospheric carbon dioxide fixation²⁵. Any negative effects of anthracene on the growth and photosynthesis of phytoplankton would be detrimental to entire aquatic ecosystems and food chains.

Materials and Methods

Algal test species and culture conditions

Euglena agilis Carter was cultured in mineral medium (pH 5)²⁶ in 1 L Erlenmeyer flasks at 25 °C under white fluorescent irradiance (PAR; 400–700 nm) of 30 µmol photons m^–2 s⁻¹ (FL400, Kum-Ho, Seoul, Korea) on a 16:8 h light:dark (LD) cycle. All experiments were performed using cells at the exponential growth phase.

Test chemicals and exposure

Anthracene (99% purity, CAS No. 120–12–7) was purchased from Sigma Aldrich (Saint Louis, MO, USA) and test solutions at the desired concentrations were prepared by serial dilution from stocks in high-performance liquid chromatography (HPLC)-grade dimethyl sulphoxide (DMSO; Sigma Aldrich). Microplate toxicity tests of 96 h in duration were conducted in 24-well cell culture plates (well diameter = 15.6 mm, growth area = 1.9 cm²; SPL Life Sciences, Gyeonggi-Do, Korea) with a test volume of 2 mL per well. Equal volumes of cell suspension and anthracene stock solutions were mixed to obtain final concentrations of 0.625, 1.25, 2.5, 5, 10 and 15 mg L⁻¹, along with untreated controls. The concentration of the carrier solvent did not exceed 0.2% v/v of the test culture volume. The initial cell density was 10 ± 0.5 × 10⁴ cells mL⁻¹ of suspension. An additional solvent toxicity test (96 h) was conducted with a maximum DMSO concentration of 0.2% v/v. Organisms were exposed to nominal concentrations of anthracene and all treatments were performed in triplicate. The well plates were covered with parafilm to avoid evaporation and mixing of the volatile toxicant.

Measurement of growth rate

Growth rates were determined by measuring the number of cells in each well on the first and final days using a hemocytometer (Marienfeld, Germany). The specific growth rate (μ) was calculated using the following formula:

$$\mathrm{\mu }=\frac{\mathrm{LN}\left(\frac{N2}{N1}\right)}{(t2-t1)}$$

where N₁ and N₂ are the number of cells at time t₁ (initial) and t₂ (final), respectively.

Estimation of photosynthetic pigments

Photosynthetic pigment content was estimated using standard protocols²⁷. Briefly, 1 mL cell suspension was collected from each replicate culture and centrifuged before extraction, and 1 mL of 90% v/v acetone was added followed by vigorous vortexing and centrifugation at 10,000 × g for 5 min at 4 °C. Supernatants were withdrawn and their optical density was measured spectrophotometrically at 470, 664 and 647 nm using an S-3100 UV/Vis spectrophotometer (Scinco, Seoul, Korea). Pigment concentrations are expressed as μg mL⁻¹ of suspension.

Measurement of chlorophyll a (Chl a) fluorescence

Chl a fluorescence was measured using a pulse amplitude modulation (PAM) imaging instrument (Walz, Germany) as a proxy for photosynthetic performance. For measurement of maximum quantum yield (F_v/F_m) and electron transport rate (ETR), samples were kept in the dark for 10–15 min and then subjected to pulsed light emitted by a diode at ~0.15 µmol photons m⁻² s⁻¹ to obtain the initial fluorescence yield (F_o), which denotes the fluorescence yield when all PSII reaction centres are open with fully oxidized plastoquinone A(QA). A saturation pulse of ~5000 µmol photons m⁻² s⁻¹ emitted by a built-in halogen lamp was then applied to produce the maximum fluorescence yield (F_m), which is induced by a short saturating pulse of actinic light that reduces all QA. The maximum PSII quantum yield (F_v/F_m) was then derived from the equation (F_m − F_o)/F_m.

