Giant Duckweed (Spirodela polyrhiza) Root Growth as a Simple and Sensitive Indicator of Copper and Chromium Contamination

Aquatic environment are often contaminated with heavy metals from various industrial sources. However, physicochemical techniques for pollutant detection are limited, thus prompting the need for additional bioassays. We investigated the use of greater duckweed (Spirodela polyrhiza) as a bioindicator of metal pollution. We exposed S. polyrhiza to four pollutants (namely, silver, cadmium, copper, and chromium) and assessed metal toxicity by measuring its frond area and the length of its regrown roots. The plant displayed significant differences in both frond size and root growth in response to the four metals. Silver was the most toxic (EC50 = 23 µg L−1) while copper the least (EC50 = 365–607 µg L−1). Direct comparisons of metal sensitivity and the reliability of the two endpoint assays showed that root growth was more sensitive (lower in terms of 50% effective concentration) to chromium, cadmium, and copper, and was more reliable (lower in terms of coefficient of variation) than those for frond area. Compared to conventional Lemna-based tests, the S. polyrhiza test is easier to perform (requiring only one 24-well plate, 3 mL of medium and a 72-h exposure). Moreover, it does not require livestock cultivation/maintenance, making it more suitable for repeated measurements. Measurements of S. polyrhiza root length may be suitable for assessment when copper and chromium in municipal and industrial wastewater exceed the environmentally permissible levels.


Introduction
Bioassays are important tools for assessing water quality and for developing ecologically relevant safety standards for water management [1].Chloroxygenic organisms (plants and protists) are frequently used as test species, highlighting the importance of primary producers for monitoring the functioning and health of ecosystems [2].
Since the 1930s, duckweeds (Lemnaceae) have been used extensively in fundamental and applied research in the environmental sciences, including in phytotoxicity testing and bioremediation [3,4].Duckweeds have many useful characteristics as test organisms, including their small size, simple structure, high surface-to-volume ratio, rapid doubling time, genetic homogeneity, and relative ease of culturing (via asexual propagation) in the Toxics 2023, 11, 788 2 of 10 laboratory.Duckweeds are commonly found in fresh water and brackish ecosystems in temperate climates globally, where they serve as important food sources for various water birds and fish, as well as habitat for small invertebrates [5].Of the five genera (Landoltia Les & D.J. Crawford, Lemna L., Spirodela Schleid, Wolffia Horkel ex Schleid, and Wolffiella Hegelm) and 57 species classified within the subclass Lemnoideae [6], most ecotoxicological studies have been performed using Lemna, particularly L. gibba (gibbous duckweed) and L. minor (lesser duckweed) [7].Lemna minor is a model organism for OECD (Organization for Economic Co-operation and Development) [8] and ISO (International Organization for Standardization) test guidelines [9], partly because it is readily available in different parts of the world [10][11][12].
Greater duckweed (Spirodela polyrhiza (L.) Schleiden) has also been the subject of extensive physiological research [13].In addition, S. polyrhiza had a high degree of genetic homogeneity based on DNA barcode analysis [14].Compared to Lemna plants, the fronds of S. polyrhiza are typically larger (~8 mm in length compared to 1-6 mm for Lemna) [15,16], making them easier to handle and more suitable for repeated measurements.
S. polyrhiza plants respond to adverse conditions by forming a special starch-rich structure, turion, which sinks to the bottom of the water and remains inactive until the environment becomes favorable for the plant's growth [17].Turions germinate and develop new vegetative fronds from two meristematic pockets [18] when favorable growth conditions are encountered.Since turions can be stored for several months while maintaining a high germination rate, the use of turions as starting material for bioassays is highly advantageous.In addition, storing turions allows bioassay procedures to be delayed for an appropriate time [19].Taking these practical characteristics into account, a Spirodela growth inhibition test using turion-derived fronds was recently standardized as ISO 20227: 'Determination of the growth inhibition effects of waste waters, natural waters and chemicals on the duckweed Spirodela polyrhiza-Method using a stock culture independent microbiotest' [20].Baudo et al. [19] found comparable sensitivities between the two duckweed genera Spirodela and Lemna when exposed to herbicides (thifensulfuron-methyl, tribenuron-methyl, metribuzin, lenacil, tritosulfuron, linuron, terbutylazine, imazamox, and metamitron), inorganic and organic compounds (3,5- ).In some cases, however, the reported effect levels of Spirodela tests for the same metal have been highly variable, highlighting the need to standardize the testing environment [21].
Standard Lemna bioassays are relatively straightforward.Plants are incubated in test vessels filled with growth medium at 25 • C under continuous illumination (usually 100 µmol m −2 s −1 photon flux density [PFD] light intensity) for 7 d (longer test periods increase potential interference from contamination), after which the inhibitory effects can be determined by measuring various endpoints, such as the number and size of fronds and wet or dry biomass [9].
A study carried out by Gopalapillai et al. [22] has demonstrated the ecological importance of root length as an appropriate endpoint for biomonitoring.The measurement of average root length was found to be more sensitive than other parameters, such as shoot size.It was the most reliable parameter with a low coefficient of variation compared to frond number or dry weight.In addition, the effect of chemicals was most predictable in the relationship between dose and root length.However, Park et al. [23,24] have developed a simpler protocol based on measurement of root regrowth in Lemna species.Compared to the conventional ISO 20079 procedure, our method is shorter in duration (3 d vs. 7 d for ISO 20079), requires only 3 mL (100-150 mL for ISO 20079) of test solution, and employs non-axenic plant material (axenic plant material for ISO 20079).In addition, inter-laboratory comparison tests based on the root regrowth of L. minor conducted by 10 international institutes showed 21.3% repeatability and 27.2% reproducibility for CuSO 4 and 21.3% repeatability and 18.6% reproducibility for wastewater, complying with repeatability and reproducibility standards for bioassays as regulatory tools [24].In the current study, we performed toxicity tests using S. polyrhiza and the conventional test species L. minor to evaluate their comparative sensitivities and reliabilities.We measured frond area and root elongation as endpoints and exposed plantlets to four metal pollutants (Ag, Cd, Cr, and Cu), considering the crucial role of these elements in the phytoremediation sector of polluted environments [25], where our study could provide a new contribution to research.We used the species sensitivity distribution to determine which of the two endpoints was appropriate for estimating the risk of metal toxicity to S. polyrhiza.We also compared the effective concentrations at which 50% inhibition occurs (EC 50 ) for root length and frond area with the Korean Nationally Permissible Standard for Wastewater Discharges (NPSWD) [26].

