Herbicide toxicity on river biofilms assessed by pulse amplitude modulated (PAM) fluorometry
Introduction
The European Union’s Water Framework Directive set the target to achieve good ecological status for all aquatic environments in Europe by 2015 (WFD, 2000/60/EC). Among environmental pollutants, pesticides are of particular concern and greatly affect water quality. In particular, aquatic organisms are often exposed to mixtures of compounds, some with complex or even unknown modes of action. This concerns especially breakdown products (Sinclair and Boxall, 2003) for which ecological risk assessment has to be improved (Artigas et al., 2012).
The Morcille River located in the Beaujolais vineyard area (Eastern France) is subjected to strong agricultural pressure, essentially exerted by vineyard treatments. In particular, high concentrations of the herbicides diuron, its main biodegradation product, 1-(3,4-dichlorophenyl)-3-methyl urea (DCPMU) and norflurazon (NFZ) have been recorded over several years at the downstream site on the river (Montuelle et al., 2010, Rabiet et al., 2010, Morin et al., 2012). Photosynthetic processes are highly sensitive to the presence of toxicants leading to an increase in their use as ecotoxicological endpoints in particular through the measurement of fluorescence parameters (Corcoll et al., 2012). Diuron is a phenylurea herbicide which blocks electron transfer from Photosystem II (PSII) to Photosystem I (PSI). It binds specifically on the D1 protein of PSII, where, it replaces the second electron acceptor (Qb) (Trebst and Draber, 1986, Zer and Ohad, 1995). Diuron thus prevents reoxidation of the primary electron acceptor (Qa) by blocking electron transfer from Qa to Qb which leads to an increase of the minimal fluorescence and a decrease in variable fluorescence (Ralph, 2000). DCPMU is the main degradation product of diuron; it is obtained by N-demethylation under aerobic conditions. The loss of the methyl group appears to cause a slight decrease in the binding affinity to the Qb receptor site of D1 (Dewez et al., 2002, Neuwoehner et al., 2010). A slightly lower toxicity of DCPMU compared to the parent compound for photosynthetic organisms is thus expected. Actually, Pesce et al. (2010a) observed a 4–18-fold lower toxicity of DCPMU compared to its parent compound on river biofilms. Nevertheless, studies assessing the effects of metabolites are still scarce. Moreover, the relative toxicity of DCPMU and diuron seems to be highly dependent on the organisms selected and the endpoints considered (Dewez et al., 2002, Gatidou and Thomaidis, 2007). NFZ is a herbicide belonging to the pyridazinone family which inhibits carotenoid biosynthesis by exerting its primary inhibitory action on the reaction catalysed by phytoene synthase (Sandmann et al., 1980). Carotenoids are photosynthetic pigments playing a crucial role in light harvesting and photoprotection (Rowan, 1989). Exposure to NFZ is expected to lead to indirect effects on fluorescence levels by inhibiting renewal of the cellular carotenoid pool essential to the correct functioning of the photosynthetic apparatus. Its effects have been well studied with regards to growth-related parameters or photosynthetic pigments in microalgae or plants (Sandmann et al., 1981, Frankart et al., 2003, Wilkinson, 1987, Wilson and Koch, 2013) but very little is known on its indirect effects on photosynthetic activity and in particular on river biofilms (Nestler et al., 2012, Kim Tiam et al., 2014).
Pulse Amplitude Modulated fluorometry (PAMF) is a powerful tool for monitoring the effects of herbicides on photosynthetic organisms based on the measurement of in vivo chlorophyll a (Chla) fluorescence (Juneau et al., 2007). It is widely used to assess the toxicity of various substances targeting PSII (Corcoll et al., 2012). PAM can be used to estimate various parameters including minimal fluorescence (F0), the effective quantum yield of photosystem II photochemistry (ФPSII) and the optimal quantum yield of PSII (Fv/Fm) which have been the most commonly used to study both short- and long-term effects of herbicides on photosynthetic activity (Corcoll et al., 2012). F0 can be used as a proxy of algal biomass since chlorophyll fluorescence can be proportional to total chlorophyll content (Serôdio et al., 1997). ФPSII gives a measure of the proportion of the PSII absorbed light that is used in photochemistry while Fv/Fm provides a relative measurement of the maximum quantum yield of PSII primary photochemistry and is commonly used for characterizing the overall physiological state of a sample (Baker, 2008). In particular, Fv/Fm and ФPSII have been shown to be appropriate when working with PSII inhibitors (e.g., Laviale et al., 2011, Laviale et al., 2010). These parameters can also potentially detect effects of compounds acting on targets other than PSII, since chemicals that inhibit any cellular process downstream of PSII (e.g., carbon assimilation or respiration, photosynthetic electron transport) will lead to increased excitation pressure on PSII. Recent investigations on such compounds recommended combining this approach with other parameters (Bonnineau et al., 2012).
PAM fluorometry also allows monitoring of the electron transport rate (ETR), which is directly derived from the measurement of ФPSII and provides a relative measurement of the linear electron transport rate (Schreiber, 2004). Changes in ETR can be followed as a function of the variation in level of ambient light (E), from limiting to saturating, thus allowing the construction of ETR versus E curves which are then used to evaluate the photoacclimation status of photosynthetic organisms (Laviale et al., 2009). Traditional steady-state light curves require from several minutes to a few hours to complete (Henley, 1993). A convenient alternative is the establishment of so-called fluorescence rapid light curves (RLCs), which can be completed within 2 min (Schreiber et al., 1997, White and Critchley, 1999). This approach has been commonly used on various photosynthetic organisms, including microalgal biofilms (Kromkamp et al., 1998, Roberts et al., 2004, Serôdio et al., 2005), but reports of the use of RLCs to highlight toxicant effects are scarce (Luís et al., 2013, Herlory et al., 2013). Nevertheless it offers promising perspectives particularly when working with toxicants with less specific modes of action than PSII inhibitors.
