Tolerance Patterns in Stream Biofilms Link Complex Chemical Pollution to Ecological Impacts.

Preventing and remedying fresh waters from chemical pollution is a fundamental societal and scientific challenge. With other nonchemical stressors potentially co-occurring, assessing the ecological consequences of reducing chemical loads in the environment is arduous. In this case study, we comparatively assessed the community structure, functions, and tolerance of stream biofilms to micropollutant mixtures extracted from deployed passive samplers at wastewater treatment plant effluents. These biofilms were growing up- and downstream of one upgraded and two nonupgraded wastewater treatment plants before being sampled for analyses. Our results showed a substantial decrease in micropollutant concentrations by 85%, as the result of upgrading the wastewater treatment plant at one of the sampling sites with activated carbon filtration. This decrease was positively correlated with a loss of community tolerance to micropollutants and the recovery of the community structure downstream of the effluent. On the other hand, downstream biofilms at the nonupgraded sites displayed higher tolerance to the extracts than the upstream biofilms. The observed higher tolerance was positively linked to micropollutant levels both in stream water and in biofilm samples, and to shifts in the community structure. Although more investigations of upgraded sites are needed, our findings point toward the suitability of using community tolerance for the retrospective assessment of the risks posed by micropollutants, to assess community recovery, and to relate effects to causes in complex environmental conditions.

Chronic Exposure Effects of Silver Nanoparticles on Stream Microbial Decomposer Communities and Ecosystem Functions.

With the accelerated use of silver nanoparticles (AgNP) in commercial products, streams will increasingly serve as recipients of, and repositories for, AgNP. This raises concerns about the potential toxicity of these nanomaterials in the environment. Here we aimed to assess the impacts of chronic AgNP exposure on the metabolic activities and community structure of fungal and bacterial plant litter decomposers as central players in stream ecosystems. Minimal variation in the size and surface charge of AgNP indicated that nanoparticles were rather stable during the experiment. Five days of exposure to 0.05 and 0.5 µM AgNP in microcosms shifted bacterial community structure but had no effect on a suite of microbial metabolic activities, despite silver accumulation in the decomposing leaf litter. After 25 days, however, a broad range of microbial endpoints, as well as rates of litter decomposition, were strongly affected. Declines matched with the total silver concentration in the leaves and were accompanied by changes in fungal and bacterial community structure. These results highlight a distinct sensitivity of litter-associated microbial communities in streams to chronic AgNP exposure, with effects on both microbial functions and community structure resulting in notable ecosystem consequences through impacts on litter decomposition and further biogeochemical processes.

Interaction of silver nanoparticles with algae and fish cells: a side by side comparison.

BACKGROUND: Silver nanoparticles (AgNP) are widely applied and can, upon use, be released into the aquatic environment. This raises concerns about potential impacts of AgNP on aquatic organisms. We here present a side by side comparison of the interaction of AgNP with two contrasting cell types: algal cells, using the algae Euglena gracilis as model, and fish cells, a cell line originating from rainbow trout (Oncorhynchus mykiss) gill (RTgill-W1). The comparison is based on the AgNP behavior in exposure media, toxicity, uptake and interaction with proteins. RESULTS: (1) The composition of exposure media affected AgNP behavior and toxicity to algae and fish cells. (2) The toxicity of AgNP to algae was mediated by dissolved silver while nanoparticle specific effects in addition to dissolved silver contributed to the toxicity of AgNP to fish cells. (3) AgNP did not enter into algal cells; they only adsorbed onto the cell surface. In contrast, AgNP were taken up by fish cells via endocytic pathways. (4) AgNP can bind to both extracellular and intracellular proteins and inhibit enzyme activity. CONCLUSION: Our results showed that fish cells take up AgNP in contrast to algal cells, where AgNP sorbed onto the cell surface, which indicates that the cell wall of algae is a barrier to particle uptake. This particle behaviour results in different responses to AgNP exposure in algae and fish cells. Yet, proteins from both cell types can be affected by AgNP exposure: for algae, extracellular proteins secreted from cells for, e.g., nutrient acquisition. For fish cells, intracellular and/or membrane-bound proteins, such as the Na+/K+-ATPase, are susceptible to AgNP binding and functional impairment.

