Ecotoxicology and Environmental Safety 111 (2015) 9–22

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Ecotoxicology and Environmental Safety

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Review Refinement of biodegradation tests methodologies and the proposed utility of new microbial ecology techniques

Agnieszka Kowalczyk a,n, Timothy James Martin b, Oliver Richard Price c, Jason Richard Snape d, Roger Albert van Egmond c, Christopher James Finnegan c, Hendrik Schäfer a, Russell James Davenport b, Gary Douglas Bending a a School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom b School of Civil Engineering and Geosciences, Newcastle University, Newcastle upon Tyne NE1 7RU, United Kingdom c Unilever, Safety & Environmental Assurance Centre, Colworth Science Park, Sharnbrook MK441LQ, United Kingdom d AstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TF, United Kingdom article info abstract

Article history: Society's reliance upon chemicals over the last few decades has led to their increased production, Received 30 April 2014 application and release into the environment. Determination of chemical persistence is crucial for risk Received in revised form assessment and management of chemicals. Current established OECD biodegradation guidelines enable 22 September 2014 testing of chemicals under laboratory conditions but with an incomplete consideration of factors that Accepted 23 September 2014 can impact on chemical persistence in the environment. The suite of OECD biodegradation tests do not characterise microbial inoculum and often provide little insight into pathways of degradation. The Keywords: present review considers limitations with the current OECD biodegradation tests and highlights novel OECD tests scientific approaches to chemical fate studies. We demonstrate how the incorporation of molecular Biodegradation microbial ecology methods (i.e., ‘omics’) may improve the underlying mechanistic understanding of Persistence biodegradation processes, and enable better extrapolation of data from laboratory based test systems to Chemical risk assessment Microbial ecology the relevant environment, which would potentially improve chemical risk assessment and decision Omics making. We outline future challenges for relevant stakeholders to modernise OECD biodegradation tests and put the ‘bio’ back into biodegradation. & 2014 Elsevier Inc. All rights reserved.

Contents

1. Introduction...... 10 2. OECD biodegradation tests ...... 10 2.1. Historical aspects and principal design of OECD test...... 10 2.2. Overview of current tests ...... 11 2.3. Limitations of current tests ...... 13 2.3.1. Ready biodegradability tests ...... 13 2.3.2. Enhanced and modified screening tests within REACH ...... 16 2.3.3. Inherent biodegradability tests ...... 16 3. Environmental realism of tests ...... 17 3.1. Effect of environmental factors on biodegradability assessment...... 17 4. Microbial ecology in biodegradation testing ...... 17 4.1. Advantages and disadvantages of ‘omics’ ...... 19 5. Conclusions ...... 19

n Corresponding author. Present address: SC Johnson, Frimley Green, Frimley, Camberley, Surrey GU16 7AJ, United Kingdom. Tel.: þ44 7758572996. E-mail address: [email protected] (A. Kowalczyk). http://dx.doi.org/10.1016/j.ecoenv.2014.09.021 0147-6513/& 2014 Elsevier Inc. All rights reserved. 10 A. Kowalczyk et al. / Ecotoxicology and Environmental Safety 111 (2015) 9–22

Acknowledgements ...... 20 References...... 20

1. Introduction Since information on the fate and behaviour of chemicals is required to make an assessment of environmental exposure, it is Ever since Silent Spring (Carson, 1962) there has been wide- crucial that chemical fate is determined in an accurate manner and spread public concern about the use of chemicals and their more important to be predictive of degradation rates in the possible impact on the environment. Silent Spring facilitated the environment. However, so far the chemical industry, regulators ban of the pesticide DDT for agricultural use in 1972 in the United and academia have encountered difficulties with extrapolating States (US EPA, 1975; Grier, 1982) and the onset of greater data from standardised laboratory tests into the environment due regulatory and public interest in the environmental consequences to discrepancies between test conditions and the complexity of of chemicals. The beginning of the chemical regulatory era within environmental conditions. Studying microbial interactions and the EU can most likely be traced back to the demise of the their functions within inocula helps to link processes associated ‘principle of microbial infallibility’ which was based on the belief with chemical biodegradation at the community level. Such that, given the opportunity and favourable conditions, any organic knowledge can be essential to better understand chemical biode- chemical would biodegrade (Painter, 1974). Ironically, synthetic gradation in OECD tests and in the environment. Application of surfactants produced in the 1950s under the concept of ‘better microbial ecology methods in existing test systems can be used to living through chemistry’ (Copley et al., 2012) were found to optimise new test guidelines. They may also have the potential in produce unsightly foaming in conventional wastewater treatment helping to develop strategies to improve the reproducibility of plants, leading to the realisation that they were not being OECD tests. degraded during treatment. The 1950s and 1960s heralded an In this review we provide an overview of the current test era of biodegradability testing development centralised predomi- methods used to assess chemical biodegradation and discuss their nantly around the aerobic biodegradability of synthetic detergents limitations. A review of the current OECD tests is essential to (Allred et al., 1964; Bunch and Chambers, 1967). outline the key issues. e.g., microbial inocula, chemical concentra- As our awareness of environmental issues associated with the tion, biodegradation pass levels. It will enable stakeholders (aca- use of chemicals has increased over the past 40–50 years, so has the demia, industry and government) to understand the major scientific understanding and regulatory requirements. However, in concerns regarding current test results and identify knowledge parallel, the global chemical industry has grown rapidly since 1970. gaps/questions. New methods can then be proposed to address Global chemical output (produced and shipped) was valued at these questions. Finally, we provide a summary of technological US$171 billion in 1970. By 2010, it had grown to $4.12 trillion developments which could improve the capacity of biodegrada- (Davies, 2009). Pesticide use has increased worldwide by 36 fold in tion tests to provide more reliable predictions of biodegradation in the last 45 years (1960–2005) (Zhang et al., 2011). Global use of the complexity of real environments. pharmaceuticals led to 112% increase in prescription drug sales recorded between 2000 and 2008 (FDA, 2013) and the global use of home and personal care products has increased by 232% and 750% between 1998 and 2013, respectively (Euromonitor, 2013). These 2. OECD biodegradation tests trends are likely to continue for the foreseeable future, as growth in emerging markets in South America, Africa and Asia continues. 2.1. Historical aspects and principal design of OECD test Various regulations have been developed to assess the adverse impacts of existing and new chemicals in the environment. Hazard A range of methods for investigating biodegradation processes data are generated to assess the persistence, bioaccumulation and have been developed to predict the fate of chemicals in the toxicity of chemicals. Exposure models and emission scenarios environment. Most efforts have focused on the fate of chemicals have been developed to predict the distribution and transport of in the aquatic environment, especially in wastewater treatment chemicals in the environment. A key component of both hazard processes (Reuschenbach et al., 2003). Testing biodegradability determination (persistence) and environmental risk assessment is under laboratory conditions aims to obtain a reliable prediction of the accurate estimation of the biodegradation of a chemical in the the likelihood of the biodegradability of chemicals in the environ- environment. ment (Pagga, 1997). Over 30 years ago, in 1981, the OECD first The degree to which any individual chemical will partition and published its guidelines for testing the biodegradation of chemicals. persist in the environment is governed by a number of intrinsic There have been several amendments and additions since, includ- and extrinsic properties/factors. These include solubility/hydro- ing updated methods for assessing ready biodegradability in 1992 phobicity, octanol/water partition coefficient (Kow), soil organic (OECD, 1992) and introduction of the CO2 headspace test (OECD 310, carbon/water partition coefficient (Koc), dissociation constant 2006). In 1990, a classification in accordance with the OECD was (pKa), as well as characteristics of the environmental compartment proposed (OECD, 2005). Three groups of tests were defined: (e.g., organic carbon content, soil/sediment particle size and viable (1) ready biodegradability (or screening), (2) inherent biodegrad- number of bacteria). Microbial biodegradation of chemicals is the ability and (3) simulation (Lapertot and Pulgarin, 2006). A list of major process that affects chemical persistence in the environ- reference chemicals for use as positive and negative controls in ment (Copley, 2009). Although several OECD biodegradation test standardized biodegradability tests (Table 1) has been proposed by guidelines have been established to determine chemical fate under regulators and industry with an agreed set of properties and different environmental scenarios, the prediction of chemical characterised set of biodegradability behaviour, which cover a range fate is still challenging (e.g. biodegradation studies conducted of environmental persistence and non-persistence (Comber and at environmentally relevant concentrations, laboratory to field Holt, 2010). These chemicals group into bins (Table 1), which align extrapolation). with OECD tiered testing (Fig. 1) and show the relationships A. Kowalczyk et al. / Ecotoxicology and Environmental Safety 111 (2015) 9–22 11

