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.

Pesticide Mixtures Cause Short-Term, Reversible Effects on the Function of Autotrophic Periphyton Assemblages.

In a laboratory experiment we investigated the effects of pesticide mixtures on the structure and function of freshwater biofilms, with focus on their photoautotrophic component. We identified 6 herbicides and 1 fungicide commonly found in Swedish streams at relatively high concentrations and created 3 ternary mixtures that were tested in concentration series ranging from observed environmental concentrations to up to 100 times higher. Biofilms were exposed to these pesticide mixtures for 8 d and then allowed to recover for another 12 d. Our results show a rapid and consistent inhibition of photosynthesis after just 24-h exposure to the highest test concentration of pesticides, as well as in some treatments with lower concentrations (i.e., 10 times the environmental level), on exposure. Interestingly, the observed effects were reversible because biofilm photosynthesis recovered rapidly and completely in clean media in all but one treatment. In contrast to the functional response, no effects were observed on the algal assemblage structure, as assessed by diagnostic pigments. We conclude that the pesticide mixtures induce a rapid but reversible inhibition of photosynthesis, without short-term effects on biofilm structure. Environ Toxicol Chem 2020;39:1367-1374. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Antimicrobial activity of pharmaceutical cocktails in sewage treatment plant effluent - An experimental and predictive approach to mixture risk assessment.

Municipal wastewater contains multi-component mixtures of active pharmaceutical ingredients (APIs). This could shape microbial communities in sewage treatment plants (STPs) and the effluent-receiving ecosystems. In this paper we assess the risk of antimicrobial effects in STPs and the aquatic environment for a mixture of 18 APIs that was previously detected in the effluent of a European municipal STP. Effects on microbial consortia (collected from a separate STP) were determined using respirometry, enumeration of culturable microorganisms and community-level physiological profiling. The mixture toxicity against selected bacteria was assessed using assays with Pseudomonas putida and Vibrio fischeri. Additional data on the toxicity to environmental bacteria were compiled from literature in order to assess the individual and expected joint bacterial toxicity of the pharmaceuticals in the mixture. The reported effluent concentration of the mixture was 15.4 nmol/l and the lowest experimentally obtained effect concentrations (EC10) were 242 nmol/l for microbial consortia in STPs, 225 nmol/l for P. putida and 73 nmol/l for V. fischeri. The lowest published effect concentrations (EC50) of the individual antibiotics in the mixture range between 15 and 150 nmol/l, whereas 0.9-190 µmol/l was the range of bacterial EC50 values found for the non-antibiotic mixture components. Pharmaceutical cocktails could shape microbial communities at concentrations relevant to STPs and the effluent receiving aquatic environment. The risk of antimicrobial mixture effects was completely dominated by the presence of antibiotics, whereas other pharmaceutical classes contributed only negligibly to the mixture toxicity. The joint bacterial toxicity can be accurately predicted from the individual toxicity of the mixture components, provided that standardized data on representative bacterial strains becomes available for all relevant compounds. These findings argue for a more sophisticated bacterial toxicity assessment of environmentally relevant pharmaceuticals, especially for those with a mode of action that is known to specifically affect prokaryotic microorganisms.

The toxicity of the three antifouling biocides DCOIT, TPBP and medetomidine to the marine pelagic copepod Acartia tonsa.

Copepods, the largest group of pelagic grazers, are at risk from exposure to antifouling biocides. This study investigated the toxicity of the antifouling biocides 4,5-dichloro-2-octyl-1,2-thiazol-3(2H)-one (DCOIT), triphenylborane pyridine (TPBP) and 4-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole (medetomidine) to the copepod Acartia tonsa, using mortality and egg production as endpoints. The toxicity ranking for mortality was as follows: DCOIT (LC50 57 nmol l(-1)) = TPBP (LC50 56 nmol l(-1)) > medetomidine (LC50 241 nmol l(-1)). Egg production was more sensitive than mortality to TPBP (EC50 3.2 nmol l(-1)), while DCOIT and medetomidine inhibited egg production at roughly the same concentrations (72 and 186 nmol l(-1) respectively). Furthermore, TPBP seems to affect egg hatching directly which was not the case for DCOIT and medetomidine. DCOIT and medetomidine might pose an environmental risk as they have been reported to occur in different exposure scenarios or analytical surveys at concentrations only 2-3 times lower than the respective EC10. Reported environmental concentrations of TPBP are few but clearly lower than the EC10 values reported here, suggesting current risk of TPBP to copepods to be moderate.

Long-term effects of the antibacterial agent triclosan on marine periphyton communities.