Linking toxicity and adaptive responses across the transcriptome, proteome, and phenotype of Chlamydomonas reinhardtii exposed to silver.

Understanding mechanistic and cellular events underlying a toxicological outcome allows the prediction of impact of environmental stressors to organisms living in different habitats. A systems-based approach aids in characterizing molecular events, and thereby the cellular pathways that have been perturbed. However, mapping only adverse outcomes of a toxicant falls short of describing the stress or adaptive response that is mounted to maintain homeostasis on perturbations and may confer resistance to the toxic insult. Silver is a potential threat to aquatic organisms because of the increasing use of silver-based nanomaterials, which release free silver ions. The effects of silver were investigated at the transcriptome, proteome, and cellular levels of Chlamydomonas reinhardtii. The cells instigate a fast transcriptome and proteome response, including perturbations in copper transport system and detoxification mechanisms. Silver causes an initial toxic insult, which leads to a plummeting of ATP and photosynthesis and damage because of oxidative stress. In response, the cells mount a defense response to combat oxidative stress and to eliminate silver via efflux transporters. From the analysis of the perturbations of the cell's functions, we derived a detailed mechanistic understanding of temporal dynamics of toxicity and adaptive response pathways for C. reinhardtii exposed to silver.

Selenium Uptake and Methylation by the Microalga Chlamydomonas reinhardtii.

Biogenic selenium (Se) emissions play a major role in the biogeochemical cycle of this essential micronutrient. Microalgae may be responsible for a large portion of these emissions via production of methylated Se compounds that volatilize into the atmosphere. However, the biochemical mechanisms underlying Se methylation in microalgae are poorly understood. Here, we study Se methylation by Chlamydomonas reinhardtii, a model freshwater alga, as a function of uptake and intracellular Se concentrations and present a biochemical model that quantitatively describes Se uptake and methylation. Both selenite and selenate, two major inorganic forms of Se, are readily internalized by C. reinhardtii, but selenite is accumulated around ten times more efficiently than selenate due to different membrane transporters. With either selenite or selenate as substrates, Se methylation was highly efficient (up to 89% of intracellular Se) and directly coupled to intracellular Se levels (R(2) > 0.92) over an intracellular concentration range exceeding an order of magnitude. At intracellular concentrations exceeding 10 mM, intracellular zerovalent Se was formed. The relationship between uptake, intracellular accumulation, and methylation was used by the biochemical model to successfully predict measured concentrations of methylated Se in natural waters. Therefore, biological Se methylation by microalgae could significantly contribute to environmental Se cycling.

Considerations of Environmentally Relevant Test Conditions for Improved Evaluation of Ecological Hazards of Engineered Nanomaterials.

Engineered nanomaterials (ENMs) are increasingly entering the environment with uncertain consequences including potential ecological effects. Various research communities view differently whether ecotoxicological testing of ENMs should be conducted using environmentally relevant concentrations-where observing outcomes is difficult-versus higher ENM doses, where responses are observable. What exposure conditions are typically used in assessing ENM hazards to populations? What conditions are used to test ecosystem-scale hazards? What is known regarding actual ENMs in the environment, via measurements or modeling simulations? How should exposure conditions, ENM transformation, dose, and body burden be used in interpreting biological and computational findings for assessing risks? These questions were addressed in the context of this critical review. As a result, three main recommendations emerged. First, researchers should improve ecotoxicology of ENMs by choosing test end points, duration, and study conditions-including ENM test concentrations-that align with realistic exposure scenarios. Second, testing should proceed via tiers with iterative feedback that informs experiments at other levels of biological organization. Finally, environmental realism in ENM hazard assessments should involve greater coordination among ENM quantitative analysts, exposure modelers, and ecotoxicologists, across government, industry, and academia.

