Effective removal of iron, nutrients, micropollutants, and faecal bacteria in constructed wetlands cotreating mine water and sewage treatment plant effluent.

Regulators in England and Wales have set new targets under the Environment Act 2021 for freshwater quality by 2038 that include halving the length of rivers polluted by harmful metals from abandoned mines and reducing phosphorus loadings from treated wastewater by 80%. In this context, an intriguing win-win opportunity exists in the removal of iron from abandoned mines and phosphate from small sewage treatment plants by coprecipitation in constructed wetlands (CWs). We investigated such a CW located at Lamesley, Northeast England, which cotreats abandoned coal mine and secondary-treated sewage treatment plant effluents. We assessed the removal of nutrients, heavy metals, organic micropollutants, and faecal coliforms by the CW, and characterized changes in the water bacteriology comprehensively using environmental DNA. The CW effectively removed ammonium-nitrogen, phosphorus, iron, and faecal coliforms by an average of 86, 74, 98, and 75%, respectively, to levels below or insignificantly different from those in the receiving river. The CW also effectively removed micropollutants such as acetaminophen, caffeine, and sulpiride by 70-100%. Molecular microbiology methods showed successful conversion of sewage and mine water microbiomes into a freshwater microbiome. Overall, the CW significantly reduced impacts on the rural water environment with minimal operational requirements.

Diversity and dynamics of bacterial communities in the drinking water distribution network of a mid-sized city in Brazil.

This study assessed the bacterial community composition of a drinking water system (DWS) serving a mid-sized city (120,000 inhabitants) in Brazil. Water samples, including raw and treated water, were collected at seven points throughout the DWS. DNA was extracted and analysed using high-throughput sequencing (Ion Torrent). Free chlorine and turbidity were measured in situ. Results showed that the highest relative abundance of 16S rRNA genes was from phyla Proteobacteria, followed by Bacteroidetes and Actinobacteria. The next most abundant phylum was Cyanobacteria, represented by Arthronema, Calothrix, and Synechococcus. An interesting observation was that the DNA-based analysis suggested a bacterial community change in the distribution network, with treated reservoir water being very different from the network samples. This suggests active microbiology within the distribution network and a tendency for bacterial diversity to decrease after chlorine disinfection but increase after pipeline distribution. In raw water, a predominance of Proteobacteria was observed with reduced Cyanobacteria, showing a negative correlation. In treated water, Proteobacteria were negatively correlated with Bacteroidetes. Finally, 16S rRNA genes from Firmicutes (especially Staphylococcus) had a high abundance in the chlorinated water, which may indicate the phylum's resistance to chlorine residuals. Opportunistic pathogens, e.g., Mycobacteria, Legionella, and Staphylococcus, were also observed.

Temperature and immigration effects on quorum sensing in the biofilms of anaerobic membrane bioreactors.

Quorum sensing (QS), a microbial communication mechanism modulated by acyl homoserine lactone (AHL) molecules impacts biofilm formation in bioreactors. This study investigated the effects of temperature and immigration on AHL levels and biofouling in anaerobic membrane bioreactors. The hypothesis was that the immigrant microbial community would increase the AHL-mediated QS, thus stimulating biofouling and that low temperatures would exacerbate this. We observed that presence of immigrants, especially when exposed to low temperatures indeed increased AHL concentrations and fouling in the biofilms on the membranes. At low temperature, the concentrations of the main AHLs observed, N-dodecanoyl-L-homoserine lactone and N-decanoyl-L-homoserine lactone, were significantly higher in the biofilms than in the sludge and correlated significantly with the abundance of immigrant bacteria. Apparently low temperature, immigration and denser community structure in the biofilm stressed the communities, triggering AHL production and excretion. These insights into the social behaviour of reactor communities responding to low temperature and influx of immigrants have implications for biofouling control in bioreactors.

Understanding the Dependence of Micropollutant Biotransformation Rates on Short-Term Temperature Shifts.

