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A high-throughput biodegradation-screening test to prioritise and evaluate chemical biodegradability Timothy James Martin, Andrew Kenneth Goodhead, Kishor Acharya, Ian M Head, Jason R. Snape, and Russell J Davenport Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 09 May 2017 Downloaded from http://pubs.acs.org on May 12, 2017

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Environmental Science & Technology

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A high-throughput biodegradation-screening test to

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prioritise and evaluate chemical biodegradability

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Timothy J. Martin† •, Andrew K. Goodhead†

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Snape‡∆, Russell J. Davenport*†

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†

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Newcastle upon Tyne, NE1 7RU, United Kingdom.

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‡

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∆

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*[email protected]; Telephone: +44 (0) 191 208 5544; Fax: +44 (0) 191 208 6502

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, Kishor Acharya†, Ian M. Head†, Jason R.

School of Civil Engineering and Geosciences, Cassie Building, Newcastle University,

AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, United Kingdom. School of Life Sciences, Gibbet Hill Campus, The University of Warwick, Coventry, CV4 7AL.

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KEYWORDS Biodegradation; Persistence; PBT; Environmental Risk Assessment

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ABSTRACT

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Comprehensive assessment of environmental biodegradability of pollutants is limited by the use

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of low-throughput systems.

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Cooperation and Development (OECD) Ready Biodegradability Tests (RBTs), where one

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sample from an environment may be used to assess a chemical’s ability to readily biodegrade or

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persist universally in that environment. This neglects the considerable inherent microbial

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variation spatially and temporally in any environment.

These are epitomized by the Organisation for Economic

Inaccurate designations of

ACS Paragon Plus Environment

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biodegradability or persistence can occur as a result. RBTs are central in assessing

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biodegradation fate of chemicals and inferring exposure concentrations in environmental risk

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assessments. We developed a colorimetric assay for the reliable quantification of suitable

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aromatic compounds in a high throughput biodegradation screening test (HT-BST). The HT-BST

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accurately differentiated and prioritised a range of structurally diverse aromatic compounds

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based on their assigned relative biodegradabilities and Quantitative Structure-Activity

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Relationship (QSAR) model outputs. Approximately 20,000 individual biodegradation tests were

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performed, returning analogous results to conventional RBTs. The effect of substituent group

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structure and position on biodegradation potential demonstrated a significant correlation

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(P methyl (CH3)= chloro (Cl) > methoxy (CH3O)= fluoro (F ) > nitro (NO2) >

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bromo (Br) for MPN data, which was similar to COOH > OH > CH3 > CH3O > Cl > NO2 > Br >

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F for Biowin2 data, where COOH has the highest potential.

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Regression analysis of the biodegradation potential (expressed as MPN counts) showed a weak

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but significant correlation with the substituent Hammett constant (R2 = 0.19, P < 0.05). When

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broken down into the different positions, there was a significant negative correlation (R2 = 0.58,

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P < 0.05) between the biodegradation potential and the Hammett constant for the substituent


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groups in position 3 (meta-position) on the ring (Figure 3). There was no significant correlation

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for positions 2 and 4 unlike those determined in previous studies for specific biodegradation rate

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30, 32

.

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DISCUSSION

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Proof of principle for HT-BSTs. The HT-BST described here, using a single activated sludge

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source of inoculum, has been shown to correctly prioritise a group of structurally diverse

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aromatic chemicals based on their previous biodegradability classification using conventional

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RBT test criteria (i.e. ability to aerobically biodegrade in an aqueous medium). The HT-BST also

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allowed us to investigate the effect of substituent group on biodegradability of the twenty-five

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mono-substituted phenols, which compared well to biodegradability from QSAR estimates and

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other experimental studies.

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The HT-BST has high throughput and replication. We typically used a 96-well plate per dilution

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of inoculum per chemical, resulting in approximately 20,000 individual biodegradation-

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screening tests during the course of this research at a fraction of the cost and time of a standard

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RBT (Table S5 SI), in addition to addressing the labour and time restrictions associated with

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conventional analyses33. This method is amenable to automation using liquid handling robots,

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unlike existing RBTs, which may further streamline the process. The format described in the

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present study could also be adapted to include multiple chemicals, multiple inocula

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concentrations, and/or multiple inocula from the same or different environmental sources on one

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plate; indeed during the standard curve production each column (i.e. 8 wells) represented a

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different chemical concentration. It opens up the possibility of explicitly investigating the


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variability of chemical biodegradation both spatially and temporally in relation to differences in

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microbial communities. The colorimetric detection of specific chemicals was accurate and

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reproducible; concentrations were statistically indistinguishable from those quantified by HPLC

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in HT-BSTs showing primary degradation, the method had a low median coefficient of variation,

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and the method detection limits were suitable for the current end-point thresholds used for RBTs.

