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Article

Metal Toxicity across Different Thallus Sections of the Green Macroalga, Ulva australis

by
Hojun Lee
1,
Geonhee Kim
2,
Stephen Depuydt
3,
Kisik Shin
4,
Taejun Han
1,5 and
Jihae Park
5,6,*
1
Bio Environmental Science and Technology (BEST) Lab, Ghent University Global Campus, 119-5, Songdomunhwa-ro, Incheon 21985, Republic of Korea
2
Department of Marine Science, Incheon National University, 119, Academy-ro, Yeonsu-gu, Incheon 22012, Republic of Korea
3
Erasmus Brussels University of Applied Sciences and Arts, Nijverheidskaai 170, 1070 Brussels, Belgium
4
Water Environmental Engineering Research Division, National Institute of Environmental Research (NIER), 42, Hwangyeong-ro, Incheon 22689, Republic of Korea
5
Department of Animal Sciences and Aquatic Ecology, Ghent University, Coupure Links 653-Block F, B-9000 Gent, Belgium
6
Centre for Environmental and Energy Research, Ghent University Global Campus, 119-5, Songdomunhwa-ro, Incheon 21985, Republic of Korea
*
Author to whom correspondence should be addressed.
Toxics 2023, 11(7), 548; https://doi.org/10.3390/toxics11070548
Submission received: 21 May 2023 / Revised: 17 June 2023 / Accepted: 20 June 2023 / Published: 22 June 2023
(This article belongs to the Special Issue Phytotoxic Activity of Metal-Based Contaminants)
Browse Figures
Versions Notes

Abstract

:
We aimed to identify functional differences between different sections of the thallus of Ulva australis and develop tissue-endpoint combinations to assess the toxicity of six metals (i.e., Ag, As, Cd, Cr, Cu, and Ni). EC50 values for these metals in three sections of the thallus of Ulva were obtained for multiple endpoints: relative growth rate (RGR), chlorophyll a fluorescence, pigment contents, and the expression of the photosynthesis-related gene, rbcL. The responses of the endpoints varied across the respective thallus sections; overall, the most toxic metals were Ag and Cu. These endpoints were the best for evaluating metal toxicity: ETRmax of the middle thallus sections for Ag toxicity; RGR of the middle thallus section for As and Cd; ETRmax of the marginal thallus section for Cr; Chl b contents of the marginal thallus section for Cu; RGR of the basal thallus section for Ni. The EC50 values for the inhibition of ETRmax in middle (0.06 mg∙L−1) and Chl b in the marginal thallus sections (0.06 mg∙L−1) were all lower than those of the quality standard for wastewater discharge values of Ag and Cu in Republic of Korea and the US, pointing to the suitability of U. australis-based endpoints for risk assessment.

Graphical Abstract

1. Introduction

Ulva australis (i.e., sea lettuce) is a green seaweed species found in temperate and subtropical coastal waters worldwide.
U. australis is morphologically simple; specifically, it has a flat, lettuce-like body that consists of two thick cell layers [1,2,3,4,5]. Overall, this green alga is thought to have a homogeneous thallus that has little functional differentiation.
However, Hiraoka and Enomoto [6] found that experimental disks excised from the thallus of U. australis showed different degrees of zooid formation depending on the part of the body from which the disk had been extracted. Han et al. [1] compared the morphology, pigmentation, photosynthesis, growth, reproduction, and UV-B sensitivity of U. australis across different thallus sections. The findings of this study were used to support the occurrence of adaptive functional differentiation among cells within the thallus. This differentiation was characterized as follows: the margins of the thallus were linked to high productivity and reproduction; the middle sections were linked to rapid growth; the basal sections were associated with the regeneration of new blade cells. This differentiation is thought to facilitate rapid adaptive responses when algae are subjected to unfavourable conditions [7]. However, to date, the morphological, physiological, and biochemical differences of the various sections (i.e., specifically, their responses to stressors other than UV radiation) of the thallus of U. australis have not been studied comprehensively.
Industrial and agricultural processes generate large amounts of wastewater; some of this water is then discharged into marine ecosystems [8]. Wastewater may contain various metals such as silver, arsenic, chromium, cadmium, copper, and nickel [9]. These metals are nonbiodegradable, toxic to non-target organisms, and can bioaccumulate via the food chain, posing a threat to the ecological integrity of oceans and human health [10]. In addition, it has been shown that exposure to high concentrations of metal can inhibit the growth, photosynthesis, and reproduction of algae. Under these conditions, algae also exhibit a heightened production of reactive oxygen species (ROS), which results in the cell membrane being damaged. This widespread and metal-exposure-driven DNA damage culminates in the development of metabolic disorders, which then eventually results in the death of the organism [11,12,13,14,15,16]. Therefore, it is necessary to create effective monitoring and management strategies, especially within the context of maintaining the ecological integrity of marine ecosystems in the ongoing Anthropocene. However, for such solutions to be developed, not only will metal pollution have to be quantified in marine ecosystems, it will have to be shown to pose a threat to marine life.
As the detection of ecologically toxic substances and the assessment of their impact on biota are becoming increasingly important across the globe, various biomarker-based methods have been proposed and implemented. Unlike approaches that entail studying contaminants in isolation, this approach directly reflects the ecological dynamics associated with the contaminants of interest; additionally, it is cheaper and easier to apply than conventional chemical analyses. Specifically, aquatic bioassays provide a basis for assessing and managing the ecological risk that contaminants, including unknown substances, may pose to the aquatic environment. Specifically, multiple endpoint bioassays provide a more comprehensive risk assessment than single endpoint tests because they can effectively detect perturbations of specific contaminants, providing important insights into the mechanisms underlying the relative sensitivity of the measured endpoints [17,18].
The green macroalga, U. australis (i.e., formerly Ulva pertusa), inhabits shallow coastal waters and provides habitat and shelter for fish and invertebrates [2,19,20]. As an important primary producer in the food chain, the disturbance of U. australis populations may greatly affect the overall balance of many marine ecosystems across the globe.
U. australis is an ideal model organism for ecotoxicity tests; for example, this species has been used to develop a test that has been applied to 75 different environmental samples containing contaminants such as metals, volatile organic compounds, herbicides, oils, dispersants, and slag waste [16,21]. The test measures the inhibition of the reproduction on the margins of the thallus by quantifying the colour changes exhibited by the thallus (i.e., caused by the release of reproductive cells) [20]. Ecotoxicity testing using U. australis has some advantages over conventional chemical techniques, namely, ease of use, sensitivity, cost, efficiency, environmental relevance, repeatability, and reproducibility [16,19,20,22,23,24,25,26].
In this study, we aimed to firstly investigate the functional differences among different sections of the thallus of Ulva australis. To achieve this aim, we evaluated the morphological, physiological, and biochemical characteristics of the different sections of the thallus. We then evaluated the sensitivity of each thallus section to six metallic toxicants (i.e., Ag, As, Cd, Cr, Cu, and Ni) to determine their respective suitability for toxicity testing. For each section, we measured various endpoints, including chlorophyll fluorescence, pigment concentration, growth, and rbcL expression. Overall, we elucidated specific thallus sections and endpoints that would be ideal for the toxicity testing of specific metals.