Rapid light curves were produced using 10 s pulses of actinic light increased stepwise from 0 to 335 (0, 1, 11, 21, 36, 56, 81, 111, 146, 186, 231, 281 and 335) μmol photons m⁻² s⁻¹. The relative electron transport rate (rETR) was calculated by multiplying the effective quantum yield (ΦPSII = F′_m − [F/F′_m], where F′_m is the maximum light-acclimated fluorescence yield and F is the light-acclimated fluorescence yield) by photon flux density (PFD) and plotting against PFD. The ETR is relative because the absorbance of light by cells was not measured. Maximum electron transport rate (ETR_max) was derived from the hyperbolic tangent formula rETR = ETR_max ∗ tanh (α/I/ETR_max), adapted from Jassby and Platt²⁸, where α indicates the electron transport rate under light-limited conditions. Alterations in Chl a fluorescence due to changes in non-photochemical quenching (NPQ) and photochemical quenching (qP) were calculated from (F_m − F′_m)/F′_m and (F′_m − F_t)/(F′_m − F′_o), respectively²⁹.

Measurement of ROS levels

The oxidant-sensing fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma Aldrich; CAS No: 4091–99–0) was used to detect intracellular ROS generation in E. agilis treated with anthracene. DCFH-DA (5 μM, final concentration) solubilized in ethanol was added to the cell suspension and incubated on a shaker at room temperature in the dark for 1 h³⁰. The fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a Spectra MAX Gemini EM microplate fluorescence reader (Molecular Devices, CA, USA). The relative production of ROS is represented as relative fluorescence units (RFU).

Statistical analyses

Data are presented as means ± 95% confidence intervals (CI). All parameters were compared across treatments with one-way analysis of variance (ANOVA, n = 3, p < 0.05) using the JMP software (JMP^® Pro version 13.1, SAS Institute, USA). Multiple comparison tests based on the least significant difference (LSD) were then carried out to find significant differences (p < 0.05) from controls and between treatments. The effective concentration at which 50% inhibition occurs (EC₅₀) was estimated by the linear interpolation method using ToxCalc 5.0 (Tidepool Science, USA). The coefficient of variation (CV), the standard deviation expressed as a percentage of the mean, was calculated to estimate the precision of test values.

Capsule

Anthracene significantly reduces growth and photosynthesis in the freshwater flagellate Euglena agilis via intracellular ROS generation.

Results and Discussion

Effect of anthracene on cell growth

The carrier solvent used in this study (DMSO) had no significant inhibitory effects on cell growth (ANOVA, df = 6, F = 1.3, P > 0.05) or photosynthetic efficiency (ANOVA, df = 5, F = 0.56, P > 0.05) of E. agilis, even at the maximum concentration of 0.2% (v/v) in the growth medium (Fig. 1). Okumura et al.³¹ previously demonstrated the suitability of DMSO as a carrier solvent in Euglenoid tests.

Figure 1.

Effect of the carrier solvent DMSO on growth and photosynthesis (F_v/F_m) of E. agilis. A maximum concentration of 2% (v/v) was tested.

Most of the toxicity data available for the effects of anthracene on freshwater microalgae are based on growth inhibition (Table 1). Growth is an important endpoint parameter that reflects the overall vitality of a population under the tested conditions. Addition of anthracene to the culture medium resulted in a concentration-dependent inhibition of the specific growth rate of E. agilis (Fig. 2). Compared with controls, the final day cell densities were significantly lower at all tested concentrations, and μ was significantly reduced from 0.53 for control cells to 0.12 at the highest anthracene dose (ANOVA, df = 6, F = 198.82, P < 0.001). The EC₅₀ value for growth was 4.28 mg L⁻¹ (Table 2), which is greater than the values previously reported for several freshwater microalgae (Table 1). At nominal concentrations exceeding 0.05 mg L⁻¹, anthracene significantly inhibits the growth of freshwater phytoplankton³². For example, the growth of Selenastrum capricornutum was extremely sensitive to anthracene, aggravated by UV radiation, with a 22 h EC₅₀ value of 3.9–37.4 μg L^−1 33. Our results suggest that anthracene itself is potentially toxic to freshwater primary producers, and could add synergistic effects with other stressors such as UV radiation³².