The first objective of this study was to compare the toxicity of diuron, DCPMU and NFZ on river biofilms, particularly of DCPMU and NFZ since few ecotoxicological data are available for these products, especially at the community level (Pesce et al., 2011). The second objective of the study was to evaluate the relevance of RLC-derived parameters as early warning endpoints of toxicity for pollutants with less specific modes of action, in comparison to more classical fluorescence-based endpoints used in toxicological studies. For this purpose, field-derived biofilms were exposed to a range of concentrations, from ecologically relevant to those acutely toxic; the evolution of biofilm responses was assessed after 1, 5, 7 and 14 days of exposure in regards to “classical endpoints” and RLC parameters.
Section snippets
Biofilm sampling and culture
Biofilms were collected in the Morcille River, located in the Beaujolais vineyard area (eastern France). Sampling took place in March 2013 at a pristine site upstream characterized by very low pesticide concentrations (Montuelle et al., 2010). Biofilm was collected by scraping streambed rocks with a razor blade and was re-suspended in river water from the same site. The biofilm was inoculated into an aquarium containing 8 L of WC culture medium (Guillard and Lorenzen, 1972) under continuous
“Classical fluorescence parameters”
F0 and Fv/Fm were measured after 30 min of dark adaptation whereas ФPSII was measured under ambient light. Fv/Fm and ФPSII are described by the following equations (Baker, 2008):Fv/Fm = (Fm − F0)/FmΦPSII = (Fm′ − Ft)/Fm′
- (1)
with F0 minimal fluorescence determined after the emission of a weak far red modulated light and Fm the maximum level of fluorescence measured during a saturating white light pulse.
- (2)
with Ft the steady-state level of fluorescence under ambient light and Fm′ the maximum level of
Rapid light curves
Rapid light curves were constructed by exposing the samples to 10 sequential actinic light levels increasing from 64 to 610 μmol m−2 s−1 (i.e., the maximum light level range provided by the PhytoPAM) after a 5 min adaptation period at 64 μmol.m−2 s−1 corresponding to the light intensity of the first step of the curve. ФPSII was calculated as described above after each illumination step of 10 s. RLCs were constructed and fitted using the Phyto-Win Software V 1.45 (Heinz Walz GmbH, Germany). For each
Principal response curve analyses
The PRCs performed with in vivo fluorescence parameters and chlorophyll data are presented in Fig. 1. For diuron (Fig. 1a) 22% of the total variance can be attributed to time, whereas, 62% can be attributed to the chemical treatment. These values were of 15% and 65% for DCPMU (Fig. 1b) and 12% and 70% for NFZ (Fig. 1c).
The parameters extracted from the Rapid Light Curves (α, ETRmax and Ik) as well as the photosynthetic yields (Fv/Fm and ФPSII) decreased with pesticide exposure, as indicated by
Relative toxicity of the compounds tested
In this study we tested the relative toxicity of two herbicides (diuron and NFZ) and one degradation product (DCPMU) for a range of concentrations (from 0.3 to 33.4 μg L−1 for diuron, from 1.0 to 1014 μg L−1 for DCPMU and from 0.6 to 585 μg L−1 for NFZ) at different exposure times. Several classical endpoints were assessed to test the effects of these three compounds on natural biofilms, photosynthetic yields (ФPSII and Fv/Fm) and minimal fluorescence (F0). Both photosynthetic yields showed that
Conclusions and perspectives
In this study, we demonstrated the usefulness of PAM fluorometry for assessing the toxicity of two pesticides with different modes of action (diuron and NFZ) and one pesticide degradation product (DCPMU). The three pesticides had significant effects on all the fluorescence parameters which were monitored: ФPSII, Fv/Fm, F0 and RLC-parameters. These effects were dose and time dependent and the observed patterns could be explained by their mode of actions: herbicides targeting PSII specifically
Acknowledgments
This work was supported by the PoToMAC (Potential Toxicity of pesticides in Continental Aquatic Environments: passive sampling and exposure/impact on biofilms) programme under the reference ANR-11-CESA-022 and the French National Agency for Water and Aquatic Environments (ONEMA). The authors would like to thank S. Moreira from Irstea Bordeaux and B. Motte and S. Pesce from Irstea Lyon, for their skilful technical assistance. They are also grateful to the two anonymous reviewers for their
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2021, Science of the Total EnvironmentCitation Excerpt :In contrast, maximum quantum yield (Fv/Fm) of freshwater phytoplankton was suppressed following glyphosate exposure (< 1.5·104 μg·L−1 (Choi et al., 2012); 5.0·102 0–1.0·103 μg·L−1 (Smedbol et al., 2017)), with responses following Michaelis-Menten saturation kinetics (Choi et al., 2012), but also showing considerable species-specific differences in sensitivity (Smedbol et al., 2017). Quantum yield alone may not be sensitive enough to assess herbicide stress in periphyton communities over short time scales (Tiam et al., 2015), in particular for herbicides that do not directly affect Photosystem II (Feckler et al., 2018). Our results support this assessment, and we recommend parallel measurement of different responses over time to effectively capture herbicide effects on algal physiology and metabolism.