2. Materials and Methods

2.1. Algal Culture

Ulva australis samples were collected from sites near Ilgwang-myeon, Gijang-gun, Busan, Korea (35.283858° N, 129.259706° E). The collected samples were then stored in an artificial seawater medium at 15 °C under a white fluorescent light (FL20SS, Royal Philips, Amsterdam, The Netherlands). The medium was prepared by dissolving commercial sea salts (Coralife, Energy Savers Unlimited, Inc., Carson, CA, USA) and nutrients (1 mM KNO3 and 0.1 mM K2HPO4) in distilled water up to a concentration of 35 g∙kg−1.

2.2. Morphological Characteristics Comparisons

Algal disks (Ø 8 mm) were cut from different sections (i.e., marginal, middle, and basal sections) of the thallus of U. australis, and microscopic measurements for thallus thickness (in cross-section), cell size (random count in surface view), and number of cells [in a randomly selected 50 µm × 50 µm (2500 µm2) square] were determined (n = 8).

2.3. Growth

Growth was evaluated by measuring the size of the U. australis disks using an image analyser Moticam (Moticam 2.0 MP, Motic, Kowloon Bay, Hong Kong), and the relative growth rate (RGR) was calculated as follows:
RGR (% d−1) = [(ln(Af) − ln(Ai))/tf)] × 100
where Ai and Af are the initial and final disk areas and tf is the test duration.

2.4. Chlorophyll a (Chl a) Fluorescence Measurements

Chl a fluorescence was measured using Imaging PAM (Heinz Walz GmbH, Effeltrich, Forchheim, Germany). After 72 h of treatment, the U. australis samples were dark-adapted for 15 min. Light pulses (0.15 µmol photons m−2∙s−1) from light-emitting diodes (LEDs) were then used to determine the initial fluorescence yield (Fo), which indicates the fluorescence yield when all reaction centres of photosystem II (PSII) are open and plastoquinone A (QA) is completely oxidized. Subsequently, a saturation pulse of approximately 5000 µmol photons m−2s−1 was applied to produce the maximum fluorescence yield (Fm), induced by a short saturating pulse of actinic light, reducing all QA molecules. The value of Fv/Fm was then calculated as follows:
Fv/Fm = (Fm − Fo)/Fm
where Fv is the variable fluorescence.
Rapid light curves were then generated using 10 s pulses of actinic light gradually increasing from 0 to 1517 μmol photons m−2s−1. The maximum electron transport rate (ETRmax) was calculated using the hyperbolic tangent equation given by Jassby and Platt [27]:
ETR = ETRmax × tanh(α × I/ETRmax)
where α and I denote ETR and irradiance, respectively, under light-limiting conditions.
Non-photochemical quenching (NPQ) was estimated using the following equation [28].
NPQ = (Fm − F′m)/F′m
where F′m denotes the maximum fluorescence yield observed under light-adapted conditions.

2.5. Pigment Contents

Chlorophyll a, chlorophyll b (Chl a and Chl b), and carotenoids were extracted from U. australis disks in 1 mL methanol (≥99.9%; Sigma–Aldrich, St. Louis, MO, USA) for 24 h in the dark at 4 °C. The absorbance of the methanol extract was measured at 666 nm (Chl a), 653 nm (Chl b), and 470 nm (carotenoids) using a spectrophotometer (Scinco S-3100 PDA UV-Vis, Scinco, Seoul, Republic of Korea). The concentrations of Chl a, Chl b, and carotenoids were estimated using the equations reported by Lichtenthaler [29].
Chl a = 15.65 × A666 − 7.34 × A653
Chl b = 27.05 × A653 − 11.21 × A666
Carotenoids (Car) = (1000 × A470 − 2.86 × Chl a − 129.2 × Chl b)/245
where A470, A653, and A666 represent the absorbances at 470 nm, 653 nm, and 666 nm, respectively.

