Mature plants of Spirodela polyrhiza (L.) Schleiden were collected from the Upo wetland located at Changnyeong-gun, Gyeongsangnam-do, Korea (35˚34'05''60N, 128˚24'06''90E). Fronds were maintained in a 1.5 L glass tank containing Steinberg medium [24] at 25˚C under continuous white light (30 - 40 µmol photons m⁻²∙s⁻¹) supplied by cool daylight fluorescence tubes (FL 20 SS/18D, Philips). The medium was replaced regularly at 7 day intervals, and its pH was adjusted to 6.9 ± 0.2 using either 1 M hydrochloric acid (HCl) or 1 M sodium hydroxide (NaOH).

To induce the production of turions, 1.0 L culture flasks were stored at 15˚C for 4 - 6 weeks. Upon induction, each turion was separated from its mother frond [25]. All turions were then collected from the bottom of the flasks and transferred to 50 mL Falcon tubes containing Steinberg medium. The Falcon tubes were covered with foil, and the stock seed banks were stored at 4˚C until needed for tests.

Prior to the tests, turions were germinated in Petri dishes (85.6 mm × 12.6 mm) containing Steinberg medium at 25˚C under continuous light (photon irradiance of 90 - 100 µmol photons m⁻²∙s⁻¹). Fronds germinated from turions were maintained under the same environmental conditions for either 3 days or 8 weeks. Just prior to experimentation, roots were cut from all fronds using stainless steel scissors and an individual rootless plant was added to each well of a 24-well plastic plate (85.4 × 127.6 mm; well diameter, 15.6 mm; SPL, Seoul, Korea). There were 4 plants per paraquat concentration, 6 concentrations per plate and three replicate plates (n = 3). Different nominal concentrations of paraquat (Table 1) were prepared by diluting the original stock solutions (CAS No. 1910-42-5) with Steinberg medium. To perform the toxicity tests, a 3.0 mL test solution was added to each well. Tests were run for 72 h in an environmentally controlled chamber maintained at 25 ± 1˚C and continuous light (100 ± 10 µmol photons m⁻²∙s⁻¹). Static tests were performed, i.e. the test solutions were not renewed during the period of exposure. A fully randomized design was used to account for any variability in environmental conditions within the culture chamber.

Data were analyzed using one-way analysis of variance (ANOVA) at a significance level of P < 0.05, after homogeneity test. To determine differences among treatments, post-hoc comparisons were performed using the least significant difference (LSD) test. Toxicity test results were reported as EC₅₀ values (effective concentration at which 50% inhibition occurs), with 95% confidence intervals estimated using the linear interpolation method (ToxCalc 5.0; Tidepool Science, California, USA).

Determination of the optimal conditions for turion germination is important since several environmental factors can simultaneously influence germination of the turions of S. polyrhiza [29], but standardization of optimal germination for toxicity testing has not yet been established [17]. The germination of S. polyrhiza under different photon irradiances, pH and temperatures is shown in Figure 1. Turions germinated in all light levels from 5 to 100 μmol photons m⁻²∙s⁻¹ but not in the dark (Figure 2(a)); turion germination is light dependent normally [30]. The maximal germination was recorded at pH 5 and 7 but at pH 3 no germination was observed (Figure 2(b)). There was 100% germination of turions at 25˚C and 30˚C but there was no evidence of germination at either 5˚C or 15˚C (Figure 2(c)).

In nature, germination of Spirodela turions is controlled by temperature and light [31], although exposure to combinations of red and far-red light, long photoperiods, gibberellin and kinetin (plant hormones) can accelerate the germination process [32] [33] [34]. In laboratory studies, turion germination was found to be controlled by exposure to temperature and light of 22˚C - 25˚C and 60 to 120 μmol photons m⁻²∙s⁻¹ for 3 - 7 days [17] [19].

It has been observed that turions float to the water surface by formation of internal oxygen bubbles prior to their germination [30] [35]. Photosynthetic activities thus appear to be involved in the germination process since turion surfacing may be geared by photosynthetic O₂ production, which is also affected by bicarbonate concentrations as a major aquatic carbon source [31]. In this respect, temperature and pH would be influential factors for initiation of germination since the amount of carbon (as CO₂ or H₂CO₃) in the water is dependent on these

parameters. It is therefore interesting to note that germination reaches its maximum at pH 7 where the availability of H₂CO₃ is at its greatest.