Toxicity Testing
Toxicity tests were run in a controlled environment culture chamber at 25 ± 1 • C, pH 6.8-7.0, with continuous light (100 ± 10 µmol photons m −2 s −1 ), as described by Park [28].The test solutions were not replaced during the exposure period (static test).We used a 24-well plastic plate (85.4 mm × 127.6 mm; 15.6 mm in diameter, SPL, Seoul, Republic of Korea) and 2.5 mL of test solution in each well.An individual rootless plantlet was added to each well with four plantlets per metal concentration and six concentrations per plate.Three replicate plates (n = 3) were exposed for 72 h.Different metal concentrations of the toxicants were generated by diluting the original stock solutions (1000 mg L −1 ) of AgNO 3 (CAS No. 7761-88-8), CdSO 4 (CAS No. 10124-36-4), K 2 Cr 2 O 7 (CAS No. 7778-50-9), and CuSO 4 (CAS No 7758-98-7) from Showa Chem.(Tokyo, Japan) in Steinberg medium.The stock solution of the test toxicant was stored in cool, dry conditions until the test solutions were prepared.The test dilutions were prepared in volumetric flasks and dispensed into the replicate test vessels, which were then left at room temperature for 1 h to allow the medium and toxicant to equilibrate.A fully randomized design was used to account for the variability in environmental conditions within the culture chamber.For our negative controls, we used identical culture mediums, test conditions, and procedures, but devoid of the test substance.

Measurement Methods
Healthy frond colonies of duckweed (dark green with two or three identical leaves attached) were selected for the experiment.Prior to exposure to the test solutions, roots were excised from fronds using stainless-steel scissors.Fronds were added to the wells under the conditions described by Park et al. [24,28].After 72 h, the fronds were transferred to a glass slide with tweezers, and the upper part of each frond was attached to the glass.By wetting the fronds, the new roots could be easily straightened by careful manipulation with tweezers.The distance between the camera and the glass slide was adjusted and fixed.Images of the frond and regrown roots were analyzed using the ImageJ software (National Institutes of Health (NIH), Bethesda, MD, USA).Frond area (FA) and the length of the longest root (root length; RL) were measured for each plantlet.