2.6. Metal Toxicity Testing

Algal disks (Ø 8 mm) were cut from different sections of the healthy thallus (i.e., marginal, middle, and basal sections) and placed in 250 mL flasks with growth medium and different concentrations of the selected metals. Metal stock solution (Junsei, Tokyo, Japan) in deionized water was acidified following the addition of 1 N hydrochloric acid (HCl) or 1 M nitric acid (HNO3) to each flask to obtain the specified final concentrations (Table 1). The pH of all test solutions, including the control, was adjusted to a pH range of 8.0 ± 0.2 using 1 M NaOH and 1 M HCl solutions. The concentrations of carrier solvents used to facilitate the dissolution of metal contaminants have not been shown to cause or intensify toxicity in Ulva. Controls consisted of artificial seawater medium without toxicants. During incubation, air bubbles were gently integrated into the medium. Thallus disks were grown for 72 h under optimal environmental conditions (i.e., photon irradiance, 100 µmol photons m−2 s−1 of white, fluorescent light with 12:12 h L:D photoperiod, salinity 30 g∙kg−1, and temperature 15 ± 1 °C), the disks treated with the metals were subsequently removed, and their growth, chlorophyll fluorescence, and pigment content were quantified.

2.7. Gene Expression (qRT-PCR)

For gene expression analysis, silver (Ag) and copper (Cu) were selected due to their high toxicity across all examined endpoints. Additionally, the exposure concentrations used in this experiment were chosen by selecting the EC50 values from among the most sensitive and reliable endpoints.
After a 72-h exposure to 0.12 mg∙L−1 of Ag and Cu, U. australis disks from different thallus sections were harvested and ground in liquid nitrogen. Total RNA was extracted from each sample using an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), and cDNA was prepared from 1 µg of total RNA using a Diastar RT Kit (SolGent Co., Ltd., Daejeon, Republic of Korea) according to the manufacturer’s instructions. The purity and concentrations of RNA and cDNA were determined using a Nanodrop UV spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
qRT-PCR was performed using a 2×RT PCR Smart Mix (with SYBR Green) (SolGent Co., Ltd., Daejeon, Republic of Korea) on a CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Amplification was performed through denaturation at 95 °C for 5 min, followed by 40 cycles of 94 °C for 30 s, 57 °C for 30 s, and 70 °C for 10 s. The primers for the reference gene (18S rRNA) and the tested gene (rbcL) were as follows: 5′ -CACGTCTTGGTGAATCATGG-3′ (18S rRNA-forward), 5′-CTTGGATGTGGTAGCCGTTT-3′ (18S rRNA-reverse), 5′- AGAAATGATGGAGCGTGGTC-3′ (rbcL-forward), 5′-TGGTCACCACCTGACATACG-3′ (rbcL -reverse).

2.8. Statistical Analyses

One-way analysis of variance (ANOVA) followed by multiple least significant difference (LSD) comparison tests were performed to test for differences between the endpoints and the sections. Results were presented as half maximal effective concentration (EC50) values with 95% confidence intervals (95% CI) and estimated using linear interpolation methods (Toxical 5.0, Tidepool Scientific Software, McKinleyville, CA, USA). The accuracy of the test was estimated by calculating the coefficient of variation (CV), which is the standard deviation expressed as a percentage of the mean.
The sensitivity and reliability of the six endpoints for metals were compared based on their EC50 and CV values, respectively. The EC50 and CV values of all endpoints for each metal were ranked in descending order regardless of the specific thallus sections; then, scores from 1 to 18 (six endpoints and three sections) were assigned to the two sequences. Mean scores were assigned to the same sequence. A lower score indicates a more sensitive and reliable endpoint.

3. Results and Discussion

3.1. Morphological, Physiological, and Biochemical Traits by Thallus Section

In descending order, the thicknesses of the different sections were as follows: basal thallus (381.40 ± 80.17 µm); middle thallus (145.93 ± 11.05 µm); the marginal thallus (89.96 ± 6.83 µm). Cell size was also greatest in basal sections (176.3 ± 4.98 µm2), followed by middle (140.94 ± 6.98 µm2) and marginal thallus sections (115.81 ± 6.22 µm2). Furthermore, the number of cells was found to be inversely proportional to cell size: 21.13 ± 1.63 cells for the marginal, 16.38 ± 1.04 cells for the middle, and 10.38 ± 0.64 cells for the basal sections (Figure 1).
Generally, U. australis is known to be morphologically simple, exhibiting little functional differentiation in its thalli. In addition, the growth points of U. australis have been shown to be randomly distributed across its thallus, causing an irregular expansion and controlled bilayer formation [4]. In contrast with these findings, our morphological measurements revealed significant differentiation across the different sections of the thallus. The findings linked to thallus thickness and cell size in this study are within the range of those reported by Han et al. [1] and, more generally for the genus, present a thickness and cell size ranging between 30–100 μm and 7–23 × 6–37 μm (length × width), respectively [30].
The relative growth rate (RGR) was greatest in the middle thallus (8.28 ± 0.64% d−1), followed by the marginal (3.35 ± 1.30% d−1) and basal thalli (0.51 ± 0.48% d−1) (Figure 1). The greater growth of the middle sections may be because U. australis can only reproduce from marginal thalli, which tend to initially grow rapidly and then stop in preparation for reproduction, as shown by Han et al. [1] and Park [26]. The slow growth of the basal sections of the thalli has been observed in a previous study [1]. The different RGRs observed between thallus sections in U. australis provide further evidence for functional differentiation along the different sections of the thallus, namely, the margins for reproduction, the middle for vegetative growth, and the base for anchoring and inter-annual carry-over.
Our assessment of photosynthetic capacity using two endpoints, Fv/Fm and ETRmax, showed that both were significantly higher in the marginal (0.73 ± 0.01 and 7.52 ± 0.70) and middle (0.74 ± 0.01 and 7.37 ± 0.95) thallus sections than in the basal sections (0.65 ± 0.01 and 4.86 ± 0.52) (Figure 1). The inter-sectional variation in photosynthetic efficiency may be linked to differences in cell size and morphology. Specifically, cell size and morphology have tended to be smallest and thinnest in the marginal thallus sections and largest and thickest in the basal thallus sections, respectively. This is in line with smaller cells having been reported to have a higher photosynthetic capacity [31,32,33]. Additionally, there is a relatively low packing effect, resulting in more efficient photon absorption in the pigments in thinner thalli [34,35].
Non-photochemical quenching (NPQ) approximates the ability of chloroplasts to dissipate excess excitation energy as heat. This process is useful for quantifying the resistance of plants to stress [36]. NPQ was highest in marginal thallus sections (0.43 ± 0.07), followed by the middle (0.31 ± 0.07) and basal thallus sections (0.27 ± 0.04) (Figure 1). In Ulva spp., the NPQ is highly correlated with the xanthophyll cycle; xanthophyll is one of the main constituents of carotenoids [37,38,39]. In this respect, it is noteworthy that the ranking of the NPQ values along the different thallus sections seems to be congruent with that of the carotenoid contents in the corresponding thallus sections.
Chlorophyll a content was greatest in the marginal thalli (0.21 ± 0.04 µg·g FW−1), followed by middle (0.10 ± 0.02 µg·g FW−1) and basal (0.05 ± 0.01 µg·g FW−1) thalli. Chl b and carotenoids were most abundant in the marginal thalli (0.13 ± 0.02 and 0.12 ± 0.02 µg·g FW−1), followed by the middle (0.06 ± 0.01 and 0.06 ± 0.01 µg·g FW−1) and basal thalli (0.03 ± 0.01 and 0.03 ± 0.004 μg·g FW−1) (Figure 1). The differences in the pigment concentrations of the different thallus sections could explain why Fv/Fm and ETRmax were higher in the marginal thallus sections compared to those in the basal thallus sections.