Temperature is a key factor that influences chemical biotransformation potential and rates, on which exposure and fate models rely to predict the environmental (micro)pollutant fate. Arrhenius-based models are currently implemented in environmental exposure assessment to adapt biotransformation rates to actual temperatures, assuming validity in the 0-30 °C range. However, evidence on how temperature shifts affect the physicochemical and microbial features in biological systems is scarce, questioning the validity of the existing modeling approaches. In this work, laboratory-scale batch assays were designed to investigate how a mixed microbial community responds to short-term temperature shifts, and how this impacts its ability to biotransform a range of structurally diverse micropollutants. Our results revealed three distinct kinetic responses at temperatures above 20 °C, mostly deviating from the classic Arrhenius-type behavior. Micropollutants with similar temperature responses appeared to undergo mostly similar initial biotransformation reactions, with substitution-type reactions maintaining Arrhenius-type behavior up to higher temperatures than oxidation-type reactions. Above 20 °C, the microbial community also showed marked shifts in both composition and activity, which mostly correlated with the observed deviations from Arrhenius-type behavior, with compositional changes becoming a more relevant factor in biotransformations catalyzed by more specific enzymes (e.g., oxidation reactions). Our findings underline the need to re-examine and further develop current environmental fate models by integrating biological aspects, to improve accuracy in predicting the environmental fate of micropollutants.

Multi-laboratory Validation of a New Marine Biodegradation Screening Test for Chemical Persistence Assessment.

Current biodegradation screening tests are not specifically designed for persistence assessment of chemicals, often show high inter- and intra-test variability, and often give false negative biodegradation results. Based on previous studies and recommendations, an international ring test involving 13 laboratories validated a new test method for marine biodegradation with a focus on improving the reliability of screening to determine the environmental degradation potential of chemicals. The new method incorporated increased bacterial cell concentrations to better represent the microbial diversity; a chemical is likely to be exposed in the sampled environments and ran beyond 60 days, which is the half-life threshold for chemical persistence in the marine environment. The new test provided a more reliable and less variable characterization of the biodegradation behavior of five reference chemicals (sodium benzoate, triethanolamine, 4-nitrophenol, anionic polyacrylamide, and pentachlorophenol), with respect to REACH and OSPAR persistence thresholds, than the current OECD 306 test. The proposed new method provides a cost-effective screening test for non-persistence that could streamline chemical regulation and reduce the cost and animal welfare implications of further higher tier testing.

Chlorination effects on DNA based characterization of water microbiomes and implications for the interpretation of data from disinfected systems.

Quantitative PCR (qPCR) and next generation sequencing (NGS) are nucleic acid based microbiology techniques that provide new insights into drinking water quality, but considerable uncertainty remains around their correct interpretation. We noticed the presence of bacterial DNA from various putative pathogens, including from faecal indicator bacteria (FIB), in disinfected water, when culturable FIB were absent. To understand these observations better we studied the effect of chlorination on conventional and DNA based microbial water quality assessments. Surface water chlorination reduced plate counts for various FIB by up to >6 log units, intact cell counts by flow cytometry by 3.3 log units, and 16S rRNA gene copies by qPCR by 1.5 and 1.6 log units for total bacteria and total coliforms, respectively. Nanopore sequencing of 16S rRNA amplicons with the portable MinION device revealed the DNA from several families containing putative pathogens appeared to be more resistant than that of other bacteria to degradation by chlorine disinfection. For instance, 16S rRNA genes assigned to the Enterobacteriaceae family, members of which are mostly the target of coliform tests, increased in relative abundance from 0.001 ± 0.0002% to 0.0036 ± 0.003% after chlorine treatment. Hence, metagenomic drinking water data needs to be interpreted with caution. Plate counts and flow cytometry in combination with DNA based analysis provide more robust insight than NGS or qPCR alone.

High Throughput Biodegradation-Screening Test To Prioritize and Evaluate Chemical Biodegradability.

Comprehensive assessment of environmental biodegradability of pollutants is limited by the use of low throughput systems. These are epitomized by the Organisation for Economic Cooperation and Development (OECD) Ready Biodegradability Tests (RBTs), where one sample from an environment may be used to assess a chemical's ability to readily biodegrade or persist universally in that environment. This neglects the considerable spatial and temporal microbial variation inherent in any environment. Inaccurate designations of biodegradability or persistence can occur as a result. RBTs are central in assessing the biodegradation fate of chemicals and inferring exposure concentrations in environmental risk assessments. We developed a colorimetric assay for the reliable quantification of suitable aromatic compounds in a high throughput biodegradation screening test (HT-BST). The HT-BST accurately differentiated and prioritized a range of structurally diverse aromatic compounds on the basis of their assigned relative biodegradabilities and quantitative structure-activity relationship (QSAR) model outputs. Approximately 20 000 individual biodegradation tests were performed, returning analogous results to conventional RBTs. The effect of substituent group structure and position on biodegradation potential demonstrated a significant correlation (P < 0.05) with Hammett's constant for substituents on position 3 of the phenol ring. The HT-BST may facilitate the rapid screening of 100 000 chemicals reportedly manufactured in Europe and reduce the need for higher-tier fate and effects tests.