Statistical Analysis
Analysis of variance (ANOVA) was performed using Microsoft Excel (2019) to test for statistical differences among treatments, with a significance level of p < 0.05.The data were first assessed to ensure that all statistical assumptions for ANOVA were met, including homogeneity of variances and normal distribution of the data.Subsequently, multiple comparison tests were carried out using the least significant difference (LSD) procedure.Average responses among treatments in each metal toxicity test were compared with one-way ANOVAs (p < 0.05).Results are reported as EC 50 , the effective concentration at which 50% inhibition occurs, with 95% confidence intervals (CIs) estimated by the linear interpolated method (ToxCalc 5.0, Tidepool Scientific, McKinleyville, CA, USA).
EC 50 values from the frond and root tests were ordered by magnitude and coefficient of variation (CV) to approximate sensitivity (i.e., the lower the EC 50 value, the more sensitive the endpoint) and the reliability (i.e., the lower the CV, the more reliable the endpoint), respectively.Mean rank values were calculated for each endpoint and metal.
Species sensitivity distribution (SSD) curves were fitted in R (R Core Team 2020) using the EC 50 values derived from the dose-response curves based on the experiment or reported values for each metal species.From each SSD EC 50 curve, the slope and two hazardous concentrations (HC 05 and HC 50 ) were derived numerically, which correspond to metal concentrations affecting 5% and 50% of the species in an assemblage, respectively.
The species sensitivity index (SSI) was then calculated as follows: SSI = log 10 (HC 50 ) − log 10 (EC 50 ), SSI is a relative index of the difference in species sensitivity: A higher SSI indicates a higher sensitivity [29].
A predicted no-effect concentration (PNEC) was also estimated to assess the risk to the environment from exposure to hazardous chemicals released using the following equation [30]: PNEC = HC 05 /AF, where HC 05 is the concentration at which the no-observable-effect concentration (NOEC) is exceeded for 5% of the species sensitivity derived from the SSD, and AF is the assessment factor (10 is used to take into account the differences between laboratory conditions and natural conditions).

Silver Toxicity
Silver is known to cause frond abscission in Lemna and Spirodela and to inhibit frond and root proliferation [19,[31][32][33][34].In this study, the mean EC 50 values for frond area (FA) and root length (RL) endpoints were 166 and 41 µg L −1 for L. minor and 23 and 23 µg L −1 for S. polyrhiza, respectively.In a previous study, EC 50 values for L. minor ranged from 78 to 140 µg L −1 , although these were based on growth rates inferred from frond number/biomass and photosynthetic pigment concentrations [31,34].Park et al. [31] reported EC 50 values of 5.3-37.6 µg L −1 for RL in L. minor.Baudo et al. [13] measured an EC 50 value of 83 µg L −1 for FA in S. polyrhiza.Thus, the FA endpoint for S. polyrhiza in this study was 7.2 times more sensitive to Ag toxicity than L. minor and 3.6 times more sensitive than other measures for Spirodela from previous studies (Table 1).For L. minor, roots were more sensitive to Ag than fronds.By contrast, the response to Ag in S. polyrhiza was similar in both organs.The root sensitivity of S. polyrhiza to Ag was similar to that of L. minor (Figures 1 and 2A).Table 1.Mean effective concentration values (EC50, EC10, NOEC, and LOEC; units: µg L −1 ) and the respective 95% confidence intervals (CI), obtained after 3 days of exposure to one of four metals (Ag, Cd, Cr, and Cu) in two duckweed species (Spirodela polyrhiza and Lemna minor).

Cadmium Toxicity
Cadmium has been shown to reduce cellular protein, carbohydrate, and chlorophyll contents in Lemna, as well as inhibit frond number, area, and biomass [32,[34][35][36][37][38].In the present study, EC 50 of Cd toxicity for FA and RL measurements in L. minor were 415 and 155 µg L −1 , respectively.The Cd EC 50 s for S. polyrhiza were 205 for FA and 122 µg L −1 for RL.Cd toxicity as measured by FA was two-fold higher in S. polyrhiza than in L. minor (Figures 1 and 2B).Toxicity measured by RL did not differ between the species (Table 1).EC 50 values measured by FA and RL in S. polyrhiza were comparable to those reported elsewhere in Lemna spp.

Chromium Toxicity
Lemnaceae plantlets appear to have a relatively high tolerance to Cr compared to other metals [39].The EC 50 s for Cr toxicity measured by FA in L. minor and S. polyrhiza were 1756 and 507 µg L −1 , respectively, and the EC 50 s for RL were 109 and 219 µg L −1 for each type of plantlet, respectively (Figure 2C).The Cr sensitivity of S. polyrhiza in the current study was higher than that of the same species reported by Baudo et al. (2130 µg L −1 ) [19].The EC 50 values for FA and RL inhibition in S. polyrhiza were similar to those of frond growth (584-35,000 µg L −1 ) and root growth (237.0-1148.3µg L −1 ) in Lemna spp.(Table 1).