Figure 2.

Effect of 96 h anthracene exposure on the specific growth rate (days⁻¹) of E. agilis. Mean and 95% confidence intervals (CI) are shown (n = 3). Vertical bar indicates LSD, least significant difference.

Table 2.

NOEC, LOEC and EC values (mg L⁻¹) plus CI and p-values for inhibition of E. agilis exposed to anthracene for 96 h.

Test Criterion	NOEC	LOEC	Mean EC10, 95% CI and CV (%)	Mean EC50, 95% CI and CV (%)	p-value
Growth rate (μ)	<0.625	0.625	0.38 (0.3–0.55) 10.10	4.28 (3.58–5.14) 6.63	<0.05
Chl a	<0.625	0.625	0.31 (0.12–0.22) 8.46	5.59 (2.84–7.98) 11.77	<0.05
Chl b	<0.625	0.625	0.34 (0.16–0.63) 16.56	8.14 (5.52–10.73) 7.99	<0.05
Carotenoids	<0.625	0.625	0.41 (0.34–0.54) 6.35	>15	<0.05
Fv/Fm	2.5	5	3.18 (0.93–4.93) 14.88	13.74 (11.90–15.65) 3.41	<0.05
rETRmax	2.5	5	2.16 (0.89–5.52) 15.57	11.80 (8.92–14.68) 7.43	<0.05

^*NOEC, no observed effect concentration; LOEC, lowest observed effect concentration; CI, confidence interval; CV, coefficient of variation.

^*Mean and 95% CI are shown (n = 3).

Growth inhibition due to PAH exposure in microalgae and higher aquatic plants has been previously reported³⁴, and the extent of growth inhibition depends on the species studied, the chemicals tested and the duration of exposure. Reduction in growth can result from an accumulation of anthracene within the lipid fraction of cells and subsequent changes in membrane properties³⁵. PAH accumulation in membranes can cause an expansion of the membrane surface area, inhibition of primary ion pumps, and an increase in proton permeability, leading to dissipation of the electrical potential and pH gradient, which ultimately results in inhibition of cellular growth³⁶. Additionally, a reduction in photosynthesis can lead to impaired growth, since these are highly interrelated phenomena, each being a function of the utilization of energy from light and nutrients. Even moderate changes in the function of the photosynthetic apparatus can lead to a marked reduction in energy production within chloroplasts³⁴.

Effect of anthracene on pigment content

Euglena contains both Chl a and b as light-harvesting pigments, along with the carotenoids, diadinoxanthin, and diatoxanthin³⁷. Despite studies on the effect of anthracene on growth and photosynthesis in algae, limited information is available on their interference with photosynthetic pigment production. Anthracene did not affect chlorophyll biosynthesis in Chlamydomonas reinhardtii strain cw92 at concentrations up to 1 mg L^−1 38, or in three Desmodesmus spp. up to 0.25 mg L^−1 37. However, in the present study, anthracene (>0.625 mg L⁻¹) had a pronounced effect on photosynthetic pigments content in E. agilis. The most abundant pigment in E. agilis was Chl a (7.14 μg mL⁻¹) followed by carotenoids (1.72 μg mL⁻¹) and Chl b (1.25 μg mL⁻¹). At the lowest test concentration (0.625 mg L⁻¹), there were significant reductions in Chl a, Chl b and total carotenoids of up to 20%, 16%, and 17%, respectively, while at the highest concentration, reductions of 58%, 64%, and 49% were observed (Fig. 3). The adverse effect on pigment content was concentration-dependent, with 96 h EC₅₀ values of 5.59 mg L⁻¹, 8.14 mg L⁻¹ and >15 mg L⁻¹ for Chl a (ANOVA, df = 6, F = 334.54, P < 0.05), Chl b (ANOVA, df = 6, F = 40.05, P < 0.05) and total carotenoids (ANOVA, df = 6, F = 130.11, P < 0.05), respectively (Table 2).