Environmental factors influence growth and photosynthetic activity of aquatic plants, with pH, light and temperature being probably the most important parameters. It has been shown that these parameters also modify the toxic effects of chemical pollutants in aquatic plants [36]. Thus, it is important that the culture conditions under which toxicity tests are carried out are fully reported/disclosed and that the tests are, preferably, carried out under standardized conditions for a particular species and endpoints to be measured. For this reason, we first determined the optimal conditions for growth of fronds and roots of S. polyrhiza (Figure 3 and Figure 4). The maximal growth of both frond and root was recorded at 100 μmol photons m⁻²∙s⁻¹ and 25˚C while no significant difference was observed in the range of pH between 4 and 10. It is interesting to note that there was some growth at low temperatures despite the lack of germination at these temperatures. Also, no growth was detected at temperatures lower than 10˚C. All these data appear to imply the existence of different control mechanisms for turion germination, frond growth and root regrowth.

Different aged fronds (3 days and 8 weeks old) showed intrinsic differences in physiological traits (Table 2). The area and fresh weight of young fronds were approximately 19.06% and 29.35% that of mature fronds. However, roots of young fronds (17.58 ± 2.98 mm) were longer than those of mature fronds (11.98 ± 3.23 mm). No statistically significant differences were detected in Chl a and Chl b contents between young and mature fronds. When Spirodela fronds become mature, there are enlargements in the mesophyll cells with prominent intercellular spaces and air chambers in the abaxial surface of maturing fronds [37]. Morphological changes in the internal structure of fronds during development may explain the significantly larger frond area and weight in mature fronds than in newly germinated fronds.

Spirodela plants have simple, short and thin roots that lack branches and hairs [38], although the root systems of young and mature fronds have not been compared. Roots appear to serve an important role in balancing and maintaining the floating body in a stable and upright position [38]. Differences in root length between young and mature fronds might be explained by the higher photosynthetic capacity of young fronds. Thus, in young plants photosynthetic products are re-directed in favor of root production that provides the smaller and lighter floating fronds stable positioning.

On the other hand, we found significantly higher Chl a and Chl b content in young than in mature fronds, which was similar to the report made by Kim [39]. As the chlorophyll contents were calculated on a fresh weight basis, lower chlorophyll in mature fronds may be ascribed to higher proportion of non-photosynthetic structures such as intracellular spaces and air chambers.

Values of F_v/F_m, rETR_max and I_K of young fronds (0.752 ± 0.034, 20.58 ± 8.28 and 122.34 ± 47.64 µmol photons m⁻²∙s⁻¹, respectively) were significantly higher than those of mature fronds (0.491 ± 0.160, 12.18 ± 6.355 and 71.22 ± 26.28 µmol

FA; relative growth rate of frond area, FW; fresh weight, RL; root regrowth length, Chl a; chlorophyll a content, Chl b; chlorophyll b content, F_v/F_m; Optimal quantum yield, rETR_max; the maximum electron transport rate, SD; standard deviation. **P < 0.001; *P < 0.05.

photons m⁻²∙s⁻¹, respectively). However, alpha values were similar between the two frond types (0.168 for young and 0.160 for mature fronds).

The maximum quantum yield of PSII chemistry (F_v/F_m) is an estimate of the photochemical conversion efficiency of PSII in the dark. Higher values of F_v/F_m indicate higher light utilization efficiency and greater ability to adapt to low light conditions [40]. Lemna spp. typically have F_v/F_m maxima of about 0.68 - 0.73 [11] [13]. Compared with Lemna spp., young fronds of S. polyrhiza were found to have similar F_v/F_m, but mature fronds showed lower F_v/F_m. F_v/F_m values appear to be in line with chlorophyll contents in that F_v/F_m was greater in young fronds with higher Chl a and Chl b than in mature fronds.

rETR is an empirical estimate of the rate of flow of electrons through the electron transport chain and rETR_max is defined as the maximum rETR at saturating irradiance [40]. Higher rETR_max in young fronds means that there is higher probability of a photochemical event resulting in more active and effective electron transport upon absorption of a photon by the antennae of PS II. The initial slope of a P vs I curve represents the light-limited phase of photosynthesis, and its gradient is affected by the efficiency with which the plant can absorb the limited light available [41]. Saturation irradiance (I_k) is defined as the point at which the extrapolated initial slope crosses rETR_max. Comparison of rETR_max, alpha and I_k showed that the number of photosynthetic units (PSUs) but not the size of PSUs differed between young and mature fronds of S. polyrhiza since ETR_max and I_k values were higher while alpha values were the same [41]. Young fronds seem to have more PSUs than mature ones, and hence the higher rETR_max and I_k values.