3.2. Effects of Metals on Six Different Endpoints of Three Different Thallus Sections

Growth is an integrative measure of plant metabolism and is therefore likely to be sensitive to the adverse effects of environmental pollutants. EC50 values based on the RGR of U. australis exposed to metals varied across thallus sections and the specific metals (Table 2) used. Specifically, in the marginal and middle thallus sections, the RGR was most sensitive to Cu (EC50 values of 0.06 and 0.05 mg∙L−1, respectively) and least sensitive to Ni (EC50 > 0.5 mg∙L−1). The RGR of basal sections was most sensitive to Ni (EC50 = 0.03 mg∙L−1) and least sensitive to As (EC50 > 12.8 mg∙L−1) (Table 2). Only a few studies have been conducted on the effect of Cu on the growth of U. australis; however, Han et al. [15] found that growth was inhibited by 50% at 0.05–0.1 mg∙L−1 Cu. This value is similar to the EC50 for marginal and middle thallus sections obtained in this study. There is limited information on the effects of Ni toxicity on the physiology of U. australis. Han et al. [24] found that the spore release of U. australis was inhibited by 50% at a Ni concentration of 0.31 mg∙L−1, which is 10-fold greater (10-fold less sensitive) than the EC50 values for the basal thallus sections observed in this study. This is because high concentrations of Ni can induce oxidative stress in algae [40,41]. It is notable that the concentration of carotenoids was lower in basal thallus sections compared to in the middle and marginal sections. This may be due to the increased sensitivity to Ni in the basal thallus sections (Table 2), as carotenoids can act as free radical scavengers in algae [26], and high concentrations of carotenoids protect cells from oxidative stress [37].
The metal sensitivity of two photosynthetic endpoints, namely, maximum potential photosystem II quantum efficiency (Fv/Fm) and maximum electron transport rate (ETRmax), also varied across specific metals and thallus sections. Fv/Fm in the marginal thallus sections was most sensitive to Ag (EC50 = 0.26 mg∙L−1) and least sensitive to As (>12.8 mg∙L−1), whereas ETRmax was most sensitive to Cu (0.08 mg∙L−1) and least sensitive to Cd (>8 mg∙L−1) (Table 2). In the middle thallus sections, the most toxic metal based on EC50 values for Fv/Fm was Cu (0.20 mg∙L−1), while the most toxic metal for ETRmax was Ag (0.06 mg∙L−1). In contrast, the least toxic for both endpoints (Table 2) was Cd (>8 mg∙L−1). In the basal part of the thallus, Ag was the most toxic, while Cd was the least toxic metal for both Fv/Fm (0.23 and >8 mg∙L−1) and ETRmax (0.07 and >8 mg∙L−1).
Most metals have an inhibitory effect on the metabolism of plants; however, certain metals are essential for photosynthesis. Nevertheless, these metals also become toxic to plants at high concentrations, negatively impacting photosynthesis [42]. For example, copper (Cu) is an important co-factor for plastocyanin and cytochrome oxidases, which are involved in key physiological processes in plants, such as photosynthesis and respiration [43]. However, excessive levels of Cu can adversely impact the photosynthetic electron transport chain and reduce photosynthetic efficiency [44]. Han et al. [15] found that Fv/Fm was significantly suppressed in the middle sections of the thalli of U. australis and U. armoricana at an exposure of 0.25 mg∙L−1. In U. australis, Kumar et al. [45] found EC50s of 0.37–0.48 mg∙L−1 Cu for Fv/Fm and 0.16–0.21 mg∙L−1 Cu for ETRmax, which are in line with our findings (Table 3).
Silver is becoming an increasingly common industrial pollutant that is ionized in water and can bind to the active sites of photosynthetic enzymes and proteins in algae, damaging their photosynthetic system [40,41,42]. In U. lactuca, a 48-h exposure to 0.03 mg∙L−1 AgNO3 resulted in a reduction in chlorophyll a fluorescence by approximately 50% compared to the control [42]. This EC50 is similar to the Ag sensitivity of ETRmax for the basal and middle thallus sections in this study. Within the context of Ag exposure, the effective quantum yield (F′v/F′m) in the middle section of U. lactuca significantly decreased in exposure to 0.02 mg∙L−1; however, it did not decrease by more than 50%, even when exposed to 0.14 mg∙L−1 [42].
Metals directly affect chlorophyll pigments and, thus, interfere with photosynthesis and plant metabolism. Cu was the most toxic for pigments in marginal thallus, whereas Ag was most toxic for those in the middle and basal thalli. The least toxic metal for pigments in all thallus sections was As (Table 2). Cu deactivates enzymes and proteins involved in photo-activity and deforms thylakoid membranes [46]. Cu may also impair the structure and function of chlorophyll by removing magnesium from both the antenna complex and the reaction centre [43]. Han et al. [15] calculated the EC50 values of photosynthetic pigments in U. armoricana and U. australis exposed to Cu for 72 h to be 0.25 mg∙L−1. This is similar to the EC50 of the middle thallus sections in this study (0.21 ± 0.03 mg∙L−1) (Table 3).