Table 1 Reference chemicals and their classification into bins. Adapted from Comber and Holt (2010)

Bin Description Half-life Chemical example (days)

1 Reference chemicals which would normally pass a RBT or modified RBT test o15 Aniline, sodium benzoate, phenol 2 Reference chemicals that would normally pass an enhanced screening biodegradability test but currently fail 16–40 4-Chloroaniline, 4-fluorophenol, 1,2,3- any other tests trimethylbenzene 3 Reference chemicals that would normally fail any biodegradability screening test whether modified RBT or 41–60 o-Terphenyl, cyclodecane, enhanced screening biodegradability test dibutylphenol 4 Reference chemicals that should never pass a modified RBT or an enhanced biodegradability screening test 460 Hexachlorobenzene, benzo(a)pyrene, hexachlorohexane

RBT – ready biodegradability tests.

Fig. 1. Relationship between screening and higher tier biodegradability tests and the bins. Adapted from Comber and Holt (2010) between the screening and higher tier tests (Comber and Holt, tests (RBTs) are considered a stringent first tier and indicate if a 2010). chemical is rapidly degradable or not. Seven methods (Table 2) permit the screening of chemicals for ready biodegradability in an 2.2. Overview of current tests aerobic aqueous medium and they are based on the removal of organic chemicals measured as dissolved organic carbon (DOC) The choice of a test should depend on the testing purpose, and (OECD 301 A, OECD 301 E), the production of the catabolic end- under REACH, the testing is graded according to production level product carbon dioxide (OECD 301 B), the determination of the and properties of a chemical (ECHA, 2012). Ready biodegradability biochemical oxygen demand (BOD) (OECD 301C, OECD 301 D, 12 A. Kowalczyk et al. / Ecotoxicology and Environmental Safety 111 (2015) 9–22

Table 2 Overview of OECD biodegradation tests.

Biodegradation OECD Pass level Incubation Chemical Inoculum source Inoculum size Test test guideline conditions concentra- duration tion

Ready OECD 301 A 70% DOC removal Aerobic 10–40 mg Activated sludge, sewage r 30 mg/l settled sewage; 28 Days biodegrad- DOC/l effluents, surface waters, r100 ml effluent/l; approx. 7 8 ability tests OECD 301 B 60% ThCO2 10–20 mg soils or mixture of these 10 –10 cells/l (screening DOC/l tests) OECD 301 C 60% ThOD 100 mg/l Fresh samples from sewage 30 mg/l settled sewage; treatment works, industrial approx. 107–108 cells/l WWTPs, soils, lakes, seas, mixed thoroughly together OECD 301 D 2–10 mg/l or Derived from secondary r5mleffluent/l; approx. 5–10 mg effluent of WWTP or 104–106 cells/l ThOD/l laboratory-scale unit predominantly domestic sewage, alternatively surface water e.g., river, lake OECD 301 E 70% DOC removal 10–40 mg Derived from secondary 0.5 ml effluent/l; approx.. DOC/l effluent of WWTP or 105 cells/l laboratory-scale unit predominantly domestic sewage OECD 301 F 60% ThOD 100 mg/l or Activated sludge, sewage r30 mg/l settled 50–100 mg effluents, surface waters, sewage; r100 ml ThOD/l soils or mixture of these effluent/l; approx.. 107– 108 cells/l OECD 310 60% ThIC 20–40 mg C/l Activated sludge, sewage 4–30 mg SS/l or 10% v/v effluents, surface waters, secondary effluent soils or mixture of these

Inherent OECD 302 A 4 20% ThBOD, ThDOC Aerobic 2 –10 mg/l Mixed settled sludges after A high concentration of Not defined (potential) removal or ThCOD (primary two weeks aeration period aerobic micro-organisms biodegrad- OECD 302 Bbiodegradation; 420% Th 50–400 mg Activated sludge 0.2–1.0 g dry matter/l ability tests BOD, ThDOC removal or DOC/l OECD 302CThCOD (ultimate 30 mg/kg Activated sludge 100 mg/kg biodegradation)

Simulation OECD 303 A Estimation of half-lives for Aerobic/ 41–100 μg/l Activated sludge 2.5 g/l dry matter; 2–10 ml/l 120 Days tests materials exhibiting first- anaerobic effluent OECD 303 Border degradation patterns. Airborne inoculation, 1 ml/l of settled sewage In the absence of first-order settled sewage OECD 307kinetics degradation times Representative soil; a sandy Microbial biomass of at

for 50%, (DT50) and 90% loam or silty loam or loamy least 1% of total organic

(DT90) may be reported sand with a pH of 5.5–8.0, carbon organic carbon content of 0.5–2.5% OECD 308 Sediments from sampling Not defined sites selected based on the history of possible agricultural, industrial or domestic inputs to the catchment and the waters upstream OECD 309 Surface water from Not defined sampling sites selected based on the history of possible agricultural, industrial or domestic inputs OECD 314 Raw wastewater, activated Not defined (A–E) sludge, anaerobic digester sludge, treated effluent- surface water mix, untreated effluent-surface water mix,

DOC – dissolved organic carbon; ThBOD – theoretical biochemical oxygen demand; ThCOD – theoretical chemical oxygen demand; ThCO2 – theoretical carbon dioxide; ThDOC – theoretical dissolved organic carbon; ThIC – theoretical inorganic carbon; ThOD – theoretical oxygen demand; WWTP – wastewater treatment plant.