Environmentally Relevant Inoculum Concentrations Improve the Reliability of Persistent Assessments in Biodegradation Screening Tests.

Standard OECD biodegradation screening tests (BSTs) have not evolved at the same rate as regulatory concerns, which now place an increased emphasis on environmental persistence. Consequently, many chemicals are falsely assigned as being potentially persistent based on results from BSTs. The present study increased test duration and increased inoculum concentrations to more environmentally relevant levels to assess their impact on biodegradation outcome and intratest replicate variability for chemicals with known environmental persistence. Chemicals were assigned to potential persistence categories based on existing degradation data. These more environmentally relevant BSTs (erBSTs) improved the reliability of persistence assignment by reducing the high variability associated with these tests and the occurrence of failures at low inoculum concentrations due to the exclusion of specific degraders. Environmental fate was determined using a reference set of 14C-labeled compounds with a range of potential environmental persistences, and full mass balance data were collated. The erBST correctly assigned five reference chemicals of known biodegradabilities to their appropriate persistence category in contrast to a standard OECD Ready Biodegradation Test (RBTs, P < 0.05). The erBST was significantly more reproducible than an OECD RBT (ANOVA, P < 0.05), with more consistent rates and extent of biodegradation observed in the erBST.

Low-temperature limitation of bioreactor sludge in anaerobic treatment of domestic wastewater.

Two strategies exist for seeding low-temperature anaerobic reactors: the use of specialist psychrophilic biomass or mesophilic bioreactor sludge acclimated to low temperature. We sought to determine the low-temperature limitation of anaerobic sludge from a bioreactor acclimated to UK temperatures (<15 °C). Anaerobic incubation tests using low-strength real domestic wastewater (DWW) and various alternative soluble COD sources were conducted at 4, 8 and 15 °C; methanogenesis and acidogenesis were monitored separately. Production of methane and acetate was observed; decreasing temperature resulted in decreased yields and increased 'start-up' times. At 4 °C methanogenesis not hydrolysis/acidogenesis was rate-limiting. The final methane yields at 4 °C were less than 35% of the theoretical potential whilst at 8 and 15 °C more than 75 and 100% of the theoretical yield was achieved respectively. We propose that the lower temperature limit for DWW treatment with anaerobic bioreactor sludge lies between 8 and 4 °C and that 8 °C is the threshold for reliable operation.

Impact of long-term temperature shifts on activated sludge microbiome dynamics and micropollutant removal.

Micropollutants removal efficiency strongly vary across different aerobic wastewater treatment plants, resulting in their frequent detection in surface and ground waters. Seasonal temperature variation is a major factor influencing plant performance, but it is still unclear how prolonged periods of temperature change impact microbiome and micropollutant biotransformation. This work investigates the effect of long-term temperature variation on the microbial dynamics in an activated sludge system, and the impact on micropollutant biotransformation. Sequencing batch reactors were used as model system and 4-40 °C temperature range was studied. 16S rRNA amplicon sequencing showed that temperature drives microbial structure (gDNA) and activity (RNA), rather than time, and this was stronger below 15 °C and above 25 °C. The microbial community was richest and more diverse at 20 °C, while rarer and more specific taxa became predominant over time, at more extreme temperatures. This suggested that less abundant taxa might be responsible for maintaining the biotransformation capability in the activated sludge at extreme temperatures. Micropollutant biotransformation rates mostly deviated from the classic Arrhenius model below 15 °C and above 25 °C, indicating that prolonged exposure to temperature changes leads to temperature-induced taxonomic shifts, resulting in the emerging of different sets of biotransformation pathways over different temperature ranges.

Accurate determination of microbial diversity from 454 pyrosequencing data.

We present an algorithm, PyroNoise, that clusters the flowgrams of 454 pyrosequencing reads using a distance measure that models sequencing noise. This infers the true sequences in a collection of amplicons. We pyrosequenced a known mixture of microbial 16S rDNA sequences extracted from a lake and found that without noise reduction the number of operational taxonomic units is overestimated but using PyroNoise it can be accurately calculated.

Effect of activated carbon amendment on bacterial community structure and functions in a PAH impacted urban soil.