Ag damages the chloroplast microstructure and chlorophyll content in algae [47]. However, studies on the effects of Ag on the pigment content of U. australis are scarce.
Table
Table 3. List of six of metal toxicity using different thallus sections of Ulva australis.
Table 3. List of six of metal toxicity using different thallus sections of Ulva australis.
EndpointsMetalsThallus SectionsTest Period (h)EC50
(mg∙L−1)		CV
(%)		References
Growth (RGR)AgMargin720.0818.67This study
Middle720.0514.38This study
Base72>0.32-This study
AsMargin722.3528.20This study
Middle720.9819.37This study
Base72>12.8-This study
CdMargin726.213.79This study
Middle721.8912.09This study
Base722.8816.33This study
CrMargin723.9432.44This study
Middle720.8728.72This study
Base720.7446.47This study
CuMargin720.0613.22This study
Middle720.0524.38This study
720.05–0.1, ca.-Han et al. [15]
Base720.124.04This study
NiMargin72>0.5-This study
Middle72>0.5-This study
Base720.0335.56This study
Fv/FmAgMargin720.262.71This study
Middle720.30-This study
Base720.232.41This study
AsMargin72>12.8-This study
Middle725.030.79This study
Base726.759.96This study
CdMargin72>8-This study
Middle72>8-This study
Base72>8-This study
CrMargin72>6.4-This study
Middle724.897.85This study
Base72>6.4-This study
CuMargin72>1.2-This study
Middle720.2023.36This study
240.486.04Kumar et al. [45]
480.375.5Kumar et al. [45]
720.25, ca.-Han et al. [15]
720.355.41Kumar et al. [45]
960.71-Kumar et al. [45]
Base720.678.37This study
NiMargin72>0.5-This study
Middle72>0.5-This study
Base72>0.5-This study
ETRmaxAgMargin720.231.80This study
Middle720.0615.27This study
Base720.0725.14This study
AsMargin725.1929.05This study
Middle723.0110.70This study
Base724.994.82This study
CdMargin72>8-This study
Middle72>8-This study
Base72>8-This study
CrMargin720.226.36This study
Middle721.2923.56This study
Base722.2228.00This study
CuMargin720.0831.47This study
Middle720.1415.05This study
240.218.98Kumar et al. [45]
480.16-Kumar et al. [45]
720.05–0.10, ca.-Han et al. [15]
720.2911.80Kumar et al. [45]
960.362.60Kumar et al. [45]
Base720.1115.68This study
NiMargin72>0.5-This study
Middle72>0.5-This study
Base72>0.5-This study
Chl aAgMargin720.224.19This study
Middle720.114.56This study
Base720.1226.01This study
AsMargin72>12.8-This study
Middle72>12.8-This study
Base72>12.8-This study
CdMargin72>8-This study
Middle72>8-This study
Base72>8-This study
CrMargin725.71-This study
Middle724.1113.08This study
Base72>6.4-This study
CuMargin720.0510.39This study
Middle720.1817.85This study
720.25, ca.-Han et al. [15]
Base720.9210.89This study
NiMargin72>0.5-This study
Middle72>0.5-This study
Base72>0.5-This study
Chl bAgMargin720.225.11This study
Middle720.123.78This study
Base720.1040.28This study
AsMargin72>12.8-This study
Middle72>12.8-This study
Base72>12.8-This study
CdMargin72>8-This study
Middle72>8-This study
Base72>8-This study
CrMargin726.04-This study
Middle724.5511.06This study
Base72>6.4-This study
CuMargin720.067.77This study
Middle720.2411.56This study
Base72>1.2-This study
NiMargin72>0.5-This study
Middle72>0.5-This study
Base72>0.5-This study
CarotenoidAgMargin720.209.74This study
Middle720.123.87This study
Base720.1134.41This study
AsMargin72>12.8-This study
Middle72>12.8-This study
Base72>12.8-This study
CdMargin72>8-This study
Middle72>8-This study
Base72>8-This study
CrMargin725.20-This study
Middle724.4810.83This study
Base72>6.4-This study
CuMargin720.0511.00This study
Middle720.219.81This study
Base721.09-This study
NiMargin72>0.5-This study
Middle72>0.5-This study
Base72>0.5-This study
AntioxidationCuMiddle720.1–0.25, ca.-Han et al. [15]
Gametophyte (length)CdMargin1440.19-Han et al. [48]
CuMargin1440.026.41Han et al. [48]
Gametophyte (No. of cells)CdMargin1440.20-Han et al. [48]
CuMargin1440.038.32Han et al. [48]
ReproductionAgMargin960.1311.41Lee et al. [16]
CdMargin72–1200.22-Han et al. [23]
CdMargin960.7217.10Lee et al. [20]
CuMargin72–1200.06-Han et al. [23]
CuMargin960.129.67Lee et al. [20]
Sporulation (visual)AgMargin960.053.11Han et al. [24]
AsMargin961.0310.54Han et al. [24]
CdMargin960.277.63Han et al. [24]
CrMargin961.502.08Han et al. [24]
CuMargin960.102.46Han et al. [24]
NiMargin960.987.30Han et al. [24]
Sporulation (image analyser)AgMargin960.052.95Han et al. [24]
AsMargin960.8610.48Han et al. [24]
CdMargin960.268.38Han et al. [24]
CrMargin961.452.90Han et al. [24]
CuMargin960.103.01Han et al. [24]
NiMargin960.954.60Han et al. [24]
SporulationCdMargin1200.3310.43, ca.Han and Choi [22]
CuMargin1200.0632.78, ca.Han and Choi [22]
Spore releaseAgMargin960.042.53Han et al. [24]
AsMargin960.4519.56Han et al. [24]
CdMargin960.1011.71Han et al. [19]
Margin960.226.55Han et al. [24]
Margin960.268.38Oh et al. [49]
CrMargin960.804.26Han et al. [24]
CuMargin960.048.40Han et al. [19]
Margin960.085.02Han et al. [24]
Margin960.103.01Oh et al. [49]
NiMargin960.314.13Han et al. [24]
Spore germinationCdMargin720.793.29Han et al. [48]
CuMargin720.023.39Han et al. [48]