OECD 301 F) (Reuschenbach et al., 2003; OECD, 1993) and by or by non-specific analysis (ultimate biodegradation). Inherent biode- measuring the inorganic carbon (IC) produced in the test bottles gradability tests include Modified SCAS Test (OECD 302 A, 1981), (OECD 310, 2006). Zahn-Wellens/EMPA Test (OECD 302 B, 1992)andModified MITI Test The second tier tests are inherent tests where inherent biodegrad- (II) (OECD 302C, 1981)(Table 2). The test procedure offers a higher ability can be measured by specific analysis (primary biodegradation) chance of detecting biodegradation compared to tests for ready A. Kowalczyk et al. / Ecotoxicology and Environmental Safety 111 (2015) 9–22 13 biodegradability and therefore, if an inherent test is negative this some tests may reduce the probability of incorporating competent could indicate a potential for environmental persistence (Comber and degraders; the artificial mineral media used in standard tests may Holt, 2010). The highest tier tests are simulation tests (Table 2)which not be suitable for supporting the growth and establishment of aim at assessing the rate and extent of biodegradation in a laboratory specific degraders. False negatives are thought to account for system designed to represent either the aerobic treatment stage of a 20–80% of RBT fails (ECETOC, 2007) and have been recognised as wastewater treatment plant (WWTP) or environmental compart- a limitation since the introduction of the tests (ECETOC, 1983). It is ments, such as fresh or marine surface water. difficult to accurately qualify the occurrence of false negatives with The majority of OECD tests are conducted under environmen- data as this information is not readily released by industry, tally unrealistic conditions e.g., the chemicals are tested at high however the variation within and between tests has previously concentrations that are unlikely to occur in the environment. been reported in academic literature. In addition, the test volumes vary from test to test, there is lack The high-levels of variation exhibited in RBTs as a result of the of consideration of the inoculum quantity or quality, and protocols previously mentioned factors were demonstrated by Gerike and regarding inoculum preparation prior to biodegradability testing Fischer (1979) who conducted a thorough assessment of pre-OECD also vary. Furthermore the tests are conducted under standardized 301 RBTs (recommendations from which were incorporated into the incubation conditions, which do not reflect highly variable envir- OECD 301 series) and reported significant differences in biodegrada- onmental conditions, for example seasonality but do allow ease tion outcome dependent upon the selected method. In the case of and conservatism in regulatory risk assessment. Other limitations 4-nitrophenol, biodegradation ranged from 0% to 100% dependent involve lack of consistency in the pass levels and test duration. upon the chosen method (Gerike and Fischer, 1979). This was supported by Thouand et al. (1995) who developed a probabilistic model which calculated a probability of degradation for 4-nitrophenol 2.3. Limitations of current tests ranging from 0% to 99% depending upon the method selected. Nyholm et al. (1992) reported that screening tests underestimated In order to provide reliable degradation data for predicting biodegradability for 4-chloroaniline, desmethyl-methyl parathion chemical fate, biodegradation tests should aim to mimic real and in some cases 4-nitrophenol, where extensive lag phases environmental exposure and represent small-scale equivalents of resulted in false negative test fails. These compounds provided a real environments. It should be noted that biodegradation tests positive biodegradation outcome in simulation tests (Nyholm et al., only provide some of the information needed to determine 1992). Žgajnar Gotvajn and Zagorc-Končan (2003) reported a test fail environmental fate of chemicals. The physico-chemical conditions for diethylene glycol in a closed bottle test (OECD 301 D) but a test and the nature of the microbial populations present in the pass in a respirometric test (OECD 301 F). In comparing two different environment are difficult to achieve in biodegradation tests and respirometric test systems, Reuschenbach et al. (2003) reported the may never be truly realised, particularly given the probability of potential for the same test to return conflicting degradation data adequately obtaining relatively rare organisms representatively in dependent upon the test system applied. A thorough literature small samples. review conducted by Comber and Holt (2010),reportederrant behaviour in standard tests and significant variations in reported 2.3.1. Ready biodegradability tests half-lives in non-standard tests for a range of compounds including RBTs are primary screening tests which have historically been 4-nitrophenol, 4-chloraniline and 4-fluorophenol. the central foundation for assessing the biodegradation of chemi- In contrast with some of the fixed test variables such as test cals in regulatory frameworks. This can be attributed to their ease duration, the biological criteria suffer from a lack of adequate of use, relatively straightforward interpretation and the amount of characterisation. Variations in the source, concentration and pre- data collated since their introduction (Aronson et al., 2006). These treatment of the inoculum have previously been shown to impact highly prescribed, standardised, and conservative regulatory tests upon the diversity of test inocula and their ability to degrade measure the relative biodegradability of chemicals. They typically chemicals (Goodhead et al., 2014), which can add to the source of measure ultimate biodegradation (the biodegradation of the variability and large number of false negatives associated with chemical to carbon dioxide, biomass, water and other inorganic RBTs (Vázquez-Rodríguez et al., 2011). The potential to produce more chemicals) but can be used to measure the primary biodegrada- reliable and reproducible tests by increasing research efforts sur- tion. A positive result in a RBT is defined as a 60% reduction in rounding the test inoculum has been identified in workshops theoretical oxygen demand (ThOD) or theoretical CO2 evolution sponsored by the European Centre for Ecotoxicology and Toxicology (ThCO2) or a 70% reduction in DOC within the 10-day window of Chemicals (ECETOC) (ECETOC, 2003, 2007). (beginning at 10% degradation) of a 28-day test. Fulfillment of these criteria can be considered as indicative of rapid and ultimate biodegradation in the environment (OECD, 2005). However, RBTs 2.3.1.1. Inoculum source, concentration and preparation. Acentral typically exhibit high levels of variation including: inter-replicate assumption in all freshwater biodegradability tests is that a variation, inter-test variation, inter-facility variation and temporal randomly selected sample from an environmental compartment will variation (Nyholm et al., 1984; Painter, 1995). As a result, they exhibit the diversity required to provide the requisite range of produce a large number of test fails, many of which may be microorganisms for assessing the biodegradation potential of a considered false negatives, whereby a chemical fails a test not particular chemical in any given environmental system. Notwithstan- necessarily due to poor biodegradability but rather because it fails ding a historical understanding of the importance microbes have in the test design. Test failure (i.e. insufficient degradation of the test biodegradation (Sweeney and Foote, 1964), the outcome is presently compound within the 10-day window of a 28-day test), may be dependent upon the potential of an arbitrarily selected mixed indicative of the persistent nature of the test compound, but it can community of relatively low concentration to degrade a chemical also occur as a result of a number of different factors: there may under standardised conditions. Typically, biodegradation is viewed only be partial degradation (less than 60% ThOD/ThCO2 or less with respect to the intrinsic properties of the chemical and rarely from than 70% DOC removal); there may be sufficient degradation but the perspective of the bacteria, namely the probability of encountering not within the