We collected urban soil samples impacted by polycyclic aromatic hydrocarbons (PAHs) from a sorbent-based remediation field trial to address concerns about unwanted side-effects of 2% powdered (PAC) or granular (GAC) activated carbon amendment on soil microbiology and pollutant biodegradation. After three years, total microbial cell counts and respiration rates were highest in the GAC amended soil. The predominant bacterial community structure derived from denaturing gradient gel electrophoresis (DGGE) shifted more strongly with time than in response to AC amendment. DGGE band sequencing revealed the presence of taxa with closest affiliations either to known PAH degraders, e.g. Rhodococcus jostii RHA-1, or taxa known to harbor PAH degraders, e.g. Rhodococcus erythropolis, in all soils. Quantification by real-time polymerase chain reaction yielded similar dioxygenases gene copy numbers in unamended, PAC-, or GAC-amended soil. PAH availability assessments in batch tests showed the greatest difference of 75% with and without biocide addition for unamended soil, while the lowest PAH availability overall was measured in PAC-amended, live soil. We conclude that AC had no detrimental effects on soil microbiology, AC-amended soils retained the potential to biodegrade PAHs, but the removal of available pollutants by biodegradation was most notable in unamended soil.

Removing noise from pyrosequenced amplicons.

BACKGROUND: In many environmental genomics applications a homologous region of DNA from a diverse sample is first amplified by PCR and then sequenced. The next generation sequencing technology, 454 pyrosequencing, has allowed much larger read numbers from PCR amplicons than ever before. This has revolutionised the study of microbial diversity as it is now possible to sequence a substantial fraction of the 16S rRNA genes in a community. However, there is a growing realisation that because of the large read numbers and the lack of consensus sequences it is vital to distinguish noise from true sequence diversity in this data. Otherwise this leads to inflated estimates of the number of types or operational taxonomic units (OTUs) present. Three sources of error are important: sequencing error, PCR single base substitutions and PCR chimeras. We present AmpliconNoise, a development of the PyroNoise algorithm that is capable of separately removing 454 sequencing errors and PCR single base errors. We also introduce a novel chimera removal program, Perseus, that exploits the sequence abundances associated with pyrosequencing data. We use data sets where samples of known diversity have been amplified and sequenced to quantify the effect of each of the sources of error on OTU inflation and to validate these algorithms. RESULTS: AmpliconNoise outperforms alternative algorithms substantially reducing per base error rates for both the GS FLX and latest Titanium protocol. All three sources of error lead to inflation of diversity estimates. In particular, chimera formation has a hitherto unrealised importance which varies according to amplification protocol. We show that AmpliconNoise allows accurate estimates of OTU number. Just as importantly AmpliconNoise generates the right OTUs even at low sequence differences. We demonstrate that Perseus has very high sensitivity, able to find 99% of chimeras, which is critical when these are present at high frequencies. CONCLUSIONS: AmpliconNoise followed by Perseus is a very effective pipeline for the removal of noise. In addition the principles behind the algorithms, the inference of true sequences using Expectation-Maximization (EM), and the treatment of chimera detection as a classification or 'supervised learning' problem, will be equally applicable to new sequencing technologies as they appear.

Coconut husk biochar amendment enhances nutrient retention by suppressing nitrification in agricultural soil following anaerobic digestate application.

Anaerobic digestate and biochar are by-products of the biogasification and pyrolysis of agricultural wastes. This study tested the hypothesis that combined application of anaerobic pig/cattle manure digestate and coconut husk (CH) biochar can improve soil nutrient conditions, whilst minimizing atmospheric and groundwater pollution risks. Microcosms simulated digestate application to agricultural soil with and without CH biochar. Ammonia volatilization and nutrient leaching were quantified after simulated heavy rainfalls. Archaeal and bacterial community and abundance changes in soils were quantified via next generation sequencing and qPCR of 16S rRNA genes. Nitrifying bacteria were additionally quantified by qPCR of functional genes. It was found that CH biochar retarded nitrate leaching via slower nitrification in digestate-amended soil. CH biochar reduced both nitrifying archaea and bacteria abundance in soil by 71-83 percent in the top 4 cm soil layer and 66-80 percent in the deeper soil layer one month after the digestate application. Methanotroph abundances were similarly reduced in the CH biochar amended soils. These findings demonstrate combined benefits of anaerobic digestate and CH biochar application which are relevant for the development of a more circular rural economy with waste minimization, renewable energy production, nutrient recycling and reduced water pollution from agricultural land.