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At environmentally relevant inoculum concentrations, the assay effectively screened for potential

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environmental persistence, correctly characterizing eight out of the ten selected chemicals, based

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on their biodegradation potential. False negatives were more frequently observed at low

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inoculum concentrations. It should be noted that the total number of bacteria present in typical

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standard OECD RBTs (A-C, F) might sometimes reach the total number present at the highest

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concentrations used in the HT-BST (~108) even though the concentrations may be different. This

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is due to the test volumes used (up to L in RBTs, hundreds of µL in HT-BST). However, the

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ratio of chemical to inoculum concentration, i.e. the food to microorganism ratio, was the same

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in the HT-BST as those used in OECD RBTs for the range of OECD inocula concentrations

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used. We have further shown previously that the variation in biodegradation at these lower

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concentrations is extremely high, which in RBTs is further exacerbated by a drastic reduction in

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the bacterial diversity of inocula preparation compared to the original activated sludge samples10.

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Other recent studies have also investigated high-throughput miniaturized biodegradation tests

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based on measuring oxygen uptake as a proxy for ultimate biodegradation of two readily and one

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inherently biodegradable chemical 17. These tests report similar limits of detection and substrate

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concentration ranges as those reported here. Unlike the current HT-BST, those based on oxygen

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uptake appear to scale differently to conventional RBTs; the biodegradation end-point achieved

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the same values but in a different amount of time, which may complicate cross-validation. The


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current HT-BST gave analogous results to conventional RBTs within the same timeframe,

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although it measured primary biodegradation after a particular time and not over time. Using

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plates that were sacrificially sampled at different time points would rectify this. Nevertheless,

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both high-throughput methods would complement each other at a cost comparable to current

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RBTs, thereby offering the opportunity to measure both primary and ultimate biodegradation in

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the same test. The assessment of primary biodegradation, as described in the present study, does

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not consider the potential formation of dead end by-products, which may or may not be toxic.

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The HT-BST described is a relatively simple screening test and must be considered as a tool in a

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broader intelligent testing strategy, as is current practice in REACH9,

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potential formation of dead-end by-products, the assay could be coupled with analytical

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chemistry, or the biodegradation of a rapidly degradable compound after the end of a study could

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be performed as a toxicity assessment.

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Regulatory implications. The prioritisation of chemicals based on hazards including

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persistency, bioaccumulation and toxicity (collectively termed PBT)6 has been advocated since

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the introduction of REACH in 2007. Of these, persistence/ biodegradation represents one of the

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greatest uncertainties and most sensitive parameters in chemical exposure models 35. Persistence

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is also a critical parameter in risk assessment: half-life combined with Kd are the two driving

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factors governing predicted environmental concentrations (PECs) in various media. There has

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been a significant drive towards increasing throughput and improving the reliability of

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bioaccumulation and toxicity tests

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required tests, as part of the replacement, reduction and refinement (3Rs) framework for humane

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animal research

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environment has received comparatively little attention, despite the need for it being highlighted

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16

10, 13, 34

. To assess the

, with a view to ultimately reducing the overall number of

. Developing methods to effectively prioritize chemical persistence in the


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in REACH documentation 5. Persistence is probably the cheapest and most straightforward of the

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hazards to characterise. Guidance recommends that chemicals should undergo persistence

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assessments before bioaccumulation and toxicity assessments to avoid unnecessary animal tests,

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and that the latter should be conducted following a chemical being assigned as potentially

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persistent, dependent upon the chemical’s properties36. However, it has been estimated that of the

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many chemicals which fail RBTs, up to 80% may be false negatives

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unnecessary bioaccumulation and toxicity tests.

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assessment would save upwards of 600 fish (Table S6 SI), and approximately $75K for every

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chemical reliably screened out earlier in the testing process37. At the current rate of failure, this

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equates to savings in the order of millions of fish and hundreds of millions of dollars.

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Such tests could also help fulfill the criteria espoused for green chemistry i.e. process

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optimisation and informed design to minimise exposure of the environment to hazardous

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substances 38. The accurate characterisation of chemicals liable to persist in the environment at

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an early stage of development has two benefits: (i) a cost-benefit opportunity to remove those

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chemicals which are unlikely to receive regulatory acceptance at an earlier stage of product

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development and (ii) prioritization of those chemicals which are designed to have a degree of

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persistence in order to perform effectively (e.g. certain pharmaceuticals which need to remain

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stable until they reach their intended point of action) for more intensive testing to determine their

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overall risk to the environment.

