Coupling pathway prediction and fluorescence spectroscopy to assess the impact of auxiliary substrates on micropollutant biodegradation.

Some bacteria can degrade organic micropollutants (OMPs) as primary carbon sources. Due to typically low OMP concentrations, these bacteria may benefit from supplemental assimilation of natural substrates present in the pool of dissolved organic matter (DOM). The biodegradability of such auxiliary substrates and the impacts on OMP removal are tightly linked to biotransformation pathways. Here, we aimed to elucidate the biodegradability and effect of different DOM constituents for the carbofuran degrader Novosphingobium sp. KN65.2, using a novel approach that combines pathway prediction, laboratory experiments, and fluorescence spectroscopy. Pathway prediction suggested that ring hydroxylation reactions catalysed by Rieske-type dioxygenases and flavin-dependent monooxygenases determine the transformability of the 11 aromatic compounds used as model DOM constituents. Our approach further identified two groups with distinct transformation mechanisms amongst the four growth-supporting compounds selected for mixed substrate biodegradation experiments with the pesticide carbofuran (Group 1: 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde; Group 2: p-coumaric acid, ferulic acid). Carbofuran biodegradation kinetics were stable in the presence of both Group 1 and Group 2 auxiliary substrates. However, Group 2 substrates would be preferable for bioremediation processes, as they showed constant biodegradation kinetics under different experimental conditions (pre-growing KN65.2 on carbofuran vs. DOM constituent). Furthermore, Group 2 substrates were utilisable by KN65.2 in the presence of a competitor (Pseudomonas fluorescens sp. P17). Our study thus presents a simple and cost-efficient approach that reveals mechanistic insights into OMP-DOM biodegradation.

Interpretable machine learning for predicting the fate and transport of pentachlorophenol in groundwater.

Pentachlorophenol (PCP) is a commonly found recalcitrant and toxic groundwater contaminant that resists degradation, bioaccumulates, and has a potential for long-range environmental transport. Taking proper actions to deal with the pollutant accounting for the life cycle consequences requires a better understanding of its behavior in the subsurface. We recognize the huge potential for enhancing decision-making at contaminated groundwater sites with the arrival of machine learning (ML) techniques in environmental applications. We used ML to enhance the understanding of the dynamics of PCP transport properties in the subsurface, and to determine key hydrochemical and hydrogeological drivers affecting its transport and fate. We demonstrate how this complementary knowledge, provided by data-driven methods, may enable a more targeted planning of monitoring and remediation at two highly contaminated Swedish groundwater sites, where the method was validated. We evaluated 6 interpretable ML methods, 3 linear regressors and 3 non-linear (i.e., tree-based) regressors, to predict PCP concentration in the groundwater. The modeling results indicate that simple linear ML models were found to be useful in the prediction of observations for datasets without any missing values, while tree-based regressors were more suitable for datasets containing missing values. Considering that missing values are common in datasets collected during contaminated site investigations, this could be of significant importance for contaminated site planners and managers, ultimately reducing site investigation and monitoring costs. Furthermore, we interpreted the proposed models using the SHAP (SHapley Additive exPlanations) approach to decipher the importance of different drivers in the prediction and simulation of critical hydrogeochemical variables. Among these, sum of chlorophenols is of highest significance in the analyses. Setting that aside from the model, tetra chlorophenols, dissolved organic carbon, and conductivity found to be of highest importance. Accordingly, ML methods could potentially be used to improve the understanding of groundwater contamination transport dynamics, filling gaps in knowledge that remain when using more sophisticated deterministic modeling approaches.

Holistic valuation of Nature-Based Solutions accounting for human perceptions and nature benefits.

When assessing strategies for implementing Nature-Based Solutions (NBS), it is paramount to identify and quantify all benefits for securing better, informed decisionmaking. Nevertheless, there appears to be a lack of primary data for linking the valuation of NBS sites with the preferences and attitudes of people interacting with them and their connection to supporting efforts to reduce biodiversity loss. This is a critical gap, as the socio-cultural context of NBS has been proven to play a big role in NBS valuation, especially for their non-tangible benefits (e.g. physical and psychological well-being, habitat enhancements, etc.). Consequently, through cocreation with the local government, we co-designed a contingent valuation (CV) survey to explore how the valuation of NBS sites may be shaped by their relationship with the users and the specific respondent and site characteristics. We applied this method to a case study of two distinct areas located in Aarhus, Denmark, with notable differences related to their attributes (e.g. size, location, time passed since construction). The esults obtained from 607 households in Aarhus Municipality show that the personal preferences of the respondent are the most relevant driver of value, surpassing both the perceptions linked to the physical features of the NBS and the socio-economic characteristics of the respondents. Specifically, the respondents attributing most importance to nature benefits were the ones assigning a higher value to the NBS and being willing to pay more for an improvement of the nature quality in the area. These findings highlight the relevance of applying a method assessing the interconnections between human perceptions and nature benefits to ensure a holistic valuation and purposeful design of NBS.