Effects of Differently Coated Silver Nanoparticles on the Photosynthesis of Chlamydomonas reinhardtii.

Various factors have been invoked to explain the toxicity of silver nanoparticles (AgNP) to microorganisms including particle size and the nature of stabilizing coatings as well as the amount of dissolved silver occurring in AgNP suspensions. In this study we have assessed the effects of nine differently coated AgNP (chitosan, lactate, polyvinylpyrrolidone, polyethelene glycol, gelatin, sodium dodecylbenzenesulfonate, citrate, dexpanthenol, and carbonate) and AgNO3 on the photosynthesis of the freshwater algae Chlamydomonas reinhardtii. We have thus examined how AgNP effects on algae relate to particle size, measured dissolved silver (Agd), and bioavailable silver (Agbioav). Agbioav was indirectly estimated in toxicity experiments by cysteine-silver complexation at the EC50. The EC50 calculated as a function of measured Agd concentrations showed for some coatings values similar to that of dissolved Ag, whereas other coated AgNP displayed lower EC50 values. In all cases, excess cysteine completely prevented effects on photosynthetic yield, confirming the role of Agd as a cause of the observed effect on the photosynthesis. Toxicity was related neither to particle size nor to the coatings. For all differently coated AgNP suspensions, the EC50 values calculated as a function of Agbioav were comparable to the value of AgNO3. Depending on the coatings Agbioav was comparable to or higher than measured Agd.

Impact of chronic lead exposure on metal distribution and biological effects to periphyton.

Chronic Pb exposure microcosm studies were carried out on two different periphyton communities over the course of 3 weeks to link Pb distribution to biological effects in periphyton. We show that three-week exposures of periphyton to 20.6 ± 0.4 µM PbT (330 nM Pb(2+)) did not have observable biological effects on photosynthesis, respiration, extracellular enzymatic activities, or biomass accrual. Metal distribution studies showed that the majority of Pb was associated with the operationally defined sorbed and non-EDTA-exchangeable fractions, and relatively little with extracellular polymeric substances (EPS). No significant effects of Pb on Fe and Mn distributions were observed, whereas higher Cu accumulation occurred from increased free Cu(2+) in the exposure medium. High Fe:C and Mn:C ratios indicated the presence of inorganic Fe and Mn material associated with the non-EDTA-exchangeable fraction, which likely sequesters Pb and explains the absence of measurable biological effects. Although no toxic effects of Pb were observed on the periphytic organisms themselves, periphyton can be a significant source of Pb to grazing organisms in aquatic ecosystems.

Effect of Ozone Treatment on Nano-Sized Silver Sulfide in Wastewater Effluent.

Silver nanoparticles used in consumer products are likely to be released into municipal wastewater. Transformation reactions, most importantly sulfidation, lead to the formation of nanoscale silver sulfide (nano-Ag2S) particles. In wastewater treatment plants (WWTP), ozonation can enhance the effluent quality by eliminating organic micropollutants. The effect of ozonation on the fate of nano-Ag2S, however, is currently unknown. In this study, we investigate the interaction of ozone with nano-Ag2S and evaluate the effect of ozonation on the short-term toxicity of WWTP effluent spiked with nano-Ag2S. The oxidation of nano-Ag2S by ozone resulted in a stoichiometric factor (number of moles of ozone required to oxidize one mole of sulfide to sulfate) of 2.91, which is comparable to the results obtained for the reaction of bisulfide (HS(-)) with ozone. The second-order rate constant for the reaction of nano-Ag2S with ozone (k = 3.1 × 10(4) M(-1) s(-1)) is comparable to the rate constant of fast-reacting micropollutants. Analysis of the ozonation products of nano-Ag2S by transmission electron microscopy (TEM) and X-ray absorption spectroscopy (XAS) revealed that ozonation dominantly led to the formation of silver chloride in WWTP effluent. After ozonation of the Ag2S-spiked effluent, the short-term toxicity for the green algae Chlamydomonas reinhardtii increased and reached EC50 values comparable to Ag(+). This study thus reveals that ozone treatment of WWTP effluent results in the oxidation of Ag2S and, hence, an increase of the Ag toxicity in the effluent, which may become relevant at elevated Ag concentrations.