confines of the 10-day window of a 28-day test; the specific degraders of the test chemical within the test inocula. chemical may have been introduced at an initial concentration OECD guidelines stipulate that inocula may be sourced from: toxic to the microbial inocula; the low inocula concentrations in activated sludge, sewage effluent, surface waters, soils, a mixture 14 A. Kowalczyk et al. / Ecotoxicology and Environmental Safety 111 (2015) 9–22 of the aforementioned, rivers, a 10 site composite sample or of chemicals in WWTPs or in the environment are usually in the derived from secondary effluent at varying concentrations. There- range of ng/l to μg/l (Ahtiainen et al., 2003; Kolpin et al., 2002). fore, OECD screening tests can involve different inocula cell Extrapolations using data from such tests at high concentrations densities which can affect chemical biodegradation. For example, may not reflect what takes place at low concentrations (Lapertot in activated sludge and river water the number of specific and Pulgarin, 2006). Toräng et al. (2003) demonstrated that direct degraders may vary from 3.7 106 to 2.5 104 cells/l, respectively use of the biodegradation rate data obtained for mecoprop and 2,4- (Thouand et al., 1995, 2011). The modified French Association dichlorophenoxyacetic acid at high (4100 μg/l)concentrationinan for Standardization (AFNOR) method specified use of 5 102 cells/l aerobic aquifer, grossly overestimated the actual degradation rates at in test assays and the modified Sturm test suggested environmentally-relevant concentrations, which were in the order of 10 2 102 cfu/l (Painter, 1995). It was reported by Thouand 0.1 μg/l. However, Kern et al. (2010) demonstrated that laboratory et al. (1996) that only 105 degrader cells/l are needed for a 99.9% biodegradation rates of pharmaceuticals and biocides and formation chance of biodegradation of 4-nitrophenol. Hence, number of cells of their transformation products reflected their removal rates during can vary by four orders of magnitude between tests. In their activated sludge treatment. current form, screening tests can be considered as much a test of Concentration of the chemical is a significant factor affecting its the inoculum as a test of chemical biodegradability. The variation behaviour in the environment and therefore, its susceptibility to in inoculum concentration also results in significant differences in microbial attack (Efroymson and Alexander, 1995), which is food to microorganism ratios (F:M), which may impact upon defined by the Michaelis–Menten kinetics of enzymatic reactions degradation kinetics: where the substrate concentration is high (Chou and Talaly, 1977). Chemicals at low concentrations do not and the microbial biomass low, there is more potential for growth serve as primary substrates but are more likely to be degraded as linked biodegradation and higher rates of CO2 evolution; as the secondary (non-growth) substrates concurrently with a variety of substrate concentration decreases and microbial biomass levels naturally occurring carbonaceous chemicals (Berg and Nyholm, increase, biodegradation kinetics change with typically less 1996). Presence of organic substrates and nutrients in those growth and thus less CO2 evolution which can impact upon the environments might lead to preferential degradation or outcome of the test (Blok and Booy, 1984; Blok et al., 1985). co-metabolism of other chemicals and lower biodegradation rates Adaptation is defined as a change in the microbial community of the primary chemical (de Lipthay et al., 2007; Markiewicz et al., that increases the rate of transformation of a test chemical as a 2011). Furthermore, presence of some chemicals may cause an result of a prior exposure to the test chemical and plays a key role inhibitory effect on microbial populations. Stasinakis et al. (2008) in chemical biodegradation (Spain and van Veld, 1983). Pretreat- reported the toxic effect of triclosan (TCS) and nonylphenol (NP) ment or preadaptation of the inocula is not permitted under OECD on activated sludge heterotrophic and autotrophic microorgan- guidelines. However, preconditioning of the inocula is permitted, isms, and a possible risk for deterioration of nitrification in including filtration, centrifugation, settling and decantation and activated sludge systems due to the presence of TCS at unrealis- aeration. These treatments have been shown to normalise tically high concentrations. observed degradation potential characteristics but reduce the Chemical concentration affects biodegradation kinetics. For efficacy of the inocula to degrade even readily biodegradable test example, Berg and Nyholm (1996) found that the percentage of chemicals (Vázquez-Rodríguez et al., 2007). This is probably due to aniline, 4-chloroaniline and pentachlorophenol removed by una- the removal of selected members of the microbial communities, dapted sludge had a tendency to be higher when the test chemical reducing overall diversity and leading to the exclusion of rare was dosed at trace concentrations (10 μg/l), than at standard specific degrader organisms (Goodhead et al., 2014) (high) concentrations (20 mg DOC/l). In contrast, degradation of Degradability and persistence are often thought of as proper- other chemicals (halobenzoate, nitroacetic acid, 2,4-dichlorophe- ties intrinsic to the chemical in question. In fact they are complex nol and isopropyl N-phenylcarbamate) can be limited at low properties influenced by a number of factors, of which the concentrations (Pitoi et al., 2011). The biodegradation of different propensity of a given chemical structure to biodegrade is only chemicals at low concentration varies, possibly due to the different one. Understanding the relationship between microbial diversity concentrations needed for the specific enzymes and chemical and biodegradation outcome, the occurrence and abundance of biodegradation pathways to be induced in bacterial cells (Grady, competent degraders and the bacterial variation between inocula 1984). will ultimately facilitate the design of more robust screening Extents and rates of microbial adaptation, important for biode- studies and enable better predictions of biodegradation. This can gradation behaviour, can also be greatly influenced by the chemi- be achieved by inoculum characterisation (e.g. cell density, micro- cal concentration. Low concentrations can be expected to increase bial community composition, functional diversity and biodegrada- the period needed for adaptation in comparison with higher tion potential) and inoculum standardisation (e.g. filtration or concentrations (Berg and Nyholm, 1996). Subsequently, many centrifugation, in order to obtain a standardized required organic chemicals persist in the environment. The reason for this inoculum size). could be that time is needed for the population of degraders to become sufficiently large or active to bring about chemical degradation or for microorganisms to synthesise and/or activate 2.3.1.2. Test chemical concentration. There is a growing focus on the the required enzymes for biodegradation (Grady, 1984). In some problem of extrapolation of biodegradability data obtained from studies, concentration thresholds have been reported below which standard laboratory tests to conditions observed in the field. OECD no adaptation could be detected. Efroymson and Alexander (1995) screening and inherent tests assess rates of biodegradation at reported a lack of phenanthrene mineralisation in a mixed culture chemical concentrations generally far in excess of those likely to at concentrations of 0.6–20 μg/l. be found in natural ecosystems (Lapertot and Pulgarin, 2006). Simulation tests for biodegradation of chemicals in surface water systems provide single scenario information about biodegradation 2.3.1.3. Inoculum. According to Thouand et al. (1996) at least three rates, and adaptation behaviour at environmentally realistic parameters must be controlled when running a biodegradation concentrations (at least below 100 μg/l and normally at or below test. These are the physico-chemical conditions (pH, temperature, 10 μg/l) (Comber and Holt, 2010). In most screening tests oxygen, concentration of mineral elements), the concentration of 10–400 mg of carbon per litre is