Triclosan is a widely used antibacterial agent that has become a ubiquitous contaminant in freshwater, estuary, and marine environments. Concerns about potential adverse effects of triclosan have been described in several recent risk assessments. Its effects on freshwater microbial communities have been well studied, but studies addressing effects on marine microbial communities are scarce. In the present study, the authors describe short- and long-term effects of triclosan on marine periphyton (microbial biofilm) communities. Short-term effects on photosynthesis were estimated after 60 min to 210 min of exposure. Long-term effects on photosynthesis, chlorophyll a fluorescence, pigment content, community tolerance, and bacterial carbon utilization were studied after exposing periphyton for 17 d in flow-through microcosms to 0.316 nM to 10,000 nM triclosan. Results from the short-term studies show that triclosan is toxic to periphyton photosynthesis. Half maximal effective concentration (EC50) values of 1080 nM and 3000 nM were estimated using (14)CO2-incorporation and pulse amplitude modulation (PAM) fluorescence measurements, respectively. After long-term triclosan exposure in flow-through microcosms, photosynthesis estimated using PAM fluorometry was not inhibited by triclosan concentrations up to 1000 nM but instead increased with increasing triclosan concentration. Similarly, at exposure concentrations of 31.6 nM and higher, triclosan caused an increase in photosynthetic pigments. At 316 nM triclosan, the pigment amounts were increased by a factor of 1.4 to 1.9 compared with the control level. Pollution-induced community tolerance was observed for algae and cyanobacteria at 100 nM triclosan and higher. Despite the widespread use of triclosan as an antibacterial agent, the compound did not have any effects on bacterial carbon utilization after long-term exposure.

A novel bioassay for evaluating the efficacy of biocides to inhibit settling and early establishment of marine biofilms.

This paper presents a novel assay that allows a quick and robust assessment of the effects of biocides on the initial settling and establishment of marine photoautotrophic biofilms including the multitude of indigenous fouling organisms. Briefly, biofilms are established in the field, sampled, comminuted and re-settled on clean surfaces, after 72h chlorophyll a is measured as an integrating endpoint to reflect both settling and growth. Eight antifoulants were used to evaluate the assay. Efficacy ranking, based on EC98 values from most to least efficacious compound is: copper pyrithione>TPBP>DCOIT>tolylfluanid>zinc pyrithione>medetomidine>copper (Cu(2+)), while ecotoxicological ranking (based on EC10 values) is irgarol, copper pyrithione>zinc pyrithione>TPBP>tolylfluanid>DCOIT>copper (Cu(2+))>medetomidine. The algaecide irgarol did not cause full inhibition. Instead the inhibition leveled out at 95% effect at 30 nmoll(-)(1), a concentration that was clearly lower than for any other of the tested biocides.

Extreme irgarol tolerance in an Ulva lactuca L. population on the Swedish west coast.

The herbicide irgarol 1051 is commonly used on ship hulls to prevent growth of algae, but as a component of self-eroding paints it can also spread in the surrounding waters and affect non-target organisms. The effect of irgarol on settlement and growth of zoospores from the marine macro algae Ulva lactuca from the Gullmar fjord on the Swedish west coast was investigated in the present study. The zoospores were allowed to settle and grow in the presence of irgarol, but neither settlement - nor growth inhibition was observed at concentrations of up to 2000 nmol l(-1). This is between 10 and 100 times higher than effect concentrations reported earlier for algae. Irgarol also induced the greening effect (4-fold increase in chlorophyll a content) in the settled zoospore/germling population, typical for photosystem II inhibitors like irgarol. This study support previous findings that irgarol constitutes a selection pressure in the marine environment.

Effects of five antifouling biocides on settlement and growth of zoospores from the marine macroalga Ulva lactuca L.

Antifouling biocides are found in the marine ecosystem were they can affect non-target organisms. In this study the effects of five antifouling biocides on the settlement and growth of Ulva lactuca zoospores were investigated. The biocides investigated were copper (Cu(2+)), 4,5-dichloro-2-n-octyl-3(2H)-isothiazolone (DCOIT), triphenylborane pyridine (TPBP), tolylfluanid and medetomidine. Full concentration-response curves where determined for each compound. EC50 values were determined for copper, DCOIT, TPBP and tolylfluanid, all of which inhibited settlement and growth in a concentration dependent manner with the following toxicity ranking; tolylfluanid (EC50 80 nmol L(-1)) ~ DCOIT (EC50 83 nmol L(-1)) > TPBP (EC50 400 nmol L(-1)) > Cu(2+) (EC50 2,000 nmol L(-1)). Medetomidine inhibited settlement and growth only at the extreme concentration of 100,000 nmol L(-1) (93% effect). The low toxicity is possibly a consequence of a lack of receptors that medetomidine can bind to in the U. lactuca zoospores.

Single-substance and mixture toxicity of five pharmaceuticals and personal care products to marine periphyton communities.