3.3. Effect of Metals on rbcL Expression

After exposure to 0.12 mg∙L−1 of Ag or Cu, the expression of rbcL increased across the middle and marginal sections of the thallus of U. australis, but not in the basal sections (Figure 2). Upon exposure to Ag, the expression level of rbcL increased five-fold (from 1.01 ± 0.14 to 5.28 ± 1.48) and four-fold (from 1.01 ± 0.11 to 3.93 ± 0.38) in the middle and marginal thallus sections, respectively, relative to the control (Figure 2). Gene expression in response to Cu increased three-fold in the marginal thallus (from 1.05 ± 0.33 to 3.50 ± 0.62) and four-fold in the middle thallus (from 1.06 ± 0.34 to 5.27 ± 0.61) (Figure 2). Conversely, the rbcL expression levels in basal sections were not significantly different from those of the control group.
Our study showed that the expression of the rbcL gene increased in the marginal and middle sections of the thalli exposed to metal toxicity. This up-regulation of gene transcription may represent a response from U. australis to these phytotoxic substances to ensure photosynthesis and survival. However, this increase could lead to more severe oxidative damage via increased ROS production [44]. Further studies are required to confirm this hypothesis. The levels of 18S rRNA can be dynamic in response to stress; therefore, the inclusion of the transcription levels of a reference gene from the U. australis genome could help isolate the effects of metal toxicity, specifically on Rubisco-related gene expression.

3.4. Sensitivity and Reliability of the Endpoints of the Three Different Thallus Sections

Figure 3 shows the pairs of the most sensitive and reliable endpoints for each metal species. For Ag, the RGR of the marginal thallus section and the ETRmax of the middle and basal thallus sections were both found to be highly sensitive and reliable. Among them, the ETRmax of the mid-section was found to be the most sensitive and reliable endpoint for the diagnosis of Ag toxicity.
With regard to As, the RGR of the marginal and middle thallus sections and the ETRmax of the basal thallus section were found to be highly sensitive and reliable. Among these, the RGR of the mid-thallus was found to be the most sensitive and reliable endpoint for the diagnosis of As toxicity.
For Cd, the RGR was found to be highly sensitive and reliable for all three different thallus sections. Among them, the RGR of the middle section was found to be the most sensitive and reliable endpoint for the diagnosis of Cd toxicity.
For Cr, the ETRmax of the marginal thallus section and the RGR of the middle and basal thallus sections were found to be highly sensitive and reliable. Among them, the ETRmax of the marginal section was found to be the most sensitive and reliable endpoint for the diagnosis of Cr toxicity.
For Cu, it was found that the Chl b of the marginal thallus section and the RGR of the middle thallus section and the ETRmax of the basal thallus section were highly sensitive and reliable. Among them, marginal Chl b was found to be the most sensitive and reliable endpoint for the diagnosis of Cu toxicity.
For Ni, the RGR of the basal section was found to be the most sensitive and reliable endpoint for the diagnosis of Ni toxicity.