applied, while the concentration the chemical to be tested, and the concentration of the bacterial A. Kowalczyk et al. / Ecotoxicology and Environmental Safety 111 (2015) 9–22 15 inoculum. While it is possible to reproduce the first two parameters in had a significant participation in the biodegradation capacity of biodegradation tests, the inoculum represents a significant diazinon-contaminated river ecosystems (Tien et al., 2011). Natural challenge for the reproducibility of test results. Microbial inocula river biofilms developed on ceramic disks showed 99.9% removal used in OECD biodegradation tests are one of the most poorly of diazinon at an initial concentration of 5 mg/l, over 26 days, in controlled variables. High variability in the biodegradability of the laboratory tests. Since biodegradation of many groups of chemicals in OECD tests is potentially due to the characteristics of chemicals requires relatively rare species to be present in the the inocula (presence or absence of biodegraders, adaptation to the tested inoculum (Thouand et al., 2011) biofilms have potential as chemical, cell density, and presence of protozoa) which control both an inoculum since they consist of complex bacterial communities, lag phase and biodegradation kinetics, which may lead to which represent natural microbial populations. problematic interpretation (Thouand et al., 1995). Unfortunately, Characterisation of microbial diversity within biofilms and its bacterial density estimation methods are rarely applied and so far changes after exposure to organic chemicals is crucial to under- there are no practical methods/protocols to measure inoculum stand the effects of chemicals on aquatic ecosystem. For instance, diversity in industrial laboratories (van Ginkel et al., 1995; Yergeau et al. (2012) determined the active community composi- Thouand et al., 2011). Therefore, there is a need for other tion within river biofilms and subtle changes in microbial com- techniques that should be used in parallel biodegradation testing munities were detected after short-term exposure to low to determine not only the overall size of inoculum but also its concentrations (0.5–1.0 μg/l) of the antibiotics erythromycin, sul- quality (number of specific degraders) and the diversity of famethoxazole and sulfamethazine. functional communities involved in biodegradation processes. Biodegradability tests may involve inocula from different ori- 2.3.1.4. Test volume. If biodegradation does occur, it is generally gins such as: river water, sea water, activated sludge and soil presumed that the bacteria responsible are rare organisms with a (Thouand et al., 2011). Since inoculum diversity is related to its minimum abundance of one competent degrader in the initial origin it should be taken into account while running tests. For inoculum. This presumption inherently concerns degradation by example, Forney et al. (2001) demonstrated that the diversity of specific degraders capable of metabolising the chemical in several activated sludges differed worldwide. This could reflect question and may not be true if, for example, co-metabolism of differences in environmental factors such as temperature, humid- the chemical as a non-growth substrate occurs first, yielding an ity and characteristics of wastewaters received by the WWTPs, but intermediate more amenable to common degradation pathways may also represent differences in the global biogeographical (Horvath, 1972). In using low density inocula, independent of inocula distribution of microbial taxa (Prosser et al., 2007). Moreover, test volumes, the probability of encountering rare organisms, even in specific degraders for non-readily degradable chemicals are more replicate samples, is reduced and their inclusion is a matter of likely to be found in the sediment of rivers and coastal areas than chance (Thouand et al., 1995; Ingerslev and Nyholm, 2000; Ingerslev in the water, whereas samples from pristine areas contain very et al., 2000). RBTs are performed typically at low volumes, which small numbers of specific degraders (Thouand et al., 2011). increases the likelihood of variation in microbial composition Franklin et al. (2000) reported higher cell numbers and microbial between tests, ultimately impacting upon the reproducibility of the diversity in contaminated zones of ground water than in pristine biodegradation outcome within and between facilities. This areas. Additionally, pristine aquifers are carbon-limited environ- phenomenon refers to the decreasing probability of encountering ments, with bacterial populations in a state of starvation and specific degraders of the test chemical within the test inocula, with reduced activity (Goldscheider et al., 2006). An inoculum with decreasing test volume, or decreasing inocula concentration high species diversity has a greater chance to contain degrading (Nyholm et al., 1992). species and therefore, to degrade test chemicals in comparison with less diverse inoculum. Margesin et al. (2003) reported that contaminated environments harbour a wide range of degrading 2.3.1.5. Test duration. Even from early development there appear microorganisms, which play a crucial role in the bioremediation to have been concerns over the robustness of RBTs with respect to processes. the arbitrary test duration assigned during development (Painter, 2.3.1.3.1. Biofilm as inoculum. There are several reasons why 1995). RBTs proceed for 28 days. In addition to passing the required biofilms should be considered in chemical fate studies (screening extent of biodegradation in this period, a ‘readily biodegradable’ and simulation studies). They are heterogenic, comprising com- chemical must also degrade at least as fast as the minimum rate plex communities of algae, cyanobacteria and bacteria embedded required to satisfy the 10-day window (see below). Painter (1995) in a polysaccharide matrix attached to substrates such as rocks, argued that the test duration might have been adopted due to sediments and aquatic plant surfaces (Vinten et al., 2011). Biofilm concerns regarding reductions in analytical sensitivity beyond 28 accumulation in riverbeds or on suspended particles contributes days, potentially impacting upon data reliability. Advancements in largely to the removal of chemicals from the water, and thus the analytical sensitivity since RBT introduction, however, would almost biofilm enhances self-purification of surface water (Muia et al., certainly negate this concern. The 28 day test duration has been 2003; Edwards et al., 1990). utilised since the early development of biodegradability testing in In rivers and streams, biofilms are the first to interact with the 1960s and it is possible that this time period has been retained dissolved chemicals such as nutrients, organic matter, and chemi- because: it has been used previously; there is a plethora of data cals. Biofilms integrate the effects of environmental conditions testing at this time point; and until recently (ECETOC, 2003, 2007)a over extended periods of time, mainly because of their rapid scientific case for change has not been presented. growth, species richness, and the physiological variety of the The ‘10-day window’ opens at 10% degradation, a time point organisms of which they are formed. They have been used for thought to indicate the cessation of the lag phase. Painter (1995) routine monitoring and could be useful as ‘early warning systems’ suggested the window may exist in reference to the 7–10 days of ecosystem disturbances (Sabater et al., 2007). Brümmer et al. typically required for primary standard degradation in the original (2000) observed stability of river biofilm community structure in OECD guidelines for assessing biodegradability of anionic surfac- spite of significant variation in chemical and biological parameters tants. Following their introduction, little research was conducted during the year. Araya et al. (2003) demonstrated the important regarding 10-day windows and few studies utilised them during contribution of natural river biofilm communities to the river their analysis at the time, making it difficult to assess their die-away test conducted under laboratory conditions. Biofilms necessity. 16 A. Kowalczyk et al. / Ecotoxicology and Environmental Safety 111 (2015) 9–22

