Fluvial Biofilms Research Articles

Saturation of intracellular phosphorus uptake and prevalence of extracellular phosphorus entrapment in fluvial biofilms after long-term P pulses

Saturation of intracellular phosphorus uptake and prevalence of extracellular phosphorus entrapment in fluvial biofilms after long-term P pulses

Contaminants of emerging concern fate and fluvial biofilm status as pollution markers in an urban river

Contaminants of emerging concern (CEC) are still under research given the vast diversity of compounds reaching freshwater ecosystems and adverse effects they might cause. In this study, the environmental fate of 73 CEC, comprising sweeteners, stimulants and several pharmaceutical therapeutic classes, and changes in fluvial biofilm photosynthetic parameters were evaluated in a semi-arid urban river receiving diffuse and point sources of pollution (Suquía river, Argentina). Out of the 37 CEC detected, 30 were quantified in surface water (n.d. – 9826 ng/L), 10 in biofilm (n.d. – 204 ng/gd.w.) and 9 in the clay fraction of sediments (n.d. – 64 ng/gd.w.). CEC distribute differently among the 3 matrices: water phase presents the biggest diversity of compounds (14 CEC families), being analgesic/anti-inflammatories the most abundant family. Antibiotics largely predominated in biofilms (7 CEC families), while the stimulant caffeine and some antibiotics where the most abundant in sediments (6 CEC families). Different CEC accumulated in biofilms and sediments upstream and downstream the city, and big shifts of biofilm community occurred downstream WWTP. The shift of biofilm community upstream (F0 > 0) and downstream the WWTP (F0 = 0) shows a sensitive response of F0 to the impact of WWTP. Biofilm photosynthetic parameters responded in less impacted urban sites (sites 1, 2 and 3), where significant correlations were found between ketoprofen and some antibiotics and biofilm parameters. The diversity and amount of CEC found in the urban section of Suquía river alert to the magnitude of point and non-point sources of pollution.

Effects of Graphene Oxide Nanosheets in Freshwater Biofilms.

Graphene oxide (GO) properties make it a promising material for graphene-based applications in areas such as biomedicine, agriculture, and the environment. Thus, its production is expected to increase, reaching hundreds of tons every year. One GO final destination is freshwater bodies, possibly affecting the communities of these systems. To clarify the effect that GO may impose in freshwater communities, a fluvial biofilm scraped from submerged river stones was exposed to a range (0.1 to 20 mg/L) of GO concentrations during 96 h. With this approach, we hypothesized that GO can: (1) cause mechanical damage and morphological changes in cell biofilms; (2) interfere with the absorption of light by biofilms; (3) and generate oxidative stress, causing oxidative damage and inducing biochemical and physiological alterations. Our results showed that GO did not inflict mechanical damage. Instead, a positive effect is proposed, linked to the ability of GO to bind cations and increase the micronutrient availability to biofilms. High concentrations of GO increased photosynthetic pigment (chlorophyll a, b, and c, and carotenoids) content as a strategy to capture the available light more effectively as a response to the shading effect. A significant increase in the enzymatic (SOD and GSTs activity) and low molecular weight (lipids and carotenoids) antioxidant response was observed, that efficiently reduced oxidative stress effects, reducing the level of peroxidation, and preserving membrane integrity. Being complex entities, biofilms are more similar to environmental communities and may provide more accurate information to evaluate the impact of GO in aquatic systems.

Interactions between microplastics and benthic biofilms in fluvial ecosystems: Knowledge gaps and future trends

Plastics, especially microplastics (<5 mm in length), are anthropogenic polymer particles that have been detected in almost all environments. Microplastics are extremely persistent pollutants and act as long-lasting reactive surfaces for additives, organic matter, and toxic substances. Biofilms are microbial assemblages that act as a sink for particulate matter, including microplastics. They are ubiquitous in freshwater ecosystems and provide key services that promote biodiversity and help sustain ecosystem function. Here, we provide a conceptual framework to describe the transient storage of microplastics in fluvial biofilm and develop hypotheses to help explain how microplastics and biofilms interact in fluvial ecosystems. We identify lines of future research that need to be addressed to better manage microplastics and biofilms, including how the sorption and desorption of environmental contaminants in microplastics affect biofilms and how microbial exchange between microplastics and the biofilm matrix affects biofilm characteristics like antibiotic resistance, speciation, biodiversity, species composition, and function. We also address the uptake mechanisms of microplastics by consumers and their propagation through the food web.