4. Conclusions

Morphological, physiological, and biochemical differences were characterised across different sections of the thallus of U. australis. The resultant trends suggest that the three different sections of the thallus are differentiated across functional axes. Specifically, marginal sections of the thallus have reproductive functions (i.e., spore formation and release), the middle sections are involved in vegetative growth, and, finally, basal sections of the thallus anchor algae support growth in subsequent growth cycles. Moreover, each thallus section responded uniquely upon exposure to the six metals (i.e., Ag, As, Cd, Cu, Cr, and Ni). Ag and Cu were consistently the most toxic metals across all the tested endpoints. Based on these results, we propose the following endpoints as the most appropriate for evaluating the toxicity of the six metals we tested: ETRmax of the middle thallus for Ag toxicity; RGR of the middle thallus for As and Cd; ETRmax of the marginal thallus for Cr; Chl b of the marginal thallus for Cu; RGR of the basal thallus for Ni.
Our study suggests that U. australis-based endpoints are generally suitable for evaluating pollution given the quality standards for wastewater discharge (QSWD) in Korea and the US. The Korean Ministry of Environment currently has no standard limit for Ag discharges but has set 1.0 mg∙L−1 for Cu discharge. In the US, the permissible limits for Ag and Cu are 0.5 mg∙L−1 and 0.25 mg∙L−1, respectively (USEPA). The EC50 values for the inhibition of ETRmax in the middle (0.06 mg∙L−1) and Chl b in the marginal thallus sections (0.06 mg∙L−1) were all lower than the QSWD values of Ag and Cu in Korea and the US. Moreover, the majority of EC50 values obtained from multiple endpoints in thallus sections exposed to Ag and Cu were lower than the QSWD values in Korea and the US. Notably, the RGR (0.03 mg∙L−1) of the basal thallus sections was sufficiently sensitive enough to detect the presence of Ni at concentrations exceeding the permissible values. Therefore, the EC50 values obtained from the bioassays could be used to establish ecologically sound and acceptable standards for wastewater discharge.
It is also important to note that the permitted levels of Ag and Cu in the effluent are higher than the safety limits for the two endpoints (i.e., ETRmax of the middle thallus section and Chl b of the marginal thallus section), highlighting the need to review the current management settings for Ag and Cu in wastewater if U. australis is to survive as a primary producer, providing energy, food, and nursery grounds for organisms of higher trophic levels in marine aquatic ecosystems.
In conclusion, our study showed that different sections of the thallus of U. australis can be used independently to assess wastewater discharge due to their different responses to different pollutants. Specifically, each thallus section provides unique endpoints that are similar to multi-species assays in detecting the inference of a specific metal toxicant, thereby providing valuable information on the level of pollution in a given environment.
There has been increasing interest in the use of multi-species tests for ecotoxicity assessment because of their greater ecological relevance compared to single-species tests. However, there are several challenges to conducting multi-species tests, including the difficulty in drawing conclusions when there are differences in sensitivity between test organisms from different trophic levels, the increased variation within organisms due to seasonal adaptation, and the challenges and costs associated with cultivation and maintenance.
On the other hand, U. australis has different characteristics depending on the section of its thallus. The marginal thallus is suitable for reproduction, the central part promotes growth, and the basal part serves as an attachment site. Therefore, by developing an ecotoxicity assessment method that incorporates different endpoints for each specific section of the U. australis thallus, it will be possible to overcome the challenges inherent in the development of multi-species test methods while addressing the limitations of single-species test methods. Here, we propose that the present U. australis method does not replace multi-species tests, but rather serves as a complementary approach to overcome their limitations. This finding is significant since it offers a more cost-effective and time-efficient method of assessing metal toxicity, which is crucial for environmental protection and public health. By using multiple endpoints (i.e., that correspond to specific sections of the thallus), this approach can facilitate a comprehensible and dependable assessment of the risks posed by metal pollution to aquatic ecosystems.

Author Contributions

Conceptualization, H.L., T.H. and J.P.; methodology, H.L. and J.P.; validation, H.L., T.H. and J.P.; formal analysis, H.L. and J.P.; investigation, H.L. and G.K.; data curation, H.L., T.H. and J.P.; writing—original draft preparation, H.L. and G.K.; writing—review and editing, S.D., K.S., T.H. and J.P.; visualization, H.L.; supervision, T.H. and J.P.; project administration, T.H.; funding acquisition, K.S. and T.H. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the National Institute of Environmental Research (NIER) and funded by the Ministry of Environment (MOE) of the Republic of Korea (NIER-2023-03-00-007).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