The 28 day test duration and 10-day window restrict the time screening studies have been identified (ECHA, 2008). Modified frame within which to observe the growth of specific competent ready biodegradability tests incorporate modifications to RBTs degraders. The relatively high chemical concentration combined aimed to address issues such as toxicity and bio-availability. with the low microbial biomass levels stipulated in some tests Proposed modifications to screening tests include the use of selects for degraders which proliferate rapidly and/or have a biosurfactants or humic acid for testing chemicals with poor water sufficiently dense population in the original sample (Thouand solubility and testing at lower substrate concentrations to circum- et al., 2011). Extending the test duration beyond 28 days in vent toxicity issues. Providing the other RBT criteria are fulfilled, persistency assessments takes into account the extensive lag they can still be used to assess ready biodegradability, whereas phases exhibited during the testing of some compounds and enhanced biodegradation screening tests should be used to assist makes allowances for poorly water soluble substances (ECHA, in persistency assessments and not as an indication of ready 2012). The need to extend screening tests beyond 28 days to biodegradability. The proposed enhancements were selected to improve chemical assessments and characterisation of persistency improve the environmental relevance of biodegradability assess- has been recognised within REACH and proposed as an enhanced ments and reduce the volume of simulation studies required method (ECHA, 2008). (ECHA, 2008). The ultimate aim is the design of a test which lies A chemical exhibiting a slower rate of degradation, for example somewhere between the more stringent primary screening studies degrading in 60 days compared to 28 days, may pose a higher risk (RBTs) and the more expensive (both fiscally and with respect to to human and environmental health, considering the impact of resources) higher tier tests (Comber and Holt, 2010). Recom- chronic, low-level chemical exposure, the effects of which are not mended enhancements focus on (1) increasing study length fully understood (Kümmerer, 2010). However, to date there is no beyond 28 days, (2) use of semi-continuous test systems, evidence to support the idea that a chemical which undergoes (3) increased vessel size and inoculum density, with environmen- complete mineralisation in a longer test is more likely to pose a tal samples not previously exposed to the test chemical and more serious threat to the environment than a chemical which (4) running two ready tests in series (ECETOC, 2007; Comber fulfils current RBT pass criteria. and Holt, 2010). The modifications and enhancements proposed RBTs are not designed to be representative of the environment, under REACH address a number of the commonly discussed they are in fact deliberately not environmentally realistic, however reasons for RBT variability and false negatives. The ultimate aim the arbitrary test durations, particularly the 10-day window, do of the amendments is to provide a more environmentally relevant not provide a full account of degradation in the environment and inoculum, which is particularly addressed by those strategies ultimately lead to more expensive, intensive testing. which ensure a more representative diversity (e.g. increasing inoculum concentration and test volume), which will allow better predictions of persistency (ECHA, 2008). 2.3.1.6. Pass threshold. A chemical is classified as readily biodegrad- able if one of the following requirements is fulfilled: a 60% reduction in ThOD (theoretical oxygen demand) or production of 2.3.3. Inherent biodegradability tests ThCO (theoretical CO ), or a 70% reduction in DOC. A ring test 2 2 The predominant focus of this critique, and of proposed performed shortly after the initial introduction of the 301 series enhancements to existing studies, is on screening tests and suggested that whilst the high DOC removal rate was justified, there particularly RBTs. In addition to their reduced complexity, in was evidence to support a reduction in ThOD and ThCO from 60% 2 comparison with inherent biodegradability studies and simulation to reflect that part of the test chemical carbon will be utilised in tests, and their ease of interpretation, RBTs are also those tests that new cell synthesis. The study found in some cases that a ThOD of most testing facilities will predominantly perform. The majority of less than 60% correlated with a DOC removal of greater than 90% experimental data available for ‘tested’ chemicals is in the form of (Painter and King, 1985). The ring test led to some modifications RBT assessments. According to the eChem portal (OECD, 2013), a which were implemented in the modified OECD guidelines (OECD, joint collaboration between the OECD and the European Chemicals 1992) but the initial pass thresholds were maintained, presumably Agency (ECHA) which collates chemical data from multiple to maintain high stringency of the tests (Weytjens et al., 1994). sources and databases, more than four times as many RBTs have Arguably a more relevant biodegradation measure would be the been performed as inherent tests when assessing chemical biode- half-life (time point at which 50% degradation is reached). Recently, gradability (4604 and 1013 respectively). environmental concerns have developed at a faster rate than these Inherent biodegradability tests are less stringent than RBTs and international standards, with a regulatory shift from screening out as such the test parameters are more relaxed. They are designed to chemicals which undergo rapid mineralisation in all environments identify chemicals which, given the opportunity, show an inherent to identifying chemicals which persist in the environment, a quality potential for degradation. Despite this difference, many of the typically assigned based on biodegradation half-lives (ECETOC, drawbacks associated with RBTs are still applicable to inherent 2007). Although screening tests are not effective as screens for tests, particularly with respect to the inoculum and the test persistence and were not originally designed to assign half-lives for duration. The inoculum is still subject to preparation methods chemicals, they are increasingly being used to do so in light of this which are thought to reduce diversity, although not as much as in shift (Aronson et al., 2006). RBTs (Thouand et al., 2011). Inocula concentrations remain vari- able between tests with no characterisation of the inoculum or 2.3.2. Enhanced and modified screening tests within REACH concentration in the majority of inherent tests. The positive aspect For the aforementioned reasons, RBTs are highly variable and with regards to the inoculum in the OECD 302 A guideline is the often produce false negative results. They are also unsuitable, in increased environmental relevance gained in some tests by using their current guise, for making assessments of persistency. Persis- inocula directly from source, rather than using unrealistically low tency is difficult to measure as it is not an intrinsic property of the microbial concentrations (in RBT/screening tests) in diluted chemical per se. It is typically inferred based on a continued inocula or artificially amended systems. concentration in the environment and a lack of observed degrada- The 302 B Zahn-Wellens inherent test is subject to the arbitrarily tion data in laboratory studies. One of the aims within REACH is to assigned 28 day test duration. However, the 302 A and 302C are effectively assess persistence for use in prioritisation, and to this given varying test durations dependent upon experimental observa- end a number of enhancements and modifications to existing tions which provides a more reflective representation of behaviour in A. Kowalczyk et al. / Ecotoxicology and Environmental Safety 111 (2015) 9–22 17 the environment. There also remain concerns, for inherent tests and (Borde et al., 2003; Li et al., 2005) due to growth of phototrophic RBTs alike, over issues such as the varying manner of endpoint organisms capable of chemical biodegradation (Roldán et al., 1998; analysis and the suitability of the artificial mineral media for Lima et al., 2003; Thomas and Hand, 2011, Davies et al., 2013). supporting the establishment and growth of competent degrader The environmental factors can have both positive and negative communities. effects on degradation rate. For instance, Eriksson et al. (2002) reported that lower temperature severely limited the biodegrada- tion of PAHs under aerobic conditions. However, Lartiges and 3. Environmental realism of tests Garrigues (1995) observed different effect of temperature on biodegradation kinetics of pesticides in different types of water There are OECD tests simulating freshwater aquatic (OECD 309, (sea water, river water, filtered river water). For example, they 2004; OECD 314, 2008) and terrestrial compartments (OECD 307, reported small influence of temperature on triazine biodegrada- 2002), based on static, semi-continuous, or continuous operation tion contrary to the carbamate carbaryl. Temperature is also an under aerobic or anaerobic conditions with different test media. important environmental variable which affects bacterial growth The test conditions are standardized as far as possible and attempt and biomass (White et al., 1991). Temperature has a strong to reflect the environmental reality of biodegradation processes. influence on the period of acclimation of microorganisms and on Although, simulation tests are applicable for use in higher tier the rate of biodegradation (Kang and Kondo, 2002; Manzano et al., biodegradation test, their complex test design, long test duration 1999) therefore, it may affect the outcome of biodegradation tests. and high costs, make them unsuitable as routine tests (Merrettig- pH may vary widely (from pH o3to49) within and between Bruns and Jelen, 2009). environmental compartments and may alter the toxicity of che- micals (Rutgers et al., 1998; Araoye, 2009) and affect the biode- 3.1. Effect of environmental factors on biodegradability assessment gradation rate (Li et al., 2008). Furthermore, chemicals can be differentially mineralised in aerobic and/or anaerobic environ- Screening tests often underestimate the potential of chemical mental compartments (Boyd et al., 1983; Battersby and Wilson, degradation in environmental systems (Guhl and Steber, 2006). 1989). In natural river systems attenuation of chemicals may be This could be due to a variety of factors, which differ between lab influenced by sediment dynamics and flow velocity which deter- and natural environments for instance, pH, light/dark cycle, mine contact of chemicals with bacteria on the sediment surface oxygen, flow rate of water body and river bed form. This section (Kunkel and Radke, 2008). The size and structure of biofilm is also is not comprehensive and Table 3 presents only some examples of affected by flow shear stress (Lau and Liu, 1993; Battin et al., 2003). environmental factors that affect chemical biodegradation in the environment, and which are not fully covered by OECD tests that are conducted under standardized laboratory conditions. Current 4. Microbial ecology in biodegradation testing OECD tests are not considered as environmentally realistic. For example, light conditions are excluded from biodegradation tests Biodegradation tests play the key role in chemical persistence to avoid the growth of algae, which may affect the biodegradation assessment upon which environmental risk assessment is per- of tested chemicals and/or make the interpretation of CO2 data formed. Current OECD tests have been designed in the manner difficult due to fixation of the CO2. Interestingly, some authors that does not consider microbial inocula and there is little under- have reported that light enhances biodegradation of chemicals standing of chemical breakdown pathways and how they relate to