Assessing the substrate specificity of a micropollutant degrading strain: generalist or specialist?

Natural dissolved organic matter (DOM) can serve as an additional substrate for organic micropollutant (OMP) degrading bacteria, thus influencing OMP biodegradation in aquatic systems. DOM biodegradation depends on the OMP degrader's ability to grow on different DOM constituents, and on its capability to compete for DOM constituents against the rest of the resident aquatic microbial community. This study aimed to investigate the growth of a model OMP degrader strain, Novosphingobium sp. KN65.2 (assumed specialist), isolated for its ability to mineralize carbofuran, on thirteen DOM constituents; compare its metabolic capabilities to those of a common freshwater strain (Pseudomonas fluorescens sp. P17) (generalist); and to evaluate competition for specific compounds. Growth experiments were carried out in pure- and mixed culture batch experiments. The DOM constituents tested included aromatic amino acids and a range of phenolic acids (lignin derivatives). The OMP degrader could biodegrade approximately half of the tested compounds. It showed a high specialization for substrates containing a hydroxyl-group in the para-position of the primary aromatic ring substituent. However, its broad substrate range enabled the strain to grow on the same number of auxiliary substrates as the generalist. Moreover, the OMP degrader was able to successfully compete against the generalist for the biodegradation of one (4-hydroxybenzaldehyde) out of three substrates (4-hydroxybenzoic acid, 4-hydroxybenzaldehyde, L-tyrosine), which were biodegraded by both strains. The study results provide insight on the substrate specificity of a model OMP degrader, which can inform development of modeling frameworks investigating the influence of DOM on OMP biodegradation.

Knowledge gaps and future research needs for assessing the non-market benefits of Nature-Based Solutions and Nature-Based Solution-like strategies.

Nature-Based Solutions (NBS) can be defined as solutions based on natural processes that meet societal challenges and simultaneously provide human well-being and biodiversity benefits. These solutions are envisioned to contribute to operationalizing sustainable development strategies, especially in the context of adaptation to climate change (e.g. flood risk reduction). In order to quantify NBS performance, ease their uptake and advocate for them as alternatives to "business-as-usual" infrastructures, a comprehensive, holistic valuation of their multiple benefits (multiple advantages and disadvantages) is needed. This entails quantifying non-market benefits for people and nature in addition to determining the (direct) cost-benefit of the risk-reduction measure. Despite the importance given to the assessment of non-tangible benefits for people and nature in the literature, systematic data collection on these dimensions seems to be missing. This study reviews publications that used stated preference methods to assess non-market human benefits of NBS and NBS-like strategies. Its aim is to highlight any biases or knowledge gaps in this kind of evaluation. Our results show that the valuation of non-tangible benefits of NBS (e.g. increased recreation and well-being, enhanced biodiversity) still suffers from a lack of common framing. Despite some steps being taken on enabling interconnected benefit assessments, unexploited opportunities concerning the integrated assessment of non-market human and nature benefits predominate. Moreover, the research to-date appears based on a case-to-case approach, and thus a shared holistic method does not emerge from the present literature, potentially delaying the uptake of NBS. We argue that future research could minimize missed opportunities by focusing on and systematically applying holistic benefits assessments. Methods based on stated preference surveys may help to ensure holistic approaches are taken, as well as contributing to their replicability and application when upscaling NBS.

DynSus: Dynamic sustainability assessment in groundwater remediation practice.