Microbial community-level physiological profiles: Considering whole data set and integrating dynamics of colour development

Abstract Community Level Physiological Profiles (CLPPs) have been used as ecological indicators from long ago as they determine physiologic and metabolic potential of microbial communities and indicate the capability to adapt their metabolism to anthropogenic stressors or to changes in environmental conditions. Also, these profiles are proxies for functional diversity or functional traits of a microbial community, usually linked to the ecosystem functioning. However, statistical data analysis of CLPPs has turned out to be the main handicap in these studies. Specifically, the interference of inoculum density, the need to choose an optimal incubation time and the different profiles obtained depending on the incubation time-point limits its usefulness. To this aim, we have developed a new approach for CLPP data analysis that considers the whole data set and dynamics of colour development along the incubation period. This approach is based on compositional data (CoDa) transformation, which reflects relative rather than absolute information, followed by repeated measures multivariate analysis of variance and data representation in a canonical variates plot. The approach has been tested by using CLPPs from real fluvial biofilm samples affected by seasonality, flow stress and different sediment depth conditions, and it has been compared to the most classic applied approaches until today (incubation fixed point or curve integration, followed by a Principal Components Analysis). Our results highlight that the proposed approach (i) makes ecological interpretation of CLPPs more sensitive, (ii) clarifies differences between CLPPs even in situations of marked seasonality while classical approaches provided confounding results, (iii) produces ecologically meaningful results (i.e. the proposed approach showed significant differences in organic matter use capabilities among flow conditions and depth), and (iv) unifies the analysis methodology for CLPPs data. With this work we provide an R-script to be used in future studies.

Near streambed flow shapes microbial guilds within and across trophic levels in fluvial biofilms

AbstractFlow is an important physical driver of biofilm communities. Here, we tested the effects of the near bed flows in two mountainous stream reaches on the structure of biofilm microbial guilds (autotrophs, heterotrophic bacteria, and heterotrophic protists) within and across trophic levels. Near bed flow velocity and turbulent kinetic energy were important physical drivers for structuring the communities within and across guilds of the multitrophic fluvial biofilms. The effects of flow were nested in a seasonal and spatial (across‐streams) context. Changes in physicochemical factors (temperature, light, dissolved carbon, and nutrients) along the reaches were alike in both streams suggesting that environmental boundary conditions at larger temporal scales were responsible for the seasonal differences of biofilm communities, whereas locally microbial diversity was shaped by near bed flow. Typically, the abundance of autotrophs increased with flow, indicating that biofilms shifted toward increasing autotrophy with increasing shear forces. Filamentous autotrophs seemed to provide protected habitats from the shear forces for smaller sized bacteria. Heterotrophic protist abundance decreased with flow leading to decreasing grazer to prey ratio. Bacteria thus benefitted from a reduction in grazing pressure at faster flowing, more turbulent sites. Our results suggest that near bed flow can impact the magnitude and direction of matter fluxes through the microbial food web and possibly affect ecosystem functioning of fluvial biofilms.

Responses of resident (DNA) and active (RNA) microbial communities in fluvial biofilms under different polluted scenarios.

Pollution from human activities is a major threat to the ecological integrity of fluvial ecosystems. Microbial communities are the most abundant organisms in biofilms, and are key indicators of various pollutants. We investigated the effects some human stressors (nutrients and heavy metals) have on the structure and activity of microbial communities in seven sampling sites located in the Ter River basin (NE Spain). Water and biofilm samples were collected in order to characterize physicochemical and biofilm parameters. The 16S rRNA gene was analysed out from DNA and RNA extracts to obtain α and β diversity. Principal coordinates analyses (PCoA) of the operational taxonomic units (OTUs) in the resident microbial community revealed that nutrients and conductivity were the main driving forces behind the diversity and composition. The effects of mining have had mainly seen on the taxonomic composition of the active microbial community, but also at the OTUs level. Remarkably, metal-impacted communities were very active, which would indicate a close link with the stress faced, that is probably related to the stimulation of detoxification.

Microbial Organic Matter Utilization in High-Arctic Streams: Key Enzymatic Controls.