Table 3 Examples of environmental factors that affect chemical biodegradation.

Variables Test chemical Degradation testþresults Reference

pH 4-Nitrophenol Degradation rate affected by pH, optimum pH¼8–9 Li et al. (2008) Pentachlorophenol (PCP) Toxicity and degradation of PCP is affected by pH Rutgers et al. (1998) Hexachlorocyclohexane Optimum conditions were found for biodegradation at pH¼8 and T¼30 1C Siddique et al. (2002)

Temperature Bisphenol A Longer biodegradation at 20 1C than at 30 1C with half-lives from 4–7 days to Kang and Kondo (2002) 2–6 days, respectively Nonylphenol polyethoxylate Biodegradation varied from 68% at 7 1C to 96% at 25 1C Manzano et al. (1999)

Light 4-Nitrophenol Light degradation (indirect photolysis) of para-nitrophenol by Rhodobacter Roldán et al. (1998) capsulatus 4-Nitrophenol Total biodegradation of para-nitrophenol by microalgae within 5 days Lima et al. (2003) 4-Nitrophenol Photocatalytic degradation of para-nitrophenol (44–80%) on nanometre size Li et al. (2005) titanium plates Salicylate, phenol, phenanthrene Photosynthesis-enhanced biodegradation of aromatic chemicals by algal– Borde et al. (2003) bacterial microcosms 2,4-Dinitrophenol (2,4-D) Total biodegradation of 2,4-D with mixed culture of phototrophic Hirooka et al. (2006) microorganisms in the dark. Other conditions caused accumulation of metabolite 2-amino-4-nitrophenol (2-ANP)

Oxygen Phenol Anaerobic degradation in digested sludge (majority monosubstituted Boyd et al. (1983) Phenol, benzoates, phthalic acid phenols degraded) Battersby and Wilson esters, pesticides, homocyclic and Anaerobic degradation of pyridine and quinoline.1- and 2-naphthol and (1989) heterocyclic ring chemicals, anthraquinone had inhibitory effect on digesting sludge monosubstituted benzenes 4-Nitrophenol Aerobic degradation of para-nitrophenol within 11 h by activated sludge Bhatti et al. (2002)

Bed form Diclofenac, bezafibrate, ibuprofen, Flow velocity and sediment dynamics (water–sediment interactions; flat Kunkel and Radke (2008) naproxen, gemfibrozil sediment vs. moving sediment) affected attenuation of acidic pharmaceuticals. Majority degraded within 2.5–18.6 days while gemfibrozil degraded after 10.5 days 18 A. Kowalczyk et al. / Ecotoxicology and Environmental Safety 111 (2015) 9–22 the complexity and dynamism/variability of environmental com- within inocula. Genetic potential for chemical biodegradation can be partments e.g., flowing rivers. Microbial community characterisa- determined in an inoculum by analysing genes that are expressed by tion helps to better understand the microbiology of samples that microbial populations during biodegradation of chemicals. Presence are used as inocula in biodegradability testing and their potential of specific genes encoding enzymes responsible for breakdown of to degrade a given chemical. This information could be used to chemicals indicates microbial potential to chemical biodegradation. understand the basis of a given test result, such as a test failure, as This can enable comparison of chemical biodegradability studies and well as for extrapolation of laboratory test results into the prediction of chemical fate. Application of microbial ecology methods environment from which the inoculum is collected. Optimisation also provides the opportunity to study chemical biodegradation of microbial inocula could also help to reduce the costs of fate pathways and to gain more insights into complex biodegradation studies and risk assessments by increasing the probability of processes conducted by microbial communities in a given environ- biodegradation and decreasing the rate of test failures, by increas- ment. Several functional gene approaches have been developed to ing the reproducibility of fate studies. study the diversity of chemical degrading microbial communities. Current OECD tests are conducted with variable inocula and Changes of key catabolic genes (catechol 2,3-dioxygenase, catechol there is no inoculum standardisation prior to biodegradability 1,2-dioxygenase and alkane-catabolic genes (alk)) and microbial testing, which has been identified as the key factor affecting the community structure were studied by Zhang et al. (2008) during reproducibility of chemical fate studies (Mezzanotte et al., 2005; the degradation of nonylphenol ethoxylates (NPEOs) and NP in Thouand et al., 2011). Molecular ‘omics’ tools are increasingly natural water microcosms. This study demonstrates that functional affordable, commonly used methods, which could be used along- genes may be used to monitor the potential for chemical biodegra- side traditional OECD methods to study chemical biodegradation dation. These methods may be applied to support biodegradation in the environment. tests by studying the functional diversity of inocula and its potential Application of advanced high throughput next-generation for biodegradation. Further development of such approaches could sequencing methods such as amplicon sequencing, metagenomics potentially identify the scope for adaptation within communities to and transcriptomics (Table 4) can be used for in depth character- degrade chemicals. Chemical fate is associated with proliferation of isation of diversity and function of a community. Metagenomics degrading bacteria which often leads to shifts (changes) in bacterial allows us to sequence genomes from complex assemblages of community composition. Shifts occurring in microbial populations microbes as a single unit in a culture-independent manner during biodegradation of chemical, in particular the abundance and (Moran, 2009) and it provides an opportunity for linking microbial diversity of specific degraders can be then correlated with chemical diversity with function (Malik et al., 2008). Metatranscriptomics fate. Whereas, lack of changes in bacterial community and low (Table 4)profiles the collective transcriptomes of a given habitat, abundance of specific degraders coupled with lack of chemical and allows actively expressed genes (mRNA) to be retrieved and biodegradation may suggest potential persistence of given chemical. sequenced to profile active metabolic processes (Stenuit et al., Moreover, metagenomics, proteomics and metabolomics allow better 2008; Moran, 2009; de Menezes et al., 2012). understanding of complex changes in microbial community func- Metatranscriptomics can be applied as a supporting tool in tions, detection and identification of existing and novel proteins order to perform inoculum characterisation, to study the micro- (enzymes) and metabolites involved in the biodegradation of biology and functional diversity of inocula. Such techniques could be chemical of interest. For example, Mackelprang et al. (2011) reported used to establish new ways of conducting biodegradation studies that metagenomics allowed examination of whole biochemical and to interpret biodegradation tests for certain chemicals, for pathways and associated processes, as opposed to individual pieces example by understanding the genetic potential for biodegradation of the metabolic matrix. Iwai et al. (2010) used gene-targeted