Decision-making processes for clean-up of contaminated sites are often highly complex and inherently uncertain. It depends not only on hydrological and biogeochemical site variability, but also on the associated health, environmental, economic, and social impacts of taking, or not taking, action. These variabilities suggest that a dynamic framework is required for promoting sustainable remediation. For this, the decision support system DynSus is presented here for integrating a predeveloped contaminant fate and transport model with a sustainability assessment tool. Implemented within a system dynamics framework, the new tool uses model simulations to provide remediation scenario analysis and handling of uncertainty in various data. DynSus was applied to a site in south Sweden, contaminated with pentachlorophenol (PCP). Simulation scenarios were developed to enable a comparison between alternative remediation strategies and combinations of these. Such comparisons are provided for selected sustainability indicators and remediation performance (in terms of concentration at the recipient). This leads to identifying the most critical variables to ensure that sustainable solutions are chosen. Simulation results indicated that although passive practices, e.g., monitored natural attenuation, were more sustainable at first (5-7 years after beginning remediation measures), they failed to compete with more active practices, e.g., bioremediation, over the entire life cycle of the project (from the beginning of remedial action to achieving the target concentration at the recipient). In addition, statistical tools (clustering and genetic algorithms) were used to further assess the available hydrogeochemical data. Taken together, the results reaffirmed the suitability of the simple analytical framework that was implemented in the contaminant transport model. DynSus outcomes could therefore enable site managers to evaluate different scenarios more quickly and effectively for life cycle sustainability in such a complex and multidimensional problem.

Natural attenuation of a chlorinated ethene plume discharging to a stream: Integrated assessment of hydrogeological, chemical and microbial interactions.

Attenuation processes of chlorinated ethenes in complex near-stream systems result in site-specific outcomes of great importance for risk assessment of contaminated sites. Additional interdisciplinary and comprehensive field research is required to enhance process understanding in these systems. In this study, several methods were combined in a multi-scale interdisciplinary in-situ approach to assess and quantify the near-stream attenuation of a chlorinated ethene plume, mainly consisting of cis-dichloroethene (cis-DCE) and vinyl chloride (VC), discharging to a lowland stream (Grindsted stream, Denmark) over a monitoring period of seven years. The approach included: hydrogeological characterisation, reach scale contaminant mass balance analysis, quantification of contaminant mass discharge, streambed fluxes of chlorinated ethenes quantified using Sediment Bed Passive Flux Meters (SBPFMs), assessment of redox conditions, temporal assessment of contaminant concentrations, microbial analysis, and compound-specific isotope analysis (CSIA). This study site exhibits a special attenuation behaviour not commonly encountered in field studies: the conversion from an initially limited degradation case (2012-16), despite seemingly optimal conditions, to one presenting notable levels of degradation (2019). Hence, this study site provides a new piece to the puzzle, as sites with different attenuation behaviours are required in order to acquire the full picture of the role groundwater-surface water interfaces have in risk mitigation. In spite of the increased degradation in the near-stream plume core, the contaminant attenuation was still incomplete in the discharging plume. A conceptualization of flow, transport and processes clarified that hydrogeology was the main control on the natural attenuation, as short residence times of 0.5-37 days restricted the time in which dechlorination could occur. This study reveals the importance of: taking an integrated approach to understand the influence of all attenuation processes in groundwater - surface water interactions; considering the scale and domain of interest when determining the main processes; and monitoring sufficiently both spatially and temporally to cover the transient conditions.

Assessing the Transport of Pharmaceutical Compounds in a Layered Aquifer Discharging to a Stream.

A groundwater plume containing high concentrations of pharmaceutical compounds, mainly sulfonamides, barbiturates, and ethyl urethane, in addition to chlorinated ethenes and benzene was investigated. The contamination originating from a former pharmaceutical industry discharges into a multilayered aquifer system and a downgradient stream. In this study, geological and hydrogeological data were integrated into a numerical flow model to examine identified trends using statistical approaches, including principal component analysis and hierarchal cluster analysis. A joint interpretation of the groundwater flow paths and contaminant concentrations in the different compartments (i.e., groundwater and hyporheic zone) provided insight on the transport processes of the different contaminant plumes to the stream. The analysis of historical groundwater concentrations of pharmaceutical compounds at the site suggested these compounds are slowly degrading. The pharmaceutical compounds migrate in both a deep semiconfined aquifer, as well as in the shallow unconfined aquifer, and enter the stream along a 2-km stretch. This contrasted with the chlorinated ethenes, which mainly discharge to the stream as a focused plume from the unconfined aquifer. The integrated approach developed here, combining groundwater flow modeling and statistical analyses of the contaminant concentration data collected in groundwater and the hyporheic zone, lead to an improved understanding of the observed distribution of contaminants in the unconfined and semiconfined aquifers, and thus to their discharge to the stream. This approach is particularly relevant for large and long-lasting contaminant sources and plumes, such as abandoned landfills and industrial production sites, where field investigations may be very expensive.

Investigating Fluorescent Organic-Matter Composition as a Key Predictor for Arsenic Mobility in Groundwater Aquifers.