Silver nanoparticle effects on stream periphyton during short-term exposures.

Silver nanoparticles (AgNP) are increasingly used as antimicrobials in consumer products. Subsequently released into aquatic environments, they are likely to come in contact with microbial communities like periphyton, which plays a key role as a primary producer in stream ecosystems. At present, however, very little is known about the effects of nanoparticles on processes mediated by periphyton communities. We assessed the effects of citrate-coated silver nanoparticles and silver ions (dosed as AgNO3) on five functional end points reflecting community and ecosystem-level processes in periphyton: photosynthetic yield, respiration potential, and the activity of three extracellular enzymes. After 2 h of exposure in experimental microcosms, AgNP and AgNO3 inhibited respiration and photosynthesis of periphyton and the activities of two of the three extracellular enzymes. Addition of a chelating ligand that complexes free silver ions indicated that, in most cases, toxicity of AgNP suspensions was caused by Ag(I) dissolved from the particles. However, these suspensions inhibited one of the extracellular enzymes (leucine aminopeptidase), pointing to a specific nanoparticle effect independent of the dissolved Ag(I). Thus, our results show that both silver nanoparticles and silver ions have potential to disrupt basic metabolic functions and enzymatic resource acquisition of stream periphyton.

Chemical Aspects of Nanoparticle Ecotoxicology.

Nanoecotoxicology strives to understand the processes and mechanisms by which engineered nanoparticles (ENP) may exert toxic effects on aquatic organisms. Detailed knowledge of the chemical reactions of nanoparticles in the media and of their interactions with organisms is required to understand these effects. The processes of agglomeration of nanoparticles, of dissolution and release of toxic metal ions, and of production of reactive oxygen species (ROS) are considered in this article. Important questions concern the role of uptake of nanoparticles in various organisms, in contrast to uptake of ions released from nanoparticles and to nanoparticle attachment to organism surfaces. These interactions are illustrated for effects of silver nanoparticles (AgNP), cerium oxide (CeO2 NP) and titanium dioxide (TiO2 NP), on aquatic organisms, including algae, biofilms, fish cells and fish embryos.

Fate and effects of microplastic particles in a periphyton-grazer system.

In the aquatic environment, microplastic particles (MP) can accumulate in microbial communities that cover submerged substrata, i.e. in periphyton. Despite periphyton being the essential food source for grazers in the benthic zones, MP transfer from periphyton to benthic biota and its ecotoxicological consequences are unknown. Therefore, in this study, we investigated the effects of 1) MP on embryonal development of freshwater gastropod Physa acuta embryos, 2) MP on adult Physa acuta individuals through dietary exposure and 3) on the MP surface properties. Embryonal development tests were carried out with spherical polyethylene MP in the size of 1-4 µm (MP). Over a period of 28 days, embryonal development and hatching rate were calculated. In the feeding experiments, periphyton was grown in the presence and absence of MP and was then offered to the adult Physa acuta for 42-152 h. The snails readily ingested and subsequently egested MP, together with the periphyton as shown by MP quantification in periphyton, snail soft body tissue and feces. No selective feeding behavior upon MP exposure was detected. The ingestion of MP had no effect on mortality, feeding and defecation rate. Yet, the reproductive output of snails, measured as the number of egg clutches and numbers of eggs per clutch, decreased after the ingestion of MPs, while the hatching success of snail embryos those parents were exposed remained unaffected. In contrast, hatching rate of snail embryos was significantly reduced upon direct MP exposure. MP optical properties were changed upon the incorporation into the periphyton and the passage through the digestive tract. Our results indicate that MP incorporated in periphyton are bioavailable to aquatic grazers, facilitating the introduction of MP into the food chain and having direct adverse effects on the grazers' reproductive fitness.

