A fluorescence-based bioassay for aquatic macrophytes and its suitability for effect analysis of non-photosystem II inhibitors |
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Authors: | Anette Küster Korinna Pohl Rolf Altenburger |
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Institution: | (1) Department Bioanalytical Ecotoxicology, UFZ — Helmholtz Centre for Environmental Research, 04318 Leipzig, Germany |
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Abstract: | Background, Goals and Scope During the last years the miniaturization of toxicity test systems for rapid and parallel measurements of large quantities
of samples has often been discussed. For unicellular algae as well as for aquatic macrophytes, fluorescence-based miniaturized
test systems have been introduced to analyze photosystem II (PSII) inhibitors. Nevertheless, high-throughput screening should
also guarantee the effect detection of a broad range of toxicants in order to ensure routinely applicable, high-throughput
measuring device experiments which can cover a broad range of toxicants and modes of action others than PSII inhibition. Thus,
the aim of this study was to establish a fast and reproducible measuring system for non-PSII inhibitors for aquatic macrophyte
species to overcome major limitations for use.
Methods A newly developed imaging pulse-amplitude-modulated chlorophyll fluorometer (I-PAM) was applied as an effect detector in short-term
bioassays with the aquatic macrophyte species Lemna minor. This multiwell-plate based measuring device enabled the incubation and measurement of up to 24 samples in parallel. The
chemicals paraquat-dichloride, alizarine and triclosan were chosen as representatives for the toxicant groups of non-PSII
herbicides, polycyclic aromatic hydrocarbons (PAHs) and pharmaceuticals and personal care products (PPCPs), which are often
detected in the aquatic environment. The I-PAM was used (i) to establish and validate the sensitivity of the test system to
the three non-PSII inhibitors, (ii) to compare the test systems with standardized and established biotests for aquatic macrophytes,
and (iii) to define necessary time scales in aquatic macrophyte testing. For validation of the fluorescence-based assay, the
standard growth test with L. minor (ISO/DIS 20079) was performed in parallel for each chemical.
Results The results revealed that fluorescence-based measurements with the I-PAM allow rapid and parallel analysis of large amounts
of aquatic macrophyte samples. The I-PAM enabled the recording of concentration-effect-curves with L. minor samples on a 24-well plate with single measurements. Fluorescence-based concentration-effect-curves could be detected for
all three chemicals after only 1 h of incubation. After 4–5 h incubation time, the maximum inhibition of fluorescence showed
an 80–100% effect for the chemicals tested. The EC50 after 24 h incubation were estimated to be 0.06 mg/L, 0.84 mg/L and 1.69
mg/L for paraquatdichloride, alizarine and triclosan, respectively.
Discussion The results obtained with the I-PAM after 24 h for the herbicide paraquat-dichloride and the polycyclic aromatic hydrocarbon
alizarine were in good accordance with median effective concentrations (EC50s) obtained by the standardized growth test for
L. minor after 7 d incubation (0.09 mg/L and 0.79 mg/L for paraquat-dichloride and alizarine, respectively). Those results were in
accordance with literature findings for the two chemicals. In contrast, fluorescence-based EC50 of the antimicrobial agent
triclosan proved to be two orders of magnitude greater when compared to the standard growth test with 7 d incubation time
(0.026 mg/L) as well as with literature findings.
Conclusion Typically, aquatic macrophyte testing is very time consuming and relies on laborious experimental set-ups. The I-PAM measuring
device enabled fast effect screening for the three chemicals tested. While established test systems for aquatic macrophytes
need incubation times of ≥ 7 d, the I-PAM can detect inhibitory effects much earlier (24 h), even if inhibition of chemicals
is not specifically associated with PSII. Thus, the fluorescence-based bioassay with the I-PAM offers a promising approach
for the miniaturization and high-throughput testing of chemicals with aquatic macrophytes. For the chemical triclosan, however,
the short-term effect prediction with the I-PAM has been shown to be less sensitive than with long-term bioassays, which might
be due to physicochemical substance properties such as lipophilicity.
Recommendations and Perspectives The results of this study show that the I-PAM represents a promising tool for decreasing the incubation times of aquatic macrophyte
toxicity testing to about 24 h as a supplement to existing test batteries. The applicability of this I-PAM bioassay on emergent
and submerged aquatic macrophyte species should be investigated in further studies. Regarding considerations that physicochemical
properties of the tested substances might play an important role in microplate bioassays, the I-PAM bioassay should either
be accompanied by evaluating physicochemical properties modeled from structural information prior to an experimental investigation,
or by intensified chemical analyses to identify and determine nominal concentrations of the toxicants tested. The chemicals
paraquat-dichloride, alizarine and triclosan were chosen as representatives for the toxicant groups of non-PSII herbicides,
PAHs and PPCPs which are often detected in the aquatic environment. Nevertheless, in order to ensure a routinely applicable
measuring device, experiments with a broader range of toxicants and samples of surface and/or waste waters are necessary.
ESS-Submission Editor: Dr. Markus Hecker (MHecker@Entrix.com) |
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Keywords: | Aquatic macrophytes bioassay Chlorophyll fluorescence high-throughput Imaging-PAM macrophytes phytotoxicity toxicity assessment |
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