Dissolved organic matter (DOM) is linked to the heterogeneous distribution of elevated arsenic (As) in groundwater used for drinking and irrigation purposes, but the relationship between DOM characteristics and arsenic mobility has yet to be fully understood. Here, DOM from groundwater sampled in the Bengal Basin region was characterized using both conventional bulk emission-excitation (EEM) spectroscopy and high-performance size-exclusion chromatography coupled to spectroscopy (HPSEC-EEM). Notably, application of the novel HPSEC-EEM approach permitted the total fluorescence of individual samples to be independently resolved into its underlying components. This allowed the external validation of the bulk-sample fluorescence decomposition and offered insight into the molecular size distribution of fluorescent DOM. Molecular size distributions were similar for the UVA fluorescent (C310 and C340) as well as the three visible fluorescent (C390, C440, and C500) components. There was a greater visible fluorescence in shallow aquifer samples (10-33 m) with high As (SH, up to 418 µg/L) than in samples from the same depth with lower As (up to 40 µg/L). This indicated a link between DOM quality and As mobility within the shallow aquifer. The deep aquifer samples (170-200 m) revealed DOM characteristics similar to SH samples but had low As concentrations (<4 µg/L), signifying that the deep aquifer is potentially vulnerable to As contamination. These findings pave the way for a more comprehensive assessment of the susceptibility of drinking water aquifers, thereby supporting the management of groundwater resources.

Linking ecological health to co-occurring organic and inorganic chemical stressors in a groundwater-fed stream system.

Freshwaters are among the most endangered ecosystems worldwide, due predominantly to excessive anthropogenic practices compromising the future provisioning of ecosystem services. Despite increased awareness of the role of multiple stressors in accounting for ecological degradation in mixed land-use stream systems, risk assessment approaches applicable in field settings are still required. This study provides a first indication for ecological consequences of the interaction of organic and inorganic chemical stressors, not typically evaluated together, which may provide a missing link enabling the reconnection of chemical and ecological findings. Specifically, impaired ecological conditions - represented by lower abundance of meiobenthic individuals - were observed in the hyporheic zone where a contaminant groundwater plume discharged to the stream. These zones were characterized by high xenobiotic organic concentrations, and strongly reduced groundwater (e.g. elevated dissolved iron and arsenic) linked to the dissolution of iron hydroxides (iron reduction) caused by the degradation of xenobiotic compounds in the plume. Further research is still needed to separate whether impact is driven by a combined effect of organic and inorganic stressors impacting the ecological communities, or whether the conditions - when present simultaneously - are responsible for enabling a specific chemical stressor's availability (e.g. trace metals), and thus toxicity, along the study stream. Regardless, these findings suggest that benthic meioinvertebrates are promising indicators for supporting biological assessments of stream systems to sufficiently represent impacts resulting from the co-occurrence of stressors in different stream compartments. Importantly, identification of the governing circumstances is crucial for revealing key patterns and impact drivers that may be needed in correctly prioritizing stressor impacts in these systems. This study further highlights the importance of stream-aquifer interfaces for investigating chemical stressor effects in multiple stressor systems. This will require holistic approaches for linking contaminant hydrogeology and eco(toxico)logy in order to positively influence the sustainable management of water resources globally.

Assessing the chemical contamination dynamics in a mixed land use stream system.

Traditionally, the monitoring of streams for chemical and ecological status has been limited to surface water concentrations, where the dominant focus has been on general water quality and the risk for eutrophication. Mixed land use stream systems, comprising urban areas and agricultural production, are challenging to assess with multiple chemical stressors impacting stream corridors. New approaches are urgently needed for identifying relevant sources, pathways and potential impacts for implementation of suitable source management and remedial measures. We developed a method for risk assessing chemical stressors in these systems and applied the approach to a 16-km groundwater-fed stream corridor (Grindsted, Denmark). Three methods were combined: (i) in-stream contaminant mass discharge for source quantification, (ii) Toxic Units and (iii) environmental standards. An evaluation of the chemical quality of all three stream compartments - stream water, hyporheic zone, streambed sediment - made it possible to link chemical stressors to their respective sources and obtain new knowledge about source composition and origin. Moreover, toxic unit estimation and comparison to environmental standards revealed the stream water quality was substantially impaired by both geogenic and diffuse anthropogenic sources of metals along the entire corridor, while the streambed was less impacted. Quantification of the contaminant mass discharge originating from a former pharmaceutical factory revealed that several 100 kgs of chlorinated ethenes and pharmaceutical compounds discharge into the stream every year. The strongly reduced redox conditions in the plume result in high concentrations of dissolved iron and additionally release arsenic, generating the complex contaminant mixture found in the narrow discharge zone. The fingerprint of the plume was observed in the stream several km downgradient, while nutrients, inorganics and pesticides played a minor role for the stream health. The results emphasize that future investigations should include multiple compounds and stream compartments, and highlight the need for holistic approaches when risk assessing these dynamic systems.