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Biodegradability and structure analysis. High throughput biodegradation tests may also enable

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the generation of extensive and reliable biodegradation half-life data that could be used to

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develop and validate in silico QSARs 39, 40. The use of QSAR methods in a regulatory capacity is

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at present fundamentally undermined by a lack of reliable and robust experimental data. We used

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, thereby resulting in

We estimate that accurate persistence


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the assay to demonstrate that there was a strong and statistically significant correlation with

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Hammett’s constant for substituents on position 3 (meta) of the phenol, indicating that

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biodegradability of phenols is influenced by the electrophilicity of the substituent group at this

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position. There was a similar but weaker relationship of the substituents on positions 2 (ortho)

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and 4 (para). This indicates that electronic effects on the 3 position are more pronounced than

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the 2 and 4 position, which are adjacent and opposite to the C1 hydroxyl group. It is interesting

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to note that for selected compounds, those with greater Gibbs free energy changes for rate

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controlling reactions had higher probabilities of degradation (4-HBA, phenol, 4-nitrophenol and

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4-chlorophenol; -251.97, -246.11, -245.61 and -207.39 kCal/mol respectively). There was thus a

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general trend for increased biodegradability as the electrophilicity of the substituted group

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decreased. The rank order of the effect of substituent groups agrees noticeably with previous

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work by Pitter et al. who showed a correlation between the Hammett constant and specific rates

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of degradation for phenolic compounds 30. Therefore, although the HT-BST is an end point, not a

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rate assay, it can be used as a good indicator of relative biodegradability.

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Future applications. The following limitations of the assay warrant further investigation: (i) the

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assay is only applicable to chemicals which can react with 4-NBTFB, although there are

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potential alternative colorimetric assays (e.g.25) which would benefit from further investigation;

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(ii) degradation products may interact with 4-NBTFB to form an azo-dye which may result in a

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false-negative assessment (although there was no evidence that this occurred); and (iii) the limit

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of detection is suitable for current standard OECD test concentrations and pass thresholds (i.e.

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the test has the capacity to resolve 70% degradation given a starting chemical concentration of

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10 mg C L-1), however, it may not be sufficiently sensitive for investigations into

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environmentally relevant chemical concentrations in the ng to µg L-1 range41. The assay is


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intended as an alternative to existing screening tests and is thus also limited by some of the

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general constraints of standard OECD RBTs. For example, this assay would not be suitable for

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volatile compounds or those with high log Kow and, in its current format, would not be suitable

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for accounting for co-metabolic processes. Modifications to existing screening studies proposed

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within REACH can also be applied to HT-BSTs to address some of these issues, however some

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compounds would still be problematic, as they are for existing screening studies. Similarly,

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potential inferences such as high DOC levels potentially impact OECD RBTs. There is a range

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of options currently prescribed within the guidelines to circumvent these issues, such as ageing

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the inocula, or washing to reduce background DOC levels1. The efficacy of these options could

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also be assessed for the HT-BST described. Modifications to the assay may allow the assessment

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of biodegradation under a range of different testing conditions, including the assessment of

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anaerobic biodegradability.

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Overall the HT-BST proposed here promises to be useful tool in the prioritization of persistent

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chemicals and for investigating the rules that govern the biodegradation rates of chemicals

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towards establishing reliable in silico methods of prioritization.

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ASSOCIATED CONTENT Supporting information. Plot of absorbance against chemical concentration for chemicals used to evaluate the 4-NBTFB colorimetric assay. RBT data for 4-nitrophenol and 4-flurophenol. Chemical structures and properties. Chemical persistence classification sources and summary. Table of coefficients of variation for replicate absorbance measurements for chemicals used to evaluate the 4-NBTFB colorimetric assay. A relative cost comparison between HT-BST and RBT. Estimated fish requirements for B and T assessments. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * Russell J. Davenport, School of Civil Engineering and Geosciences, Cassie Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, United Kingdom: [email protected]


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432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477

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Present Addresses ◊ Andrew K. Goodhead is presently affiliated with Promega Corporation, Southampton, UK. Author Contributions These authors contributed equally. All authors have given approval to the final version of the manuscript.