In the Arctic, climate changes contribute to enhanced mobilization of organic matter in streams. Microbial extracellular enzymes are important mediators of stream organic matter processing, but limited information is available on enzyme processes in this remote area. Here, we studied the variability of microbial extracellular enzyme activity in high-Arctic fluvial biofilms. We evaluated 12 stream reaches in Northeast Greenland draining areas exhibiting different geomorphological features with contrasting contents of soil organic matter to cover a wide range of environmental conditions. We determined stream nitrogen, phosphorus, and dissolved organic carbon concentrations, quantified algal biomass and bacterial density, and characterized the extracellular enzyme activities involved in catalyzing the cleavage of a range of organic matter compounds (e.g., β-glucosidase, phosphatase, β-xylosidase, cellobiohydrolase, and phenol oxidase). We found significant differences in microbial organic matter utilization among the study streams draining contrasting geomorphological features, indicating a strong coupling between terrestrial and stream ecosystems. Phosphatase and phenol oxidase activities were higher in solifluction areas than in alluvial areas. Besides dissolved organic carbon, nitrogen availability was the main driver controlling enzyme activities in the high-Arctic, which suggests enhanced organic matter mineralization at increased nutrient availability. Overall, our study provides novel information on the controls of organic matter usage by high-Arctic stream biofilms, which is of high relevance due to the predicted increase of nutrient availability in high-Arctic streams in global climate change scenarios.

Fluvial biofilms exposed to desiccation and pharmaceutical pollution: New insights using metabolomics

In many arid and semi-arid systems, biological communities in river ecosystems are submitted to flow interruption and desiccation, as well as to the impact of urban wastewaters. In this work, we studied (using a LC-LTQ-Orbitrap) the metabolomic response of biofilm communities exposed to both hydrological and chemical stressors. Fluvial biofilms were exposed to a mixture of 9 pharmaceuticals at a total concentration of 5000ng/L (mimicking concentrations and compounds found in polluted aquatic environments) and/or to seven days of desiccation, under laboratory conditions. The biosynthesis of fatty acids was the main metabolic pathway disrupted in biofilms. Endogenous biofilm's metabolites (metabolome) altered due to these stressors were identified. The metabolites that significantly changed only due to one of the stressors could be proposed as potential specific biomarkers. A biomarker of pharmaceutical exposure was the lysophosphatidic acid, which decreased a 160%, while for desiccation stearidonic acid (increased 160%), 16-Oxohexadecanoic acid (increased 340%) and palmitoleic acid (decreased 290%) were the biomarkers proposed. Besides, other metabolites showed different responses depending on the treatment, such as palmitic acid, linolenic acid, behenic acid, lignoceric acid and azelaic acid. The Carbon:Phosphorus (C:P) molar ratio increased due to all stress factors, whereas the algal community composition changed mainly due to desiccation. A possible relationship between those changes observed in structural parameters and the metabolome of biofilms was explored. Overall, our findings support the use of metabolomics to unravel at molecular level the effects from chemical and physical stressors on complex microbial communities, such as biofilms, and pinpoint biomarkers of exposure.

Antioxidant enzyme activities as biomarkers of fluvial biofilm to ZnO NPs ecotoxicity and the Integrated Biomarker Responses (IBR) assessment

The presence of ZnO nanoparticles (ZnO NPs) in natural waters has raised concerns about their environmental impacts, but the potential influences of ZnO NPs on fluvial biofilm have not been reported. In this study, the utility of antioxidant enzyme activities (AEA) as biomarkers of fluvial biofilm to ZnO NPs toxicity and a method that combines AEA into an index of “Integrated Biomarker Responses (IBR)” were studied. Compared with the absence of ZnO NPs, scanning electron microscopy (SEM) images revealed that a large amount of ZnO NPs were adsorbed onto biofilm and these NPs exerted adverse effects on the viability of bacteria in biofilm. The production of reactive oxygen species (ROS) with high concentrations (30 and 100mg/L) of ZnO NPs exposure reached to 184% and 244% of the control, while no cell leakage and membrane damage were observed. After exposure to ZnO NPs for 0.25 and 3 days, the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR), glutathione peroxidase (GSH-Px) were significantly increased, respectively. At the end of exposure period (21 days), the AEA with the presence of 1mg/L ZnO NPs exposure were comparable to the control, while most of those in high concentrations of ZnO NPs were decreased. The results of IBR showed that the biofilm can adapt to 1mg/L ZnO NPs exposure, while be seriously damaged by 30 and 100mg/L ZnO NPs after 3 and 0.25 days. IBR can be used as an appropriate evaluation system of the toxicity effects of ZnO NPs on fluvial biofim.