A Bayesian belief network approach for assessing uncertainty in conceptual site models at contaminated sites.

A key component in risk assessment of contaminated sites is in the formulation of a conceptual site model (CSM). A CSM is a simplified representation of reality and forms the basis for the mathematical modeling of contaminant fate and transport at the site. The CSM should therefore identify the most important site-specific features and processes that may affect the contaminant transport behavior at the site. However, the development of a CSM will always be associated with uncertainties due to limited data and lack of understanding of the site conditions. CSM uncertainty is often found to be a major source of model error and it should therefore be accounted for when evaluating uncertainties in risk assessments. We present a Bayesian belief network (BBN) approach for constructing CSMs and assessing their uncertainty at contaminated sites. BBNs are graphical probabilistic models that are effective for integrating quantitative and qualitative information, and thus can strengthen decisions when empirical data are lacking. The proposed BBN approach facilitates a systematic construction of multiple CSMs, and then determines the belief in each CSM using a variety of data types and/or expert opinion at different knowledge levels. The developed BBNs combine data from desktop studies and initial site investigations with expert opinion to assess which of the CSMs are more likely to reflect the actual site conditions. The method is demonstrated on a Danish field site, contaminated with chlorinated ethenes. Four different CSMs are developed by combining two contaminant source zone interpretations (presence or absence of a separate phase contamination) and two geological interpretations (fractured or unfractured clay till). The beliefs in each of the CSMs are assessed sequentially based on data from three investigation stages (a screening investigation, a more detailed investigation, and an expert consultation) to demonstrate that the belief can be updated as more information becomes available.

Legacy of a Chemical Factory Site: Contaminated Groundwater Impacts Stream Macroinvertebrates.

Legislative and managing entities of EU member states face a comprehensive task because the chemical and ecological impacts of contaminated sites on surface waters must be assessed. The ecological assessment is further complicated by the low availability or, in some cases, absence of ecotoxicity data for many of the compounds occurring at contaminated sites. We studied the potential impact of a contaminated site, characterised by chlorinated solvents, sulfonamides, and barbiturates, on benthic macroinvertebrates in a receiving stream. Most of these compounds are characterised by low or unknown ecotoxicity, but they are continuously discharged into the stream by way of a long-lasting source generating long-term chronic exposure of the stream biota. Our results show that taxonomical density and diversity of especially sediment dwelling taxa were reduced by >50 % at the sampling sites situated in the primary inflow zone of the contaminated GW. Moreover, macroinvertebrate communities at these sampling sites could be distinguished from those at upstream control sites and sites situated along a downstream dilution gradient using multidimensional scaling. Importantly, macroinvertebrate indices currently used did not identify this impairment, thus underpinning an urgent need for developing suitable tools for the assessment of ecological effects of contaminated sites in streams.

The legacy of pesticide pollution: An overlooked factor in current risk assessments of freshwater systems.

We revealed a history of legacy pesticides in water and sediment samples from 19 small streams across an agricultural landscape. Dominant legacy compounds included organochlorine pesticides, such as DDT and lindane, the organophosphate chlorpyrifos and triazine herbicides such as terbutylazine and simazine which have long been banned in the EU. The highest concentrations of legacy pesticides were found in streams draining catchments with a large proportion of arable farmland suggesting that they originated from past agricultural applications. The sum of toxic units (SumTUD.magna) based on storm water samples from agriculturally impacted streams was significantly higher when legacy pesticides were included compared to when they were omitted. Legacy pesticides did not significantly change the predicted toxicity of water samples to algae or fish. However, pesticide concentrations in bed sediment and suspended sediment samples exceeded safety thresholds in 50% of the samples and the average contribution of legacy pesticides to the SumTUC.riparius was >90%. Our results suggest that legacy pesticides can be highly significant contributors to the current toxic exposure of stream biota, especially macroinvertebrate communities, and that those communities were primarily exposed to legacy pesticides via the sediment. Additionally, our results suggest that neglecting legacy pesticides in the risk assessment of pesticides in streams may severely underestimate the risk of ecological effects.