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Toxics 11 00548 g001 550
Figure 1. Comparison of morphological, physiological, and biochemical characteristics among margins, midsections, and basal sections in the thallus of Ulva australis. The left side of the figure depicts the organization of the thallus in U. australis as observed through a microscope, with a surface view (A) and a transverse section (B) of the thallus. On the right side, the figure shows the physiological and biochemical traits among the sections of U. australis thallus, including relative growth rate (RGR), maximum potential quantum efficiency of photosystem II (Fv/Fm), maximum electron transport rate (ETRmax), non-photochemical quenching (NPQ), chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids (Car). Data represent the mean values of eight replicates, and standard deviations are shown as error bars. Statistically significant differences at p < 0.05 (one-way ANOVA, LSD) are denoted using different letters.
Figure 1. Comparison of morphological, physiological, and biochemical characteristics among margins, midsections, and basal sections in the thallus of Ulva australis. The left side of the figure depicts the organization of the thallus in U. australis as observed through a microscope, with a surface view (A) and a transverse section (B) of the thallus. On the right side, the figure shows the physiological and biochemical traits among the sections of U. australis thallus, including relative growth rate (RGR), maximum potential quantum efficiency of photosystem II (Fv/Fm), maximum electron transport rate (ETRmax), non-photochemical quenching (NPQ), chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids (Car). Data represent the mean values of eight replicates, and standard deviations are shown as error bars. Statistically significant differences at p < 0.05 (one-way ANOVA, LSD) are denoted using different letters.
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Toxics 11 00548 g002 550
Figure 2. Relative rbcL expression in different thallus sections of Ulva australis under Ag and Cu exposure. Each thallus was exposed to 0.12 mg∙L−1 of Ag (A) and Cu (B). Data are the means of four replicates (± 95% confidence intervals). Different letters indicate statistically significant differences at p < 0.05 (one-way ANOVA, LSD).
Figure 2. Relative rbcL expression in different thallus sections of Ulva australis under Ag and Cu exposure. Each thallus was exposed to 0.12 mg∙L−1 of Ag (A) and Cu (B). Data are the means of four replicates (± 95% confidence intervals). Different letters indicate statistically significant differences at p < 0.05 (one-way ANOVA, LSD).
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Figure 3. Rank of sensitivity and reliability of various endpoints for each metal species (i.e., Ag, As, Cd, Cr, Cu, and Ni). The lower the EC50 value was, the more sensitive the endpoint; moreover, the lower the coefficient of variation (CV) was, the more reliable the endpoint. Mean ranks were calculated for the endpoints, thallus sections, and metals independently using sensitivity and reliability. RGR, Relative growth rate; Fv/Fm, Maximum potential quantum efficiency of photosystem II; ETRmax, Maximum electron transport rate; Chl a, Chlorophyll a; Chl b, Chlorophyll b; Car, Carotenoid; Mar, Marginal region of Ulva australis thallus; Mid, Middle section of U. australis thallus; Bas, Basal section of U. australis thallus.
Figure 3. Rank of sensitivity and reliability of various endpoints for each metal species (i.e., Ag, As, Cd, Cr, Cu, and Ni). The lower the EC50 value was, the more sensitive the endpoint; moreover, the lower the coefficient of variation (CV) was, the more reliable the endpoint. Mean ranks were calculated for the endpoints, thallus sections, and metals independently using sensitivity and reliability. RGR, Relative growth rate; Fv/Fm, Maximum potential quantum efficiency of photosystem II; ETRmax, Maximum electron transport rate; Chl a, Chlorophyll a; Chl b, Chlorophyll b; Car, Carotenoid; Mar, Marginal region of Ulva australis thallus; Mid, Middle section of U. australis thallus; Bas, Basal section of U. australis thallus.
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Table
Table 1. Final concentration ranges for testing toxicity of each metal across different thallus sections of Ulva australis.
Table 1. Final concentration ranges for testing toxicity of each metal across different thallus sections of Ulva australis.
MetalsChemical FormulaConcentrations (mg∙L−1)CAS no.Manufacturer
CuCuSO40.0375–1.27758-98-7Showa, Japan
CrK2Cr2O70.4–6.47778-50-9Showa, Japan
NiNiSO4(NH4)2SO40.03125–0.515699-18-0Showa, Japan
AsAs2O30.8–12.81327-53-3Showa, Japan
CdCdSO40.5–810124-36-4Showa, Japan
AgAgNO30.01–0.327761-88-8Showa, Japan
Table
Table 2. EC50 values (with 95% CI; mg∙L−1) for various endpoints in each thallus section exposed to six metals.
Table 2. EC50 values (with 95% CI; mg∙L−1) for various endpoints in each thallus section exposed to six metals.
EndpointsThallus SectionsMetals
AgAsCdCrCuNi
RGRMargin0.08
(0.06–0.11)		2.35
(1.29–3.70)		6.21
(5.71–6.55)		3.94
(0.38–4.96)		0.06
(0.05–0.08)		>0.5
Middle0.05
(0.04–0.07)		0.98
(0.65–1.34)		1.89
(1.59–2.57)		0.87
(0.38–1.37)		0.05
(0.03–0.07)		>0.5
Base>0.32>12.82.88
(1.74–3.27)		0.74
(0.27–1.68)		0.12
(0.11–0.13)		0.03
(0.02–0.05)
Fv/FmMargin0.26
(0.25–0.28)		>12.8>8>6.4>1.2>0.5
Middle0.305.03
(4.96–5.11)		>84.89
(4.26–5.73)		0.20
(0.07–0.25)		>0.5
Base0.23
(0.21–0.24)		6.75
(5.96–8.20)		>8>6.40.67
(0.52–0.75)		>0.5
ETRmaxMargin0.23
(0.219–0.236)		5.19
(1.96–7.46)		>80.22
(0.21–0.26)		0.08
(0.03–0.11)		>0.5
Middle0.06
(0.041–0.076)		3.01
(2.55–3.75)		>81.29
(0.74–1.83)		0.14
(0.07–0.17)		>0.5
Base0.07
(0.04–0.11)		4.99
(4.62–5.57)		>82.22
(0.98–3.34)		0.11
(0.07–0.13)		>0.5
Chl aMargin0.22
(0.19–0.23)		>12.8>85.710.05
(0.04–0.07)		>0.5
Middle0.11
(0.10–0.12)		>12.8>84.11
(2.53–4.92)		0.18
(0.04–0.22)		>0.5
Base0.12
(0.05–0.21)		>12.8>8>6.40.92
(0.56–1.08)		>0.5
Chl bMargin0.22
(0.17–0.23)		>12.8>86.040.06
(0.05–0.07)		>0.5
Middle0.12
(0.10–0.13)		>12.8>84.55
(2.87–5.41)		0.24
(0.17–0.32)		>0.5
Base0.10
(0.01–0.21)		>12.8>8>6.4>1.2>0.5
CarMargin0.20
(0.16–0.22)		>12.8>85.200.05
(0.03–0.06)		>0.5
Middle0.12
(0.11–0.13)		>12.8>84.48
(2.68–5.27)		0.21
(0.16–0.25)		>0.5
Base0.11
(0.02–0.20)		>12.8>8>6.41.09>0.5
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MDPI and ACS Style

Lee, H.; Kim, G.; Depuydt, S.; Shin, K.; Han, T.; Park, J. Metal Toxicity across Different Thallus Sections of the Green Macroalga, Ulva australis. Toxics 2023, 11, 548. https://doi.org/10.3390/toxics11070548

AMA Style

Lee H, Kim G, Depuydt S, Shin K, Han T, Park J. Metal Toxicity across Different Thallus Sections of the Green Macroalga, Ulva australis. Toxics. 2023; 11(7):548. https://doi.org/10.3390/toxics11070548

Chicago/Turabian Style

Lee, Hojun, Geonhee Kim, Stephen Depuydt, Kisik Shin, Taejun Han, and Jihae Park. 2023. "Metal Toxicity across Different Thallus Sections of the Green Macroalga, Ulva australis" Toxics 11, no. 7: 548. https://doi.org/10.3390/toxics11070548

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