Paraquat sensitivity of S. polyrhiza fronds at both stages was dependent on the endpoints. After a 72 h exposure to paraquat, the most sensitive paraquat toxicity endpoints were root length and rETR_max in both frond types, with EC₅₀ values of 0.66 ± 0.03 and 0.76 ± 0.09 µg∙L⁻¹, respectively, in young fronds and 5.53 ± 1.5 and 2.28 ± 0.39 µg∙L⁻¹, respectively, in mature fronds (Table 3). Coefficients of variation for root re-growth and rETR_max were 4.55% and 11.84%, respectively, for young fronds and 27.12% and 17.11%, respectively, for mature fronds (Table 3). Paraquat was reported to be one of the most toxic herbicides for Lemna spp. with EC₅₀ values of 7.1 - 10.6 µg∙L⁻¹ for root regrowth and 6.6 - 8.0 µg∙L⁻¹ for rETR_max [13]. The paraquat sensitivity of Spirodela was higher in young fronds and similar in mature fronds to that of Lemna spp.

It is notable that there was statistically significant difference in the sensitivity to paraquat toxicity between young and mature fronds, which would bring to question as to which stages, young or mature fronds would then be more suitable for toxicity testing. Sensitivity of bioassay methods is important for determination of whether to use them or not for water quality risk assessment. Effective bioassays should produce results within the relevant environmental ranges.

FA; relative growth rate of frond area, FW; fresh weight, RL; root regrowth length, Chl a; chlorophyll a content, Chl b; chlorophyll b content, F_v/F_m; Optimal quantum yield, rETR_max; the maximum electron transport rate, EC₅₀; the effective concentration at which 50% inhibition occurs, CV; coefficient of values, NA; not applicable.

A current guideline for the allowable concentrations in drinking waters set by WHO is 10 µg∙L⁻¹ for paraquat. This study shows that endpoints of root length and rETR_max in both young and mature frond of S. polyrhiza are sensitive enough to detect toxic impacts of water samples containing paraquat in excess of allowable guidelines. However, the sensitivity of young fronds is almost over 10 times the permissible levels of paraquat in drinking water, and unless even a slightest presence of paraquat should be monitored and prevented, mature fronds would more successfully be employed for management decisions. Spirodela methods also show a high level of precision and reproducibility which are essential for adoption of toxicity testing methods. A desirable level of repeatability expressed by CVs is 30% or less according to Environment Canada (2007) [42]. For young and mature fronds with the endpoints of root length and rETR_max CVs for EC₅₀ values were found to lie within this acceptable range (Table 3).

The ultimate goal of bioassay testing is to provide representative and inclusive criteria of exposure conditions, thereby improving risk assessment and water quality management. In this respect, multiple rather than single endpoint assays may have greater potential for a more comprehensive risk assessment of toxicants. Such an approach allows to gain important insights into the mechanisms of toxicity and to obtain information on the relative sensitivity of measured endpoints to toxicity concentration and/or exposure duration, thereby identifying specific endpoints that can effectively detect interferences caused by certain phytotoxics [43]. In the past, little attention has been paid to the roots in Lemna since it was generally considered that root fragility made their handling for measurements difficult and that it was impractical to obtain sufficient numbers of individual plants with identical root lengths to initiate tests. However, more recently the ecotoxicological significance of the root endpoint has been re-evaluated and root length is now considered to be a sensitive, precise and ecologically significant endpoint in comparison with more traditional frond growth and biomass endpoints [36] [44].

One of the most frequently used methods for monitoring the status of the photosynthetic apparatus in plants is in vivo chlorophyll a fluorescence, a non-destructive, straightforward and rapid technique that is applicable in both laboratory and field studies. It is used as a potential indicator of exposure to environmental and chemical stresses, including herbicides. The impact of certain herbicides, such as commonly used ones like diuron, atrazine and simazine, on the photochemical activity of PSII has long been recognized [45] [46] [47] [48]. Such PSII inhibitors restrict photosynthetic activity through their binding to the D1 protein in thylakoids and blocking electron transport through the PSII reaction center with effects being manifested by changes in various chlorophyll fluorescence parameters [49]. Frond area, fresh weight, root length, chlorophylls and chlorophyll a fluorescence have never been measured simultaneously as endpoints. FigureS1 shows correlative relationships between two different endpoints. There was a strong relationship between the frond area and weight and between Chl a and Chl b to a lesser degree in both young and mature fronds. It was notable that root length rather than frond growth was highly correlated with photosynthetic performance. Photosynthetic electron transport events support the biochemical reactions needed for plant growth since the electron transport rate is closely related to the photosynthetic activity including oxygen evolution or CO₂ uptake [50]. Therefore, a direct or an indirect effect of a pollutant on photosynthetic processes is observed prior to an effect on the growth process [51]. Little relationship between PSII inhibition and frond growth may indicate possible detoxification of cells and recovery of growth, thereby the inhibition of PSII inhibition no longer reflecting the inhibition of growth [52]. In contrast, the close relationship between PSII inhibition and root re-growth may imply a direct or an indirect effect of a pollutant on photosynthetic processes observed prior to an effect on the root growth process [51].