The single-substance and mixture toxicity of five pharmaceuticals and personal care products (fluoxetine, propranolol, triclosan, zinc-pyrithione, and clotrimazole) to marine microalgal communities (periphyton) was investigated. All compounds proved to be toxic, with median effective concentration values (EC50s) between 1,800 nmol/L (triclosan) and 7.2 nmol/L (Zn-pyrithione). With an EC50 of 356 nmol/L, the toxicity of the mixture falls into this span, indicating the absence of strong synergisms or antagonisms. In fact, a comparison with mixture toxicity predictions by the classical mixture concepts of concentration addition and independent action showed a good predictability in the upper effect range. However, the mixture provoked stimulating effects (hormesis) in the lower effect range, hampering the application of either concept. An independent repetition of the mixture experiment resulted in a principally similar concentration-response curve, again with clear hormesis effects in the lower range of test concentrations. However, the curve was shifted toward higher effect concentrations (EC50 1,070 nmol/L), which likely is due to changes in the initial species composition. Clear mixture effects were observed even when all five components were present only at their individual no-observed-effect concentrations (NOECs). These results show that, even with respect to mixtures of chemically and functionally dissimilar compounds, such as the five pharmaceuticals and personal care products investigated, environmental quality standards must take possible mixture effects from low-effect concentrations of individual compounds into consideration.

Effects of three antifouling agents on algal communities and algal reproduction: mixture toxicity studies with TBT, Irgarol, and Sea-Nine.

The toxicity of three antifoulants (Sea-Nine, Irgarol, and TBT) was determined individually and in mixtures in two tests with microalgae. Effects on periphyton community photosynthesis and reproduction of the unicellular green algae Scenedesmus vacuolatus were investigated. The tested antifoulants were highly toxic in both tests. Observed mixture toxicities were compared with predictions derived from two concepts: Independent Action (IA), assumed to be more relevant for the tested mixtures that were composed of dissimilarly acting substances, and Concentration Addition (CA), regarded as a reasonable worst-case approach in predictive mixture hazard assessment. Despite the corresponding mechanistic basis, IA failed to provide accurate predictions of the observed mixture toxicities. Results show the same pattern in both assays. Mixture effects at high concentrations were slightly overestimated and effects at low concentrations were slightly underestimated. Maximum observed deviations between observed and IA-predicted concentrations amount to a factor of 4. The suggested worst-case approach using CA was protective only in effect regions above 20%. Nevertheless, the application of any concept that accounts for possible mixture effects is more realistic than the present chemical-by-chemical assessment.

Toxicity of a mixture of dissimilarly acting substances to natural algal communities: predictive power and limitations of independent action and concentration addition.

Predictive studies of chemical mixtures are typically based on experiments with single species. To study the applicability of the concepts of independent action (IA) and concentration addition (CA) on a multispecies level, the carbon fixation of natural algal communities under toxicant exposure was studied. The presented study focused on a mixture of six dissimilarly acting substances. Conceptual reasoning as well as empirical evidence from single-species tests suggest that IA is more appropriate for this type of mixture. Nonetheless, the potential of CA was also investigated, to assess whether this concept may be applicable as a reasonable worst case prediction of mixture toxicities also on a community level. IA predicted the experimental EC50 precisely. CA underestimated the EC50 by a factor of only 1.4, although the shape of the predicted concentration-response curve was clearly different from experimental data. Hence, it can be concluded that the applicability of the concepts is not restricted to the level of single species. However, some limitations of both concepts became apparent, when stimulating (hormesis-like) effects were observed fo rtwo of the mixture components. These effects were also seen in the experimental mixture toxicity data but cannot be adequately modeled by either concept.

Predictability of the mixture toxicity of 12 similarly acting congeneric inhibitors of photosystem II in marine periphyton and epipsammon communities.

Testing of single chemicals with single species is common ecotoxicological practice in contrast to contaminated environments where highly diverse biological communities are exposed to highly diverse mixtures of chemical compounds. We, therefore, investigated whether mixture toxicity approaches that have been used successfully for single species, might also be applied on a community level of biological complexity. Twelve inhibitors of photosystem II, selected by QSAR and chemometrical approaches as the structurally most similar from a congeneric group of phenylurea herbicides, were tested singly and as mixtures on two types of marine microalgal communities, periphyton and epipsammon. Inhibition of photosynthesis was measured in short-term tests using incorporation of radiolabelled carbon (14C) to estimate photosynthetic rates. Two basic concepts, concentration addition (CA) and independent action (IA), were used to predict the toxicities of the mixtures. Congeneric and similar-acting substances such as the phenylureas are expected to comply with CA rather than IA. The aim of the present study was to evaluate whether these concepts can be used to predict mixture toxicity also to periphyton and epipsammon photosynthesis, i.e. at the level of natural communities. We found that deviations between observed and predicted mixture toxicity were relatively small but that CA predictions were the more accurate ones. The predictions proved to be robust, when based on single substance information even from different seasons, years, and sites. We conclude that the concept of CA for predicting mixture toxicity applies also at the community level of algal testing; at least when a physiological short-term effect indicator is used that matches the mechanism of action of the substances.