Figure 3.

Effect of exposure to anthracene for 96 h on photosynthetic pigmentscontent in E. agilis; (a) Chl a, (b) Chl b, and (c) total carotenoids. Mean and 95% confidence intervals (CI) are shown (n = 3). Vertical bars indicate LSD, least significant difference.

The molecular mechanism of the reduction in pigment levels may involve the accumulation of lipophilic anthracene in thylakoid membranes³⁹, resulting in conformational changes in their structure and composition. In general, reduced pigmentation under chemical stress results from inhibition of enzymes related to chlorophyll synthesis, degradation of chlorophyll and DNA damage⁴⁰, or accelerated degradation of pigments due to increased ROS formation at various positions in the photosynthetic electron transport chain. Moreover, carotenoids prevent photo-oxidative destruction of chlorophylls⁴¹ and, therefore, a reduction in carotenoids could have additional serious consequences on chlorophyll molecules. The simultaneous reduction in all three photosynthetic pigments suggests that the major target of anthracene toxicity is the thylakoid compartment of chloroplasts. These results also indicate that in addition to causing a severe reduction in growth, anthracene exposure may reduce photosynthetic performance via the destruction of pigments responsible for harvesting available photons.

Inhibition of photosynthesis

Anthracene is a strong inhibitor of phytoplankton photosynthesis in vivo^32,35,38,42. We conducted in vivo Chl a fluorescence measurements as an intriguing tool to reveal the toxic effects of anthracene on the photosynthetic machinery of E. agilis (Fig. 4). The quantum yield and quantum efficiency parameters are indicators of the efficiency of solar energy absorption, which decreases under chemical stress, implying that stress negatively impacts photon absorption and conversion of solar energy during photosynthesis⁴³. We found that at higher anthracene concentrations (>5 mg L⁻¹), there were significant reductions in dark fluorescence (F₀; ANOVA, df = 6, F = 40.90, P < 0.05), which reflects emission by excited Chl a molecules in the antennae structure of PSII, and in maximal fluorescence (F_m; ANOVA, df = 6, F = 33.54, P < 0.05), which can be attributed to severe loss of pigments and/or inactivation of PSII reaction centres. It is evident that at higher anthracene doses, reduction in the number of cells and pigment levels resulted in an overall decline in the light-harvesting by E. agilis. Moreover, no significant variation (ANOVA, df = 3, F = 2.01, P > 0.05) in F₀ was observed between 0–2.5 mg L^–1 anthracene, despite significant reductions in the concentration of Chl a (Fig. 3a), suggesting that pigment molecules associated with PSII reaction centres are less affected. Instead, anthracene may pose a more serious threat to the pigment pool of PSI. This interpretation is supported by the findings of Huang et al.⁴⁴, who suggested that PSI is the primary site of action of anthracene. However, Chl a fluorescence measurements in plants and algae have suggested inhibition of the cytochrome-b6/f complex and/or photo-oxidative damage to PSII as additional modes of anthracene toxicity^6,38.

Figure 4.

Effect of exposure to anthracene for 96 h on Chl a fluorescence parameters of E. agilis. (a) Minimum fluorescence (F_o). (b) Maximum fluorescence (F_m). (c) Maximum quantum yield of PSII (F_v/F_m), (d) Non-photochemical quenching (NPQ), (e) Maximum relative electron transport rate (rETR_max). Mean and 95% confidence intervals (CI) are shown (n = 3). Vertical bars indicate LSD, least significant difference.