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Funding Sources Funding for this work was received from EPSRC (including a Challenging Engineering award, EP/I025782/1), RCUK (EP500501/1), BBSRC (BB/H530897/1) and AstraZeneca Global SHE Research Programme. ACKNOWLEDGEMENT This work represents an AstraZeneca Global Environment contribution to the Innovative Medicines Initiative (IMI) under grant agreement no. 115735 – iPiE: Intelligent led assessment of Pharmaceuticals in the Environment. ABBREVIATIONS 1-A-2-N-4 SA, 1-amino-2-naphthalene-4-sulphonic acid; 2-A-1-NSA, 2-amino-1-naphthalene sulfonic acid; 2-N, 2-naphthol; 3-R’s, replacement, reduction and refinement framework; 4-A-5H, 2, 7-NSA, 4-amino-5-hydroxy-2, 7-naphthalene sulfonic acid; 4-CP, 4-chlorophenol; 4-HBA, 4-hydroxybenzoic acid; 4-HQ, 4-hydroquinone; 4-NBTFB, 4-nitrobenzenediazonium tetrafluoroborate; 4-NP, 4-nitrophenol; AS, activated sludge; BSA, benzene sulphinic acid; BST, biodegradation screening test; DOC, dissolved organic carbon; HPLC, high performance liquid chromatography; HT-BST, high throughput biodegradation screening test; MPN, most probable number; MS, mass spectrometry; OECD, Organisation for Economic Co-operation and Development; P, phenol; PBT, persistent, bioaccumulatory and toxic; Pexp, experimentally derived probability of biodegradation; PQSAR, QSAR derived probability of biodegradation; QSAR, quantitative structure-activity relationship; RBT, ready biodegradability test; REACH, registration, evaluation, authorization and restriction of chemicals; vPvB, very persistent and very bioaccumulative REFERENCES 1. OECD, Guidelines for the testing of chemicals, section 3, degradation and accumulation. Test No. 301: Ready Biodegradability. 1992. 2. Vazquez-Rodriguez, G.; Goma, G.; Rols, J. L., Toward a standardization of the microbial inoculum for ready biodegradability testing of chemicals. Water Sci. Technol. 2000, 42, (5-6), 43-46. 3. Geiss, F.; Del Bino, G.; Belech, G.; Norager, O.; Orthmann, E.; Mosselmans, G.; Powell, J.; Roy, R.; Smyrniotis, T.; Town, W. G., The EINECS inventory of existing chemical substances on the EC market. Toxicol. Environ. Chem. 1992, 37, (1-2), 21-33. 4. Binetti, R.; Costamanga, F. M.; Marcello, I., Exponential growth of new chemicals and evolution of information relevant to risk control. Ann. Ist. Super. Sanita 2008, 44, (1), 13-15. 5. ECHA, Guidance on information requirements and chemical safety assessment. Chapter R.11: PBT assessment. Version 2.0. 2014. 6. ECHA REACH. http://echa.europa.eu/web/guest/regulations/reach (27/10/2013),


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a. Probability of degradation

1.0

0.8

0.6

Phenol 4-HBA 4-HQ

0.4

0.2

0.0

Maximum cell concentration in OECD RBT (301 A-F) 104

105

106

107

108

109

108

109

108

109

Cells/mL

b. Probability of degradation

1.0

0.8 4-CP 4-NP 2-N BSA

0.6

0.4

0.2

0.0

104

105

106

107

Cells/mL

c. Probability of degradation

1.0

0.8

0.6

2-A1-NSA 1-A2N-4 4-A5-H2,7-N

0.4

0.2

0.0

104

105

106

107

Cells/mL



Figure 1. Probability of degradation for the selected chemicals at different activated sludge inoculum concentrations, displayed as cells/mL. Chemicals are clustered into 3 groups: (a) readily biodegradable; (b) inherently biodegradable or variable reported biodegradation and (c) not readily biodegradable. These assessments were based on regulatory data accessed via eChem portal 22. The standard OECD 301 series inoculum concentrations are marked on the plot by the shaded area.


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104


OH Position 2 (ortho-) Position 3 (meta-)

log10 MPN/mL

103

Position 4 (para-)

Br NO2 Fl CH3O Cl CH3 OH COOH

102

101

100 Position 2 (ortho-)

Position 3 (meta-)

Position 4 (para-)

Figure 2. Most Probable Number (MPN) estimates of specific degrader abundance for phenolic compounds derived from the number of wells exhibiting a positive biodegradation outcome, volume contained within negative wells and the total volume contained within all wells. Error bars indicate the standard deviation, which was low for all compounds.



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4.0 COOH

3.5

R-Sq (adj) (adj) 0.579 R-Sq 57.9% yy = 2.723 1.239 = 2.723 – 1.239 xx

OH

log MPN

3.0 CH3

Cl

2.5 CH3O

Fl

2.0

1.5

NO2

Br

1.0 -0.5

0.0

0.5

1.0

1.5

Substituent constants

Figure 3. Regression analysis showing a strong and significant negative correlation (R2 = 0.58, P70 – 100% in 1-20 days) Not readily (6-24% in 14 days) Not readily (0-8% in 14-29 days) No data Low probability of degradation¥ Inherently (13-54% in 28 days) Inherently§ (28-100% in 414 days) Variable (0-100% in 7-28 days) Readily (76.5% in 4 days) Readily (70% in 14 days)


OECD Environmental Range Range

96 tests 28 days

20,000 tests 28 days ……..

Probability of degradation

One test 28 days

Ready (not P)

Inherent (not P)

Persistent (P) Increasing: inoculum concentration; environmental relevance; confidence in persistence assignment


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