Exposure of silver-nanoparticles and silver-ions to lung cells in vitro at the air-liquid interface.

BACKGROUND: Due to its antibacterial properties, silver (Ag) has been used in more consumer products than any other nanomaterial so far. Despite the promising advantages posed by using Ag-nanoparticles (NPs), their interaction with mammalian systems is currently not fully understood. An exposure route via inhalation is of primary concern for humans in an occupational setting. Aim of this study was therefore to investigate the potential adverse effects of aerosolised Ag-NPs using a human epithelial airway barrier model composed of A549, monocyte derived macrophage and dendritic cells cultured in vitro at the air-liquid interface. Cell cultures were exposed to 20 nm citrate-coated Ag-NPs with a deposition of 30 and 278 ng/cm2 respectively and incubated for 4 h and 24 h. To elucidate whether any effects of Ag-NPs are due to ionic effects, Ag-Nitrate (AgNO3) solutions were aerosolised at the same molecular mass concentrations. RESULTS: Agglomerates of Ag-NPs were detected at 24 h post exposure in vesicular structures inside cells but the cellular integrity was not impaired upon Ag-NP exposures. Minimal cytotoxicity, by measuring the release of lactate dehydrogenase, could only be detected following a higher concentrated AgNO3-solution. A release of pro-inflammatory markers TNF-α and IL-8 was neither observed upon Ag-NP and AgNO3 exposures as well as was not affected when cells were pre-stimulated with lipopolysaccharide (LPS). Also, an induction of mRNA expression of TNF-α and IL-8, could only be observed for the highest AgNO3 concentration alone or even significantly increased when pre-stimulated with LPS after 4 h. However, this effect disappeared after 24 h. Furthermore, oxidative stress markers (HMOX-1, SOD-1) were expressed after 4 h in a concentration dependent manner following AgNO3 exposures only. CONCLUSIONS: With an experimental setup reflecting physiological exposure conditions in the human lung more realistic, the present study indicates that Ag-NPs do not cause adverse effects and cells were only sensitive to high Ag-ion concentrations. Chronic exposure scenarios however, are needed to reveal further insight into the fate of Ag-NPs after deposition and cell interactions.

Colloidal stability of carbonate-coated silver nanoparticles in synthetic and natural freshwater.

To gain important information on fate, mobility, and bioavailability of silver nanoparticles (AgNP) in aquatic systems, the influence of pH, ionic strength, and humic substances on the stability of carbonate-coated AgNP (average diameter 29 nm) was systematically investigated in 10 mM carbonate and 10 mM MOPS buffer, and in filtered natural freshwater. Changes in the physicochemical properties of AgNP were measured using nanoparticle tracking analysis, dynamic light scattering, and ultraviolet-visible spectroscopy. According to the pH-dependent carbonate speciation, below pH 4 the negatively charged surface of AgNP became positive and increased agglomeration was observed. Electrolyte concentrations above 2 mM Ca(2+) and 100 mM Na(+) enhanced AgNP agglomeration in the synthetic media. In the considered concentration range of humic substances, no relevant changes in the AgNP agglomeration state were measured. Agglomeration of AgNP exposed in filtered natural freshwater was observed to be primarily controlled by the electrolyte type and concentration. Moreover, agglomerated AgNP were still detected after 7 days of exposure. Consequently, slow sedimentation and high mobility of agglomerated AgNP could be expected under the considered natural conditions. A critical evaluation of the different methods used is presented as well.

Intracellular silver accumulation in Chlamydomonas reinhardtii upon exposure to carbonate coated silver nanoparticles and silver nitrate.