Copper Toxicity
Comparing the sensitivity of the two species to Cu for the relevant endpoints, the difference in FA between the two species is inconclusive based on the current data.For RL, Spirodela appears to be less sensitive than Lemna.However, previously reported EC 50 values based on frond number and frond weight for Lemna spp.range from 160-616 µg L −1 .On the other hand, Park et al. [31] calculated values between 221-470 µg L −1 for three Lemna species (L.gibba, L. minor, and L. paucicostata) when measuring RL.Based on these comparisons, the Cu sensitivity of S. polyrhiza is similar to that of Lemna, although it appears that roots are generally more sensitive to Cu than fronds (Figures 1 and 2D).

Applications for Wastewater Management
For S. polyrhiza, the most sensitive assays (based on mean rank of the EC 50 values) were measures of Ag toxicity using either RL or FA as endpoints.For L. minor, measuring Ag toxicity with RL was the most sensitive assay.The most reliable endpoint-metal combinations (based on the mean rank of the CV) were either FA-Ag or RL-Cd for S. polyrhiza.Other assays, including RL for Ag, Cd, Cr, and Cu, had intermediate levels of sensitivity for both species.Assays based on RL and FA (except FA-Ag) had moderate levels of reliability for S. polyrhiza.
Several of the toxicity assays for Spirodela tested here were able to detect pollutants at concentrations below the NPSWD in Republic of Korea.The EC 50 values for Cr and Cu using RL were below NPSWD (500 µg L −1 and 3000 µg L −1 , respectively) while only the EC 50 for Cu was below NPSWD when using FA.The Korean Ministry of the Environment does not currently have a standard limit for Ag discharge.While both sensitivity and reliability are important criteria for evaluating laboratory tests, they must ultimately be compared to the costs and precision of in situ water quality tests.
In Republic of Korea, water quality monitoring is typically conducted via direct chemical analysis.This method has several drawbacks, including complex procedures for sample preparation, the need for expensive analytical equipment, and interference from secondary pollutants during analysis.To compensate for these shortcomings, the use of EC 50 values obtained from bioassays should be considered to establish more ecologically meaningful permissible standards for wastewater discharge.

Predicted No-Effect Concentrations for Four Metals
The constructed SSD model used a log-normal distribution, and the simulation curves for the four metals in freshwater ecosystems are shown in Figure 1.In addition, to assess the potential risk to the aquatic environment, we compared the relevant literature for Spirodela and Lemna with the PNEC values derived from the SSD curves in this study with the metal tolerance limits in Korean river water as summarized in Table 2. To ensure the safety of aquatic plants like duckweed from metal pollutants in their environment, the concentration of metals in the water (e.g., river water) where these organisms live must be kept below a specific threshold, i.e., the corresponding PNECs for these organisms.Currently, the allowable limits for metals in river water set by the Korean Ministry of Environment are registered as Cd < 5 µg L −1 and Cr(VI) < 50 µg L −1 (there are no criteria for Ag and Cu).Comparing the PNECs calculated from the results of the current study and the literature with the permissible levels of Cd and Cr shown in Table 2, it can be seen that the established limits for both Cd and Cr(VI) in river water are higher than the safety limits for the two aquatic plants.Therefore, the current management settings for Cd and Cr in river water need to be reviewed if Spirodela and Lemna are to survive as primary producers providing energy, food and nursery grounds for organisms of higher trophic levels.

EVIEW 5 of 10 Figure 1 .
Figure 1.Mean ranks of sensitivity and reliability for each of the two endpoints (frond area and root regrowth length) in two duckweeds exposed to four metals (Ag, Cd, Cr, and Cu).

Figure 1 .
Figure 1.Mean ranks of sensitivity and reliability for each of the two endpoints (frond area and root regrowth length) in two duckweeds exposed to four metals (Ag, Cd, Cr, and Cu).

Figure 1 .
Figure 1.Mean ranks of sensitivity and reliability for each of the two endpoints (frond area and root regrowth length) in two duckweeds exposed to four metals (Ag, Cd, Cr, and Cu).

Table 2 .
Summary statistics for SSDs fit to two duckweed test results (units: µg L −1 ).05 ; the slope, PNEC; predicted no-effect concentration, NPSRW; Korean Nationally Permissible Standard for River Water.There are no criteria for Ag and Cu in the permissible limits of river water in Republic of Korea. HC