Fluvial biofilm responses to joint changes in nutrients, temperature, turbidity and water velocity: an ex situ experiment

The aim of this study was to explore the responses of the epipelic biofilm of a Pampean stream with little impact from human activity to two environmental conditions, with joint modifications in nutrients, temperature, water velocity and turbidity. The experiment was conducted using artificial channels and lasted five weeks. The biological variables measured included chlorophyll-a content, bacterial biomass, ash-free dry weight, total carbohydrate concentration, total respiratory activity, and biofilm composition. Results show that the species’ composition of the biofilm was affected, although no other structural or metabolic variables measured were. These results highlight the importance of including structural parameters to measure rapid changes in water quality, even when analyzing the effects of co-occurring variables.

Effects of low arsenic concentration exposure on freshwater fish in the presence of fluvial biofilms

Arsenic (As) is a highly toxic element and its carcinogenic effect on living organisms is well known. However, predicting real effects in the environment requires an ecological approach since toxicity is influenced by many environmental and biological factors. The purpose of this paper was to evaluate if environmentally-realistic arsenic exposure causes toxicity to fish. An experiment with four different treatments (control (C), biofilm (B), arsenic (+As) and biofilm with arsenic (B+As)) was conducted and each one included sediment to enhance environmental realism, allowing the testing of the interactive effects of biofilm and arsenic on the toxicity to fish. Average arsenic exposure to Eastern mosquitofish (Gambusia holbrooki) was 40.5±7.5μg/L for +As treatment and 34.4±1.4μg/L for B+As treatment for 56days. Fish were affected directly and indirectly by this low arsenic concentration since exposure did not only affect fish but also the function of periphytic biofilms. Arsenic effects on the superoxide dismutase (SOD) and glutathione reductase (GR) activities in the liver of mosquitofish were ameliorated in the presence of biofilms at the beginning of exposure (day 9). Moreover, fish weight gaining was only affected in the treatment without biofilm. After longer exposure (56days), effects of exposure were clearly seen. Fish showed a marked increase in the catalase (CAT) activity in the liver but the interactive influence of biofilms was not further observed since the arsenic-affected biofilm had lost its role in water purification. Our results highlight the interest and application of incorporating some of the complexity of natural systems in ecotoxicology and support the use of criterion continuous concentration (CCC) for arsenic lower than 150μg/L and closer to the water quality criteria to protect aquatic life recommended by the Canadian government which is 5μg As/L.

Effects of ZnO nanoparticles and Zn2 + on fluvial biofilms and the related toxicity mechanisms

Zinc oxide nanoparticles (ZnO NPs) used in consumer products are largely released into the environment through the wastewater stream. The health hazard of ZnO NPs and the contribution of dissolved Zn2+ in toxicity of ZnO NPs has attracted extensive worldwide attention. In this study, the toxic effects of ZnO nanoparticles (ZnO NPs) and the effects of dissolved Zn2+ on fluvial biofilms were investigated. At the end of the exposure time (21days), scanning electron microscopy (SEM) images and bioaccumulation experiments revealed that large quantities of ZnO NPs were adsorbed on the biofilm. The algal biomasses were significantly decreased by six- and eleven-fold compared with the control (1.43μg/L) by exposure to concentrations of 100mg/L ZnO NPs and 7.85mg/L Zn2+, respectively. Moreover, under the same exposure conditions, the quantum yields presented contents of 53.33 and 33.33% relative to the control, and a shift in the community composition that manifested as a strong reduction in diatoms was observed from 7days and reached 15.63 and 6.25% of the control after 21days of exposure, respectively. The reductions in bacteria viability and reactive oxygen species (ROS) production were noticeably enhanced following exposure to 100mg/L ZnO NPs and 7.85mg/L Zn2+, respectively. Additionally, the acute and rapid toxicity of Zn2+ and the increasing toxicity of the ZnO NPs with increased bioaccumulation were noted in the exposure experiment.