The intracellular silver accumulation ({Ag}(in)) upon exposure to carbonate coated silver nanoparticles (AgNP, 0.5-10 µM, average diameter 29 nm) and silver nitrate (20-500 nM) was examined in the wild type and in the cell wall free mutant of the green alga Chlamydomonas reinhardtii at pH 7.5. The {Ag}(in) was measured over time up to 1 h after a wash procedure to remove silver ions (Ag(+)) and AgNP from the algal cell surface. The {Ag}(in) increased with increasing exposure time and with increasing AgNP and AgNO(3) concentrations in the exposure media, reaching steady-state concentrations between 10(-5) and 10(-3) mol L(cell)(-1). According to estimated kinetic parameters, high Ag(+) bioconcentration factors were calculated (>10(3) L L(cell)(-1)). Higher accumulation rate constants were assessed in the cell wall free mutant, indicating a protective role of the cell wall in limiting Ag(+) uptake. The bioavailability of AgNP was calculated to be low in both strains relative to Ag(+), suggesting that AgNP internalization across the cell membrane was limited.

Practical considerations for conducting ecotoxicity test methods with manufactured nanomaterials: what have we learnt so far?

This review paper reports the consensus of a technical workshop hosted by the European network, NanoImpactNet (NIN). The workshop aimed to review the collective experience of working at the bench with manufactured nanomaterials (MNMs), and to recommend modifications to existing experimental methods and OECD protocols. Current procedures for cleaning glassware are appropriate for most MNMs, although interference with electrodes may occur. Maintaining exposure is more difficult with MNMs compared to conventional chemicals. A metal salt control is recommended for experiments with metallic MNMs that may release free metal ions. Dispersing agents should be avoided, but if they must be used, then natural or synthetic dispersing agents are possible, and dispersion controls essential. Time constraints and technology gaps indicate that full characterisation of test media during ecotoxicity tests is currently not practical. Details of electron microscopy, dark-field microscopy, a range of spectroscopic methods (EDX, XRD, XANES, EXAFS), light scattering techniques (DLS, SLS) and chromatography are discussed. The development of user-friendly software to predict particle behaviour in test media according to DLVO theory is in progress, and simple optical methods are available to estimate the settling behaviour of suspensions during experiments. However, for soil matrices such simple approaches may not be applicable. Alternatively, a Critical Body Residue approach may be taken in which body concentrations in organisms are related to effects, and toxicity thresholds derived. For microbial assays, the cell wall is a formidable barrier to MNMs and end points that rely on the test substance penetrating the cell may be insensitive. Instead assays based on the cell envelope should be developed for MNMs. In algal growth tests, the abiotic factors that promote particle aggregation in the media (e.g. ionic strength) are also important in providing nutrients, and manipulation of the media to control the dispersion may also inhibit growth. Controls to quantify shading effects, and precise details of lighting regimes, shaking or mixing should be reported in algal tests. Photosynthesis may be more sensitive than traditional growth end points for algae and plants. Tests with invertebrates should consider non-chemical toxicity from particle adherence to the organisms. The use of semi-static exposure methods with fish can reduce the logistical issues of waste water disposal and facilitate aspects of animal husbandry relevant to MMNs. There are concerns that the existing bioaccumulation tests are conceptually flawed for MNMs and that new test(s) are required. In vitro testing strategies, as exemplified by genotoxicity assays, can be modified for MNMs, but the risk of false negatives in some assays is highlighted. In conclusion, most protocols will require some modifications and recommendations are made to aid the researcher at the bench.

Function and regulation of the glutathione peroxidase homologous gene GPXH/GPX5 in Chlamydomonas reinhardtii.