Table 4 Application and potential of high-throughput technologies in biodegradation studies. Adapted from Stenuit et al. (2008)

Technique Area of application Potential Application in fate studies and risk assessment

Fingerprinting Community structure Relate microbial community analysis to the Will enable more realistic exposure assessments and enable better techniques based on and dynamics metabolic function of specific groups of extrapolation from in vitro test systems to in situ conditions amplicon sequencing bacteria

Metatranscriptomics Community function Identification of the biodegradation Optimisation of inoculum characterisation, which will reduce the Metaproteomics potential and the function of specific rate of test failures and increase the reproducibility of fate studies Metabolomics microbial communities

Biosensors Presence of undefined Monitoring ‘real-time’ the performance of a Understand genetic potential for biodegradation within inocula chemicals bioremediation process Provide new methods and insights to study chemical Nature and biodegradation pathways concentration of the Provide in silico tools to better characterise and predict chemicals biodegradation based on chemical structure and microbial Toxicity of the communities potential to degrade chemicals for living organisms Decrease in chemical concentration, and/or transformation

Cultivation (þgenome Use of exogenous New metabolic functions of microbial Understanding interactions between chemicals and sequencing), biocatalysts communities environmental factors that may modulate microbial responses metagenomics Search for new catabolic activities A. Kowalczyk et al. / Ecotoxicology and Environmental Safety 111 (2015) 9–22 19 metagenomics to study the diversity of aromatic dioxygenase genes assessment and there is the need to develop tools that take involved in the turnover of more recalcitrant organic chemicals. Such account of the complexity of exposed environments and enable genes can be used in the assessment of inoculum biodegradation assessment of site-specific effects (SCHER, SHENIHR, SCCS, 2013). potential, and hence in prediction of chemical fate in the environ- Although simulation of complex environmental systems under ment. These approaches are useful for assessment of chemical laboratory conditions is still challenging, better understanding of persistence since they provide insight into processes such as inocu- ecosystems can be achieved through the application of microbial lum adaptation and can also address evolutionary issues. Introduc- ecology and systems biology. tion of new chemicals into the environment often leads to General knowledge about the processes operating within a development of novel chemical biodegradation pathways and is microbial community is not sufficient to determine the role of associated with the presence of specificdegradingbacteria.‘Omics’ individual members and their interactions within the community. will help us to understand such processes and their consequences for For biodegradation studies it is crucial to understand how microbial ecosystems. relationships could be affected by the environment and new In metaproteomics (Table 4), complex mixtures of proteins from chemicals entering the environment, and vice versa (Zengler and an environmental sample are separated and fractions of interest are Palsson, 2012). Also, the biodegradation processes are framed in a analysed by high-throughput mass spectrometry-based analytical complex web of metabolic and regulatory interactions which are platforms (Stenuit et al., 2008). Exploring the differential expression extremely difficult to approach with traditional molecular methods. of a wide variety of proteins and screening of the entire genome for The recent accumulation of knowledge about the biochemistry and proteins that interact with particular mineralisation regulatory genetics of the biodegradation process, and creation of structured factors is helpful to get insights into biodegradation (Vieites et al., databases, has recently opened the door to systems biology (Trigo 2009). For instance, profiles of enzymes involved in biodegradation et al., 2009). Linking microbial functions with processes occurring in pathways, which are expressed by microbial inocula in the presence the environment can be useful for designing novel test systems of a test chemical or specific metabolites, can be used to identify the simulating environmental conditions. ‘Omics’ techniques make it potential for chemical degradation and microbial responses to possible to understand the reasons underlying test failure and contaminated environments. Therefore, proteomics plays an essen- success. For instance, it can be determined how laboratory condi- tial role in determining the physiological changes of microorgan- tions affect the inoculum potential (i.e., presence and diversity of isms under specific environmental influences, while functional specific degraders) for chemical biodegradation. ‘Omics’ could also proteomics (based on the presence of enzymes involved in chemical help us to understand interactions between chemicals and environ- biodegradation pathways) would predict metabolism of chemicals mental factors e.g., temperature, salinity, organic matter content, and by degrading organisms (Chauhan and Jain, 2010). can indicate how environmental factors might modulate microbial Another approach, environmental metabolomics (Table 4) has responses to chemicals. This information can be applied in chemical many advantages for studying organism–environment interactions fate studies to simulate the right conditions for adaptation of inocula and for assessing organism function and health at the molecular and chemical biodegradation. Improving test conditions can help to level (Bundy et al., 2009). The role of metabolomics and proteo- decrease the rate of test failures and may improve laboratory to field mics in understanding mechanisms of microbial aromatic degra- extrapolations. dation was described by Seo et al. (2009). They reported that metabolomics can be used to profile degradation products of PAHs 4.1. Advantages and disadvantages of ‘omics’ and primary metabolites in response to PAH exposures. Keum et al. (2008) studied comparative metabolic responses of Sinorhi- Although, high throughput techniques are emerging tools in zobium sp. C4 during degradation of phenanthrene. Comprehen- biodegradation studies they are still relatively new techniques. sive profiling of metabolite profiles, including polar metabolites, One of the biggest disadvantages of ‘omics’ is their blind applica- fatty acids and polyhydroxyalkanoates was performed through tion. While there is a place for discovery science using high- untargeted metabolome analyses. Large metabolome differences throughput techniques, progress is usually achieved in studies that were observed between the cultures grown with phenanthrene are applied with a clear hypothesis in mind. The major concerns and natural carbon sources. Metaproteomics and metabolomics arising around ‘omics’ are also linked with constant development can be used to determine the biodegradation potential of inocula of technologies and urge to bring new improvements and applica- applied in fate studies, in determination of chemical fate pathways tions by commercial enterprises. This requires constant develop- in the environment, in accurate assessment of primary or ultimate ment and education of scientists in order to be on top of those biodegradation based on metabolites. Metabolomics can be approaches. Another challenge is the interpretation and manage- applied to identify signature metabolites which are indicative of ment of massive amount of data, which are generated by high chemical biodegradation (Parisi et al., 2009), and hence, can be throughput technologies. Therefore, development of efficient sta- used to predict degradation pathways and the residual concentra- tistical algorithms, pipelines and other bioinformatic tools is tion of chemicals in receiving environments. crucial to obtain good quality data and to perform interpretation. Biosensors also have the potential to be applied in biodegrada- However, the costs of generating high throughput data and data tion studies (Table 4). Microorganisms are increasingly used as analysis are becoming more affordable which increases their avail- specific sensing devices for measuring chemical concentrations in ability and applicability for biodegradation studies (Shendure and the environment. These biosensors measure gene expression Ji, 2008; Desai et al., 2010). induced by the presence of a chemical of interest, which serves as a measure of its bioavailable concentration (Stiner and Halverson, 2002). As such, biosensors can detect biologically 5. Conclusions relevant chemical concentrations. 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