Seasonal biostabilization and erosion behavior of fluvial biofilms under different hydrodynamic and light conditions

Biofilm growth may considerably influence the erodibility of river bed sediments and is, thus, an important morphological feature. The extent to which the sediment is stabilized depends on many environmental conditions and even though it is well accepted that these conditions are responsible for the variance of results obtained from field studies, little is known about their impact on biostabilization. In this article, the results from five long-term (4–8 weeks) experiments are presented, investigating the impact of hydrodynamics and light intensity on biostabilization in different seasons. Biofilms are cultivated in a sophisticated setup of six identical straight flumes by circulating natural river water over glass beads with diameters between 0.1 and 0.2mm. During the course of the experiments biofilm samples are eroded regularly to determine the critical shear stress and to learn more about the erosional behavior of biostabilized sediments. The results indicate a large variance of biostabilization between seasons with the highest values in spring and the lowest in late autumn. Presumably, this is explained by the differences in biofilm growth and composition resulting in mechanically diverse responses to the increased bed shear stress. Higher bed shear stress during cultivation delayed biofilm growth possibly due to reduced net attachment and growth of formerly advected microbes on the sediment surface. Unexpectedly, no substantial biostabilization effect is detected in experiments where biofilms are cultivated in darkness. Another focus in this article is the analysis of the mechanical behavior observed during erosion of the biofilms, that is shown to be an important aspect for the understanding of freshwater biostabilization caused by young growing biofilms.

Modeling the Effects of Hydrodynamic Regimes on Microbial Communities within Fluvial Biofilms: Combining Deterministic and Stochastic Processes.

To fully understand the effects of hydrodynamics on a microbial community, the roles of niche-based and neutral processes must be considered in a mathematical model. To this end, a two-dimensional model combining mechanisms of immigration, dispersal, and niche differentiation was first established to describe the effects of hydrodynamics on bacterial communities within fluvial biofilms. Deterministic factors of the model were identified via the calculation of Spearman's rank correlation coefficients between parameters of hydrodynamics and the bacterial community. It was found that turbulent kinetic energy and turbulent intensity were considered as a set of reasonable predictors of community composition, whereas flow velocity and turbulent intensity can be combined together to predict biofilm bacterial biomass. According to the modeling result, the bacterial community could get its favorable assembly condition with a flow velocity ranging from 0.041 to 0.061 m/s. However, the driving force for biofilm community assembly changed with the local hydrodynamics. Individuals reproduction within the biofilm was the main driving force with flow velocity less than 0.05 m/s, while cell migration played a much more important role with velocity larger than 0.05 m/s. The developed model could be considered as a useful tool for improving the technologies of water environment protection and remediation.

Fluvial biofilms from upper and lower river reaches respond differently to wastewater treatment plant inputs

We examined how wastewater treatment plant (WWTP) inputs affect fluvial biofilm functioning and structure developing on river surface sediments in two different reaches characterized by different anthropogenic pressures. The upper reach includes one site before and one after Palautordera WWTP (Pref and P, respectively); the lower reach includes one site before and one after Tordera WWTP (Tref and T, respectively). We performed an ex situ translocation experiment which resulted in four treatments (Pref-P, Pref-T, Tref-T, Tref-P) and in three different impact grades according to a translocation gradient defined by water chemical changes between sites, being Tref-T the lowest translocation gradient, Pref-P and Tref-P the medium translocation gradient, and Pref-T the highest translocation gradient. After receiving WWTP inputs, upper reach communities (Pref-P and Pref-T treatments) had a decrease in live bacteria and an inhibition of extracellular enzyme activities, while lower reach communities (Tref-T and Tref-P treatments) showed an increase of enzyme activities. Treatments with the highest and lowest translocation gradients resulted in different metabolic fingerprints. Results suggest that the origin and sensitivity of the receiving community determines its functional response and also reinforce the idea that the impact level is an influential aspect to consider when analyzing community responses to anthropogenic impacts.

Short-term arsenic exposure reduces diatom cell size in biofilm communities.