F_v/F_m, an estimate of the photochemical conversion efficiency of PSII in the dark, has been widely used to assess the acute toxicity of aromatic hydrocarbons in freshwater plants and phytoplankton^6,35. An F_v/F_m value of ~0.55 relative units (RU) was recorded in our control E. agilis population, comparable to the value reported previously for Euglena gracilis²¹. F_v/F_m did not significantly vary up to 1.25 mg L⁻¹ anthracene (Fig. 4c). However, at higher concentrations, F_v/F_m was declined (ANOVA, df = 6, F = 63.18, P < 0.05) by 17% (5 mg L⁻¹), 36% (10 mg L⁻¹), and 55% (15 mg L⁻¹) with an EC₅₀ of 13.74 mg L⁻¹ (Table 2). Toxicity of anthracene on F_v/F_m in microalgae taxa has not been reported previously, so direct comparison of the sensitivity of E. agilis with other species is not possible. Nevertheless, in the macrophyte Lemna gibba, F_v/F_m appeared to be a more sensitive biomarker of anthracene toxicity, with a 4 h EC₅₀ value of 2 mg L^−1 44.

We noted that the reduction in F_v/F_m at >2.5 mg L⁻¹ anthracene was accompanied by a significant loss of NPQ (Fig. 4d). Although values were not statistically significant, NPQ tended to increase up to 1.25 mg L⁻¹ and then decreased significantly thereafter (ANOVA, df = 3, F = 44.74, P < 0.05). This decline in Chl a fluorescence quenching can be attributed to impairment of electron transport downstream from PSII and an elevated reduction of the PQ pool⁴⁵. NPQ is produced through the generation of an H⁺ electrochemical gradient across the thylakoid membranes⁴⁶ and is an indicator of absorbed energy that is dissipated through heat loss and other non-photochemical mechanisms. We assume that a severe reduction in the photosynthetic process at high anthracene levels likely reduces the magnitude of the pH gradient, thereby affecting NPQ values, consistent with the view of González–Moreno et al.⁴⁷ who reported similar observations on fluorescence quenching in E. gracilis under salt stress. Severe reduction in F_v/F_m of E. gracilis exposed to NaCl had resulted in a diminution of the pH gradient across the thylakoid membranes and subsequently, up to 95% reduction in NPQ⁴⁷.

In the present study, anthracene reduced the ETR across PSII at higher concentrations (Fig. 4e). rETR is an empirical estimate of the rate of the flow of electrons through the electron transport chain. rETR_max was not significantly affected up to 1.25 mg L⁻¹ anthracene, whereas 5, 10 and 15 mg L⁻¹ doses resulted in 18%, 46%, and 56% reductions, respectively, with an EC₅₀ value of 11.8 mg L⁻¹ (Table 2). Thus, the threshold value of anthracene for a significant reduction in ETR was higher than that for growth inhibition. In aquatic plants, PAHs inhibit photosynthetic electron transport at concentrations below which growth and CO₂ fixation are inhibited⁴⁴. For PAHs in general, the target of their toxicity to photosynthesis is the electron transport downstream from PSII, specifically at cytochrome-b6f. Inhibition of electron transport blocks reoxidation of the reduced plastoquinone pool (PQH₂) and the absorbed energy cannot be used in photochemistry⁶. A probable consequence of inhibition of the electron transport chain at PSII is the transfer of energy from triplet chlorophylls to oxygen, forming singlet oxygen species, which induces oxidative damage of cells⁴⁸. The generation of free radicals and subsequent intracellular oxidative stress is a prominent mechanism of anthracene toxicity in freshwater phytoplankton³⁸.