Sources, occurrence and predicted aquatic impact of legacy and contemporary pesticides in streams.

We couple current findings of pesticides in surface and groundwater to the history of pesticide usage, focusing on the potential contribution of legacy pesticides to the predicted ecotoxicological impact on benthic macroinvertebrates in headwater streams. Results suggest that groundwater, in addition to precipitation and surface runoff, is an important source of pesticides (particularly legacy herbicides) entering surface water. In addition to current-use active ingredients, legacy pesticides, metabolites and impurities are important for explaining the estimated total toxicity attributable to pesticides. Sediment-bound insecticides were identified as the primary source for predicted ecotoxicity. Our results support recent studies indicating that highly sorbing chemicals contribute and even drive impacts on aquatic ecosystems. They further indicate that groundwater contaminated by legacy and contemporary pesticides may impact adjoining streams. Stream observations of soluble and sediment-bound pesticides are valuable for understanding the long-term fate of pesticides in aquifers, and should be included in stream monitoring programs.

A catchment scale evaluation of multiple stressor effects in headwater streams.

Mitigation activities to improve water quality and quantity in streams as well as stream management and restoration efforts are conducted in the European Union aiming to improve the chemical, physical and ecological status of streams. Headwater streams are often characterised by impairment of hydromorphological, chemical, and ecological conditions due to multiple anthropogenic impacts. However, they are generally disregarded as water bodies for mitigation activities in the European Water Framework Directive despite their importance for supporting a higher ecological quality in higher order streams. We studied 11 headwater streams in the Hove catchment in the Copenhagen region. All sites had substantial physical habitat and water quality impairments due to anthropogenic influence (intensive agriculture, urban settlements, contaminated sites and low base-flow due to water abstraction activities in the catchment). We aimed to identify the dominating anthropogenic stressors at the catchment scale causing ecological impairment of benthic macroinvertebrate communities and provide a rank-order of importance that could help in prioritising mitigation activities. We identified numerous chemical and hydromorphological impacts of which several were probably causing major ecological impairments, but we were unable to provide a robust rank-ordering of importance suggesting that targeted mitigation efforts on single anthropogenic stressors in the catchment are unlikely to have substantial effects on the ecological quality in these streams. The SPEcies At Risk (SPEAR) index explained most of the variability in the macroinvertebrate community structure, and notably, SPEAR index scores were often very low (<10% SPEAR abundance). An extensive re-sampling of a subset of the streams provided evidence that especially insecticides were probably essential contributors to the overall ecological impairment of these streams. Our results suggest that headwater streams should be considered in future management and mitigation plans. Catchment-based management is necessary because several anthropogenic stressors exceeded problematic thresholds, suggesting that more holistic approaches should be preferred.

Integrated assessment of the impact of chemical stressors on surface water ecosystems.

The release of chemicals such as chlorinated solvents, pesticides and other xenobiotic organic compounds to streams, either from contaminated sites, accidental or direct application/release, is a significant threat to water resources. In this paper, different methods for evaluating the impacts of chemical stressors on stream ecosystems are evaluated for a stream in Denmark where the effects of major physical habitat degradation can be disregarded. The methods are: (i) the Danish Stream Fauna Index, (ii) Toxic Units (TU), (iii) SPEAR indices, (iv) Hazard Quotient (HQ) index and (v) AQUATOX, an ecological model. The results showed that the hydromorphology, nutrients, biological oxygen demand and contaminants (pesticides and trichloroethylene from a contaminated site) originating from groundwater do not affect the good ecological status in the stream. In contrast, the evaluation by the novel SPEAR(pesticides) index and TU indicated that the site is far from obtaining good ecological status - a direct contradiction to the ecological index currently in use in Denmark today - most likely due to stream sediment-bound pesticides arising from the spring spraying season. In order to generalise the findings of this case study, the HQ index and AQUATOX were extended for additional compounds, not only partly to identify potential compounds of concern, but also to determine thresholds where ecological impacts could be expected to occur. The results demonstrate that some commonly used methods for the assessment of ecological impact are not sufficient for capturing - and ideally separating - the effects of all anthropogenic stressors affecting ecosystems. Predictive modelling techniques can be especially useful in supporting early decisions on prioritising hot spots, serving to identify knowledge gaps and thereby direct future data collection. This case study presents a strong argument for combining bioassessment and modelling techniques to multi-stressor field sites, especially before cost-intensive studies are conducted.