Arsenic (As) pollution in water has important impacts for human and ecosystem health. In freshwaters, arsenate (As(V)) can be taken up by microalgae due to its similarity with phosphate molecules, its toxicity being aggravated under phosphate depletion. An experiment combining ecological and ecotoxicological descriptors was conducted to investigate the effects of As(V) (130μgL(-1) over 13days) on the structure and function of fluvial biofilm under phosphate-limiting conditions. We further incorporated fish (Gambusia holbrooki) into our experimental system, expecting fish to provide more available phosphate for algae and, consequently, protecting algae against As toxicity. However, this protection role was not fully achieved. Arsenic inhibited algal growth and productivity but not bacteria. The diatom community was clearly affected showing a strong reduction in cell biovolume; selection for tolerant species, in particular Achnanthidium minutissimum; and a reduction in species richness. Our results have important implications for risk assessment, as the experimental As concentration used was lower than acute toxicity criteria established by the USEPA.

Experimental evaluation of the contribution of acidic pH and Fe concentration to the structure, function and tolerance to metals (Cu and Zn) exposure in fluvial biofilms.

An indoor channel system was colonised with fluvial biofilms to study the chronic effects of high Fe and SO4(2-) concentrations and acidic pH, the water chemistry in the surrounding streams of Aljustrel mining area (Alentejo, Portugal), and their contribution to community (in)tolerance to metal toxicity by short-term experiments with Cu and Zn. Biofilms were subjected to four different treatments during 8 weeks: high Fe and SO4(2-) concentrations (1 mg Fe l(-1)+ 700 mg SO4(2-) l(-1)) and acidic pH, high Fe and SO4(2-) at alkaline pH; lower Fe and SO4(2-) at acidic pH: and lower Fe and SO4(2-) concentrations at alkaline pH as negative control. During chronic exposure, acidic pH affected growth negatively, based on low values of algal biomass and the autotrophic index, high values of the antioxidant enzyme activities and low diversity diatom communities, dominated by acidophilic species (Pinnularia aljustrelica) in acidic treatments, being the effects more marked with high Fe and SO4(2-). Co-tolerance to metals (Cu and Zn) was also shown in biofilms from the acidic treatments, contrasting with the higher sensitivity observed in the alkaline treatments. We can conclude that the Aljustrel mining area acidic environment limits algal growth and exerts a strong selection pressure on the community composition which is in turn, more tolerant to metal exposure.

Pollution-induced community tolerance to non-steroidal anti-inflammatory drugs (NSAIDs) in fluvial biofilm communities affected by WWTP effluents

We assessed the tolerance acquired by stream biofilms to two non-steroidal anti-inflammatory-drugs (NSAIDs), ibuprofen and diclofenac. Biofilms came from a stream system receiving the effluent of a wastewater treatment plant (WWTP). The response of biofilms from a non-polluted site (upstream the WWTP) was compared to that of others downstream with relevant and decreasing levels of NSAIDs. Experiments performed in the laboratory following the pollution-induced community tolerance (PICT) approach determined that both algae and microbial communities from biofilms of the sites exposed at the highest concentrations of ibuprofen and diclofenac acquired tolerance to the mixture of these NSAIDs occurring at the sites. It was also observed that the chronic pollution by the WWTP effluent affected the microbial metabolic profile, as well as the structure of the algal community. The low (at ngL−1 level) but chronic inputs of pharmaceuticals to the river ecosystem result in tolerant communities of lower diversity and altered microbial metabolism.

Single and mixture effects of pesticides and a degradation product on fluvial biofilms

The Morcille River located in the Beaujolais vineyard area (Eastern France) is subjected to strong vine-growing pressure leading to the contamination by a range of herbicides and fungicides of the surrounding freshwater environment. Particularly high concentrations of norflurazon, desmethyl norflurazon and tebuconazole were recorded in spring 2010 at the downstream site of the river. Despite their occurrence in rivers, scarce toxicity data are available for these products, in particular in the case of desmethyl norflurazon (main norflurazon degradation product). Furthermore, the toxicity data are generally available only for single compounds and are issued from single species toxicity tests, leading to a lack of ecological relevance. Consequently, this study was undertaken to evaluate the toxic effects of norflurazon, desmethyl norflurazon and tebuconazole singly and in a ternary mixture on fluvial biofilm. Toxicity tests were performed in microplates for 48 h. Photosynthetic endpoints were measured using pulse amplitude-modulated fluorometry; diatom densities and taxonomic composition were determined. After 48 h of exposure, significant effects on optimal quantum yield (F v/F m) for desmethyl norflurazon and mixture were observed.