Microalgae-Based Fluorimetric Bioassays for Studying Interferences on Photosynthesis Induced by Environmentally Relevant Concentrations of the Herbicide Diuron

Abstract

The widespread agricultural use of the phenylurea herbicide Diuron (DCMU) requires the investigation of ecotoxicological risk in freshwater and soil ecosystems in light of potential effects on non-target primary producers and a heavier effect on higher trophic levels. We used microalgae-based fluorimetric bioassays for studying the interferences on the photosynthesis of a freshwater and soil model green microalga (Chlamydomonas reinhardtii) induced by environmentally relevant concentrations of the herbicide DCMU. Measurements of steady-state chlorophyll a (Chl-a) fluorescence emission spectra were performed; as well, the kinetics of the Chl-a fluorescence transient were recorded. Percentage indexes of interference on photosynthesis were calculated after comparison of steady-state and kinetic Chl-a fluorescence measurements of DCMU-exposed and control C. reinhardtii cell suspensions. The results obtained after 30 min exposure to the herbicide DCMU confirmed a significant inhibitory effect of DCMU 2 μg/L, and no significant differences between %ι values for DCMU 0.2 μg/L and 0.02 μg/L exposures. Positive %ε values from kinetic measurements of the Chl-a fluorescence transient confirmed the same interfering effect of 2 μg/L DCMU on PSII photochemistry in the exposed C. reinhardtii cell suspensions. Negative values of %ε observed for 0.2 and 0.02 μg/L DCMU exposures could be attributable to a presumptive 'stimulatory-like' effect in the photochemistry of photosynthesis. Short-term exposure to sub-μg/L DCMU concentration (≤0.2 μg/L) affects the photosynthetic process of the model microalga C. reinhardtii. Similar environmental exposures could affect natural communities of unicellular autotrophs, with hardly predictable cascading secondary effects on higher trophic levels.

Keywords: Kautsky effect; chlorophyll fluorescence; ecosystem health; ecotoxicology; herbicides; microalgae.

Figure 1

Schematic representation of experimental protocol used for the in vivo steady-state Chl-a fluorescence emission spectra.

Figure 2

Schematic representation of experimental protocol used in the kinetic bioassays.

Figure 3

The comparison of F_{684 nm} values between exposed and control microalgae cell suspensions after 30 min exposure to DCMU. RSD ≤ 15%. Two–way ANOVA without replication testing was applied for statistical differences between means values of F_{684 nm exp} and F_{684 nm blk}. Statistical significance of the results is indicated with asterisks (* p < 0.05; ** p < 0.01).

Figure 4

OJIP phase of the Kautsky curves recorded for (a) 0.02 μg/L DCMU, (b) 0.2 μg/L DCMU, and (c) 2 μg/L DCMU–exposed microalgae cell suspensions (green line). Blank microalgae cell suspensions are in the blue line. Fo (minimal fluorescence, first reliable fluorescence value); F_M (maximal fluorescence value at the peak of the OJIP curve under saturating illumination); F_{8 ms} (fluorescence value at 8 milliseconds). Baseline correction was performed using curves from TAP growth medium.

Figure 5

The percentage variation of V_{8 ms} index (%ε) after 30 min exposure to DCMU. RSD ≤ 15%. Two-way ANOVA without replication testing was applied for statistical differences between means values of V_{8 ms exp} and V_{8 ms blk}. Statistical significance of the results is indicated with asterisks (** p < 0.01).