Experimental and Genomic Evaluation of the Oestrogen Degrading Bacterium Rhodococcus equi ATCC13557.

Rhodococcus equi ATCC13557 was selected as a model organism to study oestrogen degradation based on its previous ability to degrade 17α-ethinylestradiol (EE2). Biodegradation experiments revealed that R. equi ATCC13557 was unable to metabolise EE2. However, it was able to metabolise E2 with the major metabolite being E1 with no further degradation of E1. However, the conversion of E2 into E1 was incomplete, with 11.2 and 50.6% of E2 degraded in mixed (E1-E2-EE2) and E2-only conditions, respectively. Therefore, the metabolic pathway of E2 degradation by R. equi ATCC13557 may have two possible pathways. The genome of R. equi ATCC13557 was sequenced, assembled, and mapped for the first time. The genome analysis allowed the identification of genes possibly responsible for the observed biodegradation characteristics of R. equi ATCC13557. Several genes within R. equi ATCC13557 are similar, but not identical in sequence, to those identified within the genomes of other oestrogen degrading bacteria, including Pseudomonas putida strain SJTE-1 and Sphingomonas strain KC8. Homologous gene sequences coding for enzymes potentially involved in oestrogen degradation, most commonly a cytochrome P450 monooxygenase (oecB), extradiol dioxygenase (oecC), and 17ß-hydroxysteroid dehydrogenase (oecA), were identified within the genome of R. equi ATCC13557. These searches also revealed a gene cluster potentially coding for enzymes involved in steroid/oestrogen degradation; 3-carboxyethylcatechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde hydrolase, 3-alpha-(or 20-beta)-hydroxysteroid dehydrogenase, 3-(3-hydroxy-phenyl)propionate hydroxylase, cytochrome P450 monooxygenase, and 3-oxosteroid 1-dehydrogenase. Further, the searches revealed steroid hormone metabolism gene clusters from the 9, 10-seco pathway, therefore R. equi ATCC13557 also has the potential to metabolise other steroid hormones such as cholesterol.

Increased cell numbers improve marine biodegradation tests for persistence assessment.

Currently available OECD biodegradation screening tests (BSTs) are not particularly suited for persistence screening. Their duration can be much less than international half-life thresholds for persistence and they are variable and stringent, therefore prone to false negatives. The present study extended test durations beyond 28 days and increased biomass concentrations for marine BSTs to better represent the microbial diversity inherent in the sampled environment. For this so-called environmentally relevant BST (erBST) marine cell concentrations were nominally increased 100-fold by tangential flow filtration. The marine erBST was validated against a standard BST using five 14C labeled reference compounds with a range of biodegradation potentials (aniline, 4-fluorophenol, 4-nitrophenol, 4-chloroaniline and pentachlorophenol) in a modified OECD 301B test. A full mass balance was collated to follow chemical fate in the tests. The erBST was more accurate and less variable than the comparator BST in assigning the reference compounds to their expected biodegradation classifications (non-persistent or potentially persistent). According to the REACH non-persistence criterion of ≥60% biodegradation over 60 days, the erBST correctly classified 60% of chemical replicates according to their expected biodegradation classification and had a coefficient of variation of 21% between replicates. In contrast, the BST correctly assessed 40% of reference chemicals in regards to their expected biodegradation classification with a coefficient of variation of 36%. All non-persistent chemicals showed increased degradation in the erBST, except for 4-chloroaniline, which did not degrade in either BST or erBST. Both tests showed no false positive results, correctly classifying the negative control pentachlorophenol as potentially persistent. Next, it is recommended to further validate the marine erBST in an inter-laboratory study incorporating different seawater sources to fully assess its variability and reliability.

A comparative assessment of conventional and molecular methods, including MinION nanopore sequencing, for surveying water quality.