When DCFH-DA fluorescence emission in anthracene exposed E. agilis cells was measured, a significant increase was observed at all dosages (ANOVA, df = 6, F = 81.11, P < 0.05), indicating a rise in intracellular ROS levels (Fig. 5). ROS level at 2.5 mg L⁻¹ anthracene was almost double than that in the controls. The subsequent reduction in fluorescence at high anthracene (>5 mg L⁻¹) can be attributed to reduced cell growth and diminished enzyme activities, although values were still significantly higher (p < 0.5) than in controls. The major site of ROS production in photosynthetic organisms is the disrupted electron transport chain across PSII⁴⁹. We report here, for the first time in freshwater microalga taxa, the significant elevation of intracellular ROS levels under anthracene stress. In Euglena spp., ROS play a significant role in metal toxicity⁴⁵, UV damage and defense mechanisms⁵⁰. However, ROS, generated by chemical stressors, trigger adverse effects through multifaceted actions inside the cell. They attack thylakoid lipids and initiate peroxyl radical chain reactions, eventually destroying membranes and pigment-protein complexes⁴⁵. Moreover, in chloroplasts, ROS cause lipid peroxidation, which results in the disruption of photosynthetic pigments, and the inactivation and degradation of RuBisCo and other components of the Calvin cycle⁵¹. Our results correspond to Babu et al.⁵², who found that 1,2-dihydroanthraquinone, a photoproduct of anthracene, inhibited photosynthetic electron transport, leading to the overproduction of O₂⁻ and subsequent oxidation of proteins, membranes, and pigments in Lemna gibba.

Figure 5.

Effect of exposure to anthracene for 96 h on DCFH-DA fluorescence intensity. The excitation wavelength was 485 nm and the emission wavelength was 530 nm. Mean and 95% confidence intervals (CI) are shown (n = 3). Vertical bar indicates LSD, least significant difference.

DCFH-DA is more suitable to estimate total ROS production rather than as a probe for a particular type of ROS⁵³. The superoxide anion radicals (O₂⁻) produced in the electron transport chain is a precursor for many other ROS species. They are rapidly converted to hydrogen peroxide (H₂O₂) and subsequently to hydroxy radicals (OH^•) by enzymatic reactions⁵⁴. DCFH-DA does not directly react with O₂⁻ but can be oxidized to highly fluorescent DCF by H₂O₂ and OH^• radicals⁵⁵. Thus, DCFH-DA probing of ROS revealed the overall cellular redox status of E. agilis under anthracene stress. Higher ROS also reflect the inefficiency of both photochemical pathways and protective regulatory mechanisms to process the excitation energy at PSII. Our data suggest that ROS generation and consequential oxidative stress play a pivotal role in acute anthracene toxicity in the model organism, E. agilis. We detected significant ROS levels under optimal PAR irradiation, where photo-modification of the parent compound is less likely. Under high oxidative damage, Euglena relies on the activation of antioxidant enzymes such as ascorbate peroxidase (APX) and glutathione peroxidase (GPX)⁵⁶, and biosynthesis of antioxidant metabolites such as reduced glutathione (GSH) and its derivatives⁵⁷. Furthermore, some canonical metabolites act as indicators of oxidative damage, such as malondialdehyde (MDA)⁵⁸. Thus, antioxidant/oxidant responses upon anthracene exposure may represent a promising area for further investigation.

We further analyzed the three photochemical quantum yields of PSII measured by imaging PAM to describe the response of PSII photochemistry to anthracene (Fig. 6). Y(II) represents the fraction of excitation energy converted photochemically at PSII. The remaining fraction, 1–Y(II), is the sum of the yields of regulated dissipation, referred to as Y(NPQ), and unregulated dissipation, indicated by Y(NO)⁵⁹. As stated before, anthracene reduced the F_v/F_m in a dose-dependent manner, and the photon energy requirements for a complete reduction of Q_A decrease drastically. This explains the gradual reduction in Y(II) with increasing anthracene doses and a corresponding increase in Y(NO) (Fig. 6). Y(NO) denotes the excess energy, the fraction of absorbed energy used for the generation of free radicals (ROS) via an apparent catalytic transfer of electrons occurred from the reduced PQ pool to O₂. The higher quantum yield of non-regulated non-photochemical energy loss of PSII (Y(NO)) is a significant stress response, suggesting potential damage to the photosynthetic apparatus exerted by anthracene. Moreover, the declining Y(NPQ) is an indicator of the failure in regulated non-photochemical quenching mechanisms to process the excess energy at PSII. These PSII quantum yield parameters collectively indicates the reduced efficiency of photochemical energy regulation imposed by anthracene exposure.