When exposed to strong sunlight, photosynthetic organisms encounter photooxidative stress by the increased production of reactive oxygen species causing harmful damages to proteins and membranes. Consequently, a fast and specific induction of defense mechanisms is required to protect the organism from cell death. In Chlamydomonas reinhardtii, the glutathione peroxidase homologous gene GPXH/GPX5 was shown to be specifically upregulated by singlet oxygen formed during high light conditions presumably to prevent the accumulation of lipid hydroperoxides and membrane damage. We now showed that the GPXH protein is a thioredoxin-dependent peroxidase catalyzing the reduction of hydrogen peroxide and organic hydroperoxides.Furthermore, the GPXH gene seems to encode a dual-targeted protein, predicted to be localized both in the chloroplast and the cytoplasm, which is active with either plastidic TRXy or cytosolic TRXh1. Putative dual-targeting is achieved by alternative transcription and translation start sites expressed independently from either a TATA-box or an Initiator core promoter. Expression of both transcripts was upregulated by photooxidative stress even though with different strengths. The induction required the presence of the core promoter sequences and multiple upstream regulatory elements including a Sp1-like element and an earlier identified CRE/AP-1 homologous sequence. This element was further characterized by mutation analysis but could not be confirmed to be a consensus CRE or AP1 element. Instead, it rather seems to be another member of the large group of TGAC-transcription factor binding sites found to be involved in the response of different genes to oxidative stress.

Kinetics of cadmium accumulation in periphyton under freshwater conditions.

The aim of the present study was to investigate the kinetics of cadmium (Cd) accumulation (total and intracellular) in periphyton under freshwater conditions in a short-term microcosm experiment. Periphyton was precolonized in artificial flow-through channels supplied with natural freshwater and then exposed for 26.4 h to nominal Cd concentrations of 5 and 20 nM added to natural freshwater. Labile Cd in water determined with diffusion gradient in thin films was 60 to 69% of total dissolved Cd in the exposure channels and 11% in the control channel. Intracellular Cd concentrations in periphyton increased rapidly and linearly during the first 71 min. Initial intracellular uptake rates were 0.05 and 0.18 nmol of Cd/g of dry weight x min in the 5 nM and 20 nM exposures, respectively. The subsequent intracellular uptake was slower, approaching steady state at the end of Cd exposure. Uptake kinetics of Cd was slower when compared to experiments with planktonic algal cultures, probably due to diffusion limitations. Intracellular Cd uptake during the entire exposure was modeled with a nonlinear, one-compartment model from which uptake and clearance rate constants, as well as bioconcentration factors, were obtained. The release of Cd from periphyton after the end of Cd exposure was slow when compared to the initial uptake rates.

Evaluation of Phototrophic Stream Biofilms Under Stress: Comparing Traditional and Novel Ecotoxicological Endpoints After Exposure to Diuron.

Stream biofilms have been shown to be among the most sensitive indicators of environmental stress in aquatic ecosystems and several endpoints have been developed to measure biofilm adverse effects caused by environmental stressors. Here, we compare the effects of long-term exposure of stream biofilms to diuron, a commonly used herbicide, on several traditional ecotoxicological endpoints (biomass growth, photosynthetic efficiency, chlorophyll-a content, and taxonomic composition), with the effects measured by recently developed methods [community structure assessed by flow cytometry (FC-CS) and measurement of extracellular polymeric substances (EPS)]. Biofilms grown from local stream water in recirculating microcosms were exposed to a constant concentration of 20 µg/L diuron over a period of 3 weeks. During the experiment, we observed temporal variation in photosynthetic efficiency, biomass, cell size, presence of decaying cells and in the EPS protein fraction. While biomass growth, photosynthetic efficiency, and chlorophyll-a content were treatment independent, the effects of diuron were detectable with both FC and EPS measurements. This demonstrates that, at least for our experimental setup, a combination of different ecotoxicological endpoints can be important for evaluating biofilm environmental stress and suggests that the more recent ecotoxicological endpoints (FC-CS, EPS protein content and humic substances) can be a useful addition for stream biofilm ecotoxicological assessment.