Nucleic acid based techniques, such as quantitative PCR (qPCR) and next generation sequencing (NGS), provide new insights into microbial water quality, but considerable uncertainty remains around their correct interpretation. We demonstrate, for different water sources in informal settlements in the Kathmandu Valley, Nepal, significant Spearman rank correlations between conventional and molecular microbiology methods that indicate faecal contamination. At family and genera level, 16S rRNA amplicon sequencing results obtained with the low-cost, portable next generation sequencer MinION from Oxford Nanopore Technologies had significant Spearman rank correlations with Illumina MiSeq sequencing results. However, method validation by amplicon sequencing of a MOCK microbial community revealed the need to ascertain MinION sequencing results for putative pathogens at species level with complementary qPCR assays. Vibrio cholerae hazards were poorly associated with plate count faecal coliforms, but flagged up by the MinION screening method, and confirmed by a qPCR assay. Plate counting methods remain important to assess viability of faecal coliforms in disinfected water sources. We outline a systematic approach for data collection and interpretation of such complementary results. In the Kathmandu Valley, there is high variability of water quality from different sources, including for treated water samples, illustrating the importance of disinfection at the point of use.

Molecular evaluation of microalgal communities in full-scale waste stabilisation ponds.

Waste stabilisation ponds (WSPs) are widely used across the world as a passive wastewater treatment for domestic wastewaters, but little is known about their ecology, especially their phototrophic communities. This study uses molecular methods and flow cytometry to assess the cyanobacterial and eukaryotic communities longitudinally throughout two systems, one treating domestic wastewater and the other mixed industrial/domestic wastewaters. More variation was seen between the systems than between different stages in the treatment processes for both eukaryotic and cyanobacterial communities. Chlorella species and Planktophrix cyanobacteria dominated both treatment systems. Arthrospira cyanobacteria were detected only in the industrial/domestic system. The balance between non-photosynthetic and photosynthetic organisms is rarely considered, though both play vital roles in WSP functioning. Flow cytometry showed that the facultative and first maturation pond in the industrial system contained a lower proportion of photosynthetic organisms compared to the domestic system. This is reflected in the species richness data and low dissolved oxygen levels detected. All data indicated that both systems are significantly different from one another and that variation longitudinally throughout the systems is lower. A more systematic study is needed to determine if it is the wastewater source rather than the initial inoculum that drives community composition.

A quantitative structure-biodegradation relationship (QSBR) approach to predict biodegradation rates of aromatic chemicals.

The objective of this work was to develop a QSBR model for the prioritization of organic pollutants based on biodegradation rates from a database containing globally harmonized biodegradation tests using relevant molecular descriptors. To do this, we first categorized the chemicals into three groups (Group 1: simple aromatic chemicals with a single ring, Group 2: aromatic chemicals with multiple rings and Group3: Group 1 plus Group 2) based on molecular descriptors, estimated the first order biodegradation rate of the chemicals using rating values derived from the BIOWIN3 model, and finally developed, validated and defined the applicability domain of models for each group using a multiple linear regression approach. All the developed QSBR models complied with OECD principles for QSAR validation. The biodegradation rate in the models for the two groups (Group 2 and 3 chemicals) are associated with abstract molecular descriptors that provide little relevant practical information towards understanding the relationship between chemical structure and biodegradation rates. However, molecular descriptors associated with the QSBR model for Group 1 chemicals (R2 = 0.89, Q2loo = 0.87) provided information on properties that can readily be scrutinised and interpreted in relation to biodegradation processes. In combination, these results lead to the conclusion that QSBRs can be an alternative tool to estimate the persistence of chemicals, some of which can provide further insights into those factors affecting biodegradation.

Improving the biodegradability in seawater test (OECD 306).

Growth and extensive urbanisation of the human population has been accompanied by increased manufacture and use of chemical compounds. To classify the fate and behaviour of these compounds in the environment, a series of international standardised biodegradation screening tests (BSTs) were developed over 30 years ago. In recent years, regulatory emphasis (e.g. REACH) has shifted from measuring biodegradation towards prioritisations based on chemical persistence. In their current guise, BSTs are ineffective as screens for persistence. The marine BST OECD 306 in particular is prone to high levels of variation and produces a large number of fails, many of which can be considered false negatives. An ECETOC funded two-day workshop of academia, industry and regulatory bodies was held in 2015 to discuss improvements to the marine BSTs based on previous research findings from the Cefic LRI ECO11 project and other foregoing studies. During this workshop, methodological improvements to the OECD 306 test were discussed, in addition to clarifying guidance on testing and interpretation of results obtained from marine BSTs (such as pass criteria, lag phases, freshwater read across and complex substances). Methodologically: (i) increasing bacterial cell concentrations to better represent the bacterial diversity inherent in the sampled environments; and (ii) increasing test durations to investigate extended lag phases observed in marine assessments, were recommended to be validated in a multi-institutional ring test.