Figure 6.

Effect of exposure to anthracene on overall energy conversion at PSII in terms of three quantum yields; (i) photochemical quantum yield of photosystem II, Y(II); (ii) quantum yield of non-photochemical fluorescence quenching due to downregulation of the light harvesting function, Y(NPQ); (iii) quantum yield of non-photochemical fluorescence quenching other than that caused by down-regulation of the light harvesting function, Y(NO). Values are given as % relative to untreated controls.

On the downside of our methodology, anthracene has a higher n-octanol/water partition coefficient (K_OW) of 4.54⁶⁰ and, therefore, multi-well plate assay is likely to underestimate the toxicity potential, because a loss of exposure concentration due to lipophilicity is expected for hydrophobic compounds with K_OW > 4⁶¹. We controlled the evaporative loss of the toxicant by sealing the well plates, however, loss in nominal concentration due to physicochemical properties of the tested PAH may have adversely affected the toxicity thresholds reported here. Nevertheless, our data confirmed the mode of anthracene toxicity in E. agilis through Chl a fluorescence technique and the role of ROS in the overall toxicity response.

Conclusions

Microalgae play a pivotal role in primary production in aquatic ecosystems, hence microalgal ingestion in polluted water bodies is a major route by which toxic chemicals can enter the food chain. The results of the present study confirm that anthracene exerts phytotoxic effects on E. agilis by disrupting growth, pigmentation and photosynthesis. Any severe reduction in these parameters will be followed by a loss of ecological competence and diminished survival of the entire E. agilis population, which could have a devastating impact on associated food chains.

Five principal conclusions derived from this study are:

1. The addition of anthracene resulted in a concentration-dependent reduction in cellular growth which appears to be highly related to a reduction in photosynthesis.

2. Anthracene had a pronounced negative effect on photosynthetic pigment content and a simultaneous reduction in all three photosynthetic pigments suggests that the major target of anthracene toxicity is the thylakoid compartment of chloroplasts. These results also indicate that in addition to causing a severe reduction in growth, anthracene exposure may reduce photosynthetic performance via the destruction of pigments responsible for harvesting available photons.

3. Toxicity of anthracene on Y(II), rETR_max and non-photochemical quenching parameters in microalgae taxa has for the first time been reported in the current study. The PSII quantum yield parameters collectively indicate the reduced efficiency of photochemical energy regulation, impairment of electron transport downstream from PSII, and an elevated reduction of the PQ pool imposed by anthracene exposure.

4. There was a significant increase in DCFH-DA fluorescence emission in anthracene exposed E. agilis cells, indicating a rise in the intracellular ROS levels. A probable source of generation of ROS would be an inhibition of the electron transport chain at PSII which would have transferred energy from triplet chlorophylls to oxygen, forming singlet oxygen species. A corresponding increase in Y(NO) with increasing anthracene also confirmed that the fraction of absorbed energy might have been used for the generation of free radicals (ROS) via an apparent catalytic transfer of electrons that occurred from the reduced PQ pool to O₂.

5. Growth and Chl a content of E. agilis may serve as sensitive risk assessment parameters of anthracene toxicity in water management since EC₅₀ values for both overlap with anthracene levels (8.3 mg L⁻¹) permitted by the US Environmental Protection Agency (USEPA).

Author contributions

S.K. designed and performed the experiment and wrote the draft manuscript. J.P contributed to construction of tables and figures, performance of statistics, making the final revisions, and writing the responses to the reviewers. The paper was reviewed and approved by both authors prior to submission for peer review.

Data availability

Data can be obtained by contacting the corresponding author.

Competing interests

The authors declare no competing interests.