Increased cell numbers improve marine biodegradation tests for persistence assessment

Science of the Total Environment 706 (2020) 135621

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Increased cell numbers improve marine biodegradation tests for persistence assessment Amelie Ott a,⁎, Timothy J. Martin a,1, Jason R. Snape a,b,c, Russell J. Davenport a a b c

Newcastle University, School of Engineering, Cassie Building, Newcastle upon Tyne NE1 7RU, UK AstraZeneca Global Environment, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TF, UK University of Warwick, School of Life Sciences, Gibbet Hill Campus, Coventry CV4 7AL, UK

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Current biodegradation screening tests were not designed for persistence assessment. • These current tests often give false negative results and are highly variable. • This study validated a new test to assess chemical degradation in seawater. • New test incorporates increased bacterial cell numbers and runs beyond 60 days. • New test is less variable and provides more reliable persistence assessment.

a r t i c l e

i n f o

Article history: Received 6 September 2019 Received in revised form 15 November 2019 Accepted 17 November 2019 Available online 09 December 2019 Editor: Jose Julio Ortega-Calvo Keywords: Seawater Risk assessment Tangential flow filtration Biomass concentration OECD 306 REACH

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

⁎ Corresponding author. E-mail address: [email protected] (A. Ott). 1 Present address: Department for Environment, Food and Rural Affairs, Lancaster House, Newcastle upon Tyne NE4 7YJ, UK.

https://doi.org/10.1016/j.scitotenv.2019.135621 0048-9697/© 2019 Elsevier B.V. All rights reserved.

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A. Ott et al. / Science of the Total Environment 706 (2020) 135621

control pentachlorophenol as potentially persistent. Next, it is recommended to further validate the marine erBST in an inter-laboratory study incorporating different seawater sources to fully assess its variability and reliability. © 2019 Elsevier B.V. All rights reserved.

Abbreviations 4-CA 4-FP 4-NP AS ANI BST CICAD CV DAPI dt50 ECHA erBST GLP ITS LSC OECD PCP RBT SB t50 TFF tL

4-chloroanline 4-fluorophenol 4-nitrophenol activated sludge aniline biodegradation screening test Concise International Chemical Assessment Document coefficient of variation 4′,6-diamidino-2-phenylindole =t50 − tL European Chemicals Agency environmentally relevant biodegradation screening test Good Laboratory Practice Integrated Assessment and Testing Strategy liquid scintillation counting Organisation for Economic Co-operation and Development pentachlorophenol ready biodegradability test sodium benzoate time to reach 50% degradation tangential flow filtration lag time, time to reach 10% degradation

1. Introduction Biodegradation screening tests (BSTs) are stringent tests which aim to provide a conservative assessment of chemical fate, and in doing so screen out chemicals which are easily degraded in all environments (OECD, 1992a). A series of BSTs have been developed and approved by the Organisation for Economic Co-operation and Development (OECD) including ready biodegradability tests (OECD 301 and 310), biodegradability in seawater tests (OECD 306) and inherent biodegradation tests (OECD 302). While the original OECD biodegradation testing hierarchy did not list the OECD 306 test as a ready biodegradation test (OECD, 1992b), more recent OECD publications (OECD, 2006) and regulatory guidance documents (ECHA, 2017a) appear to withdraw this distinction (Ott et al., 2019). Consequently, for this study, ready biodegradability tests (RBTs) are defined to include test methods OECD 301, 306 and 310 in accordance with the terminology used by the European Chemicals Agency (ECHA) in the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) regulation (ECHA, 2017a). BSTs have historically formed the foundation on which regulatory frameworks have been developed to protect the environment. This has been largely due to their relative low cost and somewhat straightforward implementation and interpretation. In recent years, BSTs, have not evolved at the same rate as regulatory concerns, which now place an increased emphasis on environmental persistence as defined by a series of half-life thresholds (Regulation (EC) No 1907, 2006). As part of REACH, the Integrated Assessment and Testing Strategy (ITS) follows a tiered approach to conclude on persistence (ECHA, 2017a). Within this strategy, RBTs represent the first tier to screen for nonpersistent or potentially persistent chemicals (ECHA, 2017a), despite the fact that these tests were not developed for this purpose: some RBT durations are shorter than proposed half-life thresholds for persistence (Regulation (EC) No 1907, 2006); RBTs are notoriously highly variable, particularly with respect to lag phase and biodegradation outcome (Painter, 1995); RBTs regularly result in test failure, with

many failures thought to be false negatives (ECETOC, 2007). Kowalczyk et al. (2015) identified the inadequacies of existing RBTs, suggesting that the high variability observed may be due to characteristics of the inocula such as the presence or absence of competent degraders, adaptation to the test chemical and bacterial cell concentrations; characteristics which control both the lag phase and biodegradation kinetics. False assignments of potential persistence in RBTs result in additional higher tier tests and limit the effective prioritisation of persistent chemicals from the thousands of new and existing chemicals being registered under the EU's REACH regulation. A number of enhancements to existing RBTs have been identified (ECETOC, 2013, 2007, 2003) and included in the original REACH endpoint specific guidance document (ECHA, 2008) to enable a more effective prioritisation on persistence. The modifications for these so-called “enhanced screening tests” included methods to increase total cell numbers within a test by increasing biomass concentrations and test volumes, as well as extending test durations to better capture adaptation and growth-linked biodegradation. The option of increasing biomass concentrations was omitted from the latest version of the REACH endpoint specific guidance (ECHA, 2017b) on the basis that “there is already some flexibility of the inoculum concentration given in Ready Biodegradability Tests. Going beyond the limits defined will change the ratio of substance to inoculum in a way that is deemed to be too favourable” (ECHA, 2017b). However, it has been shown that inocula concentrations in current RBTs inadequately represent environmental cell numbers and misrepresent their diversity, both of which affects the variability of RBTs (Goodhead et al., 2014; Kowalczyk et al., 2015; Ott et al., 2020). The latest version of the guidance was drafted at about the same time that evidence was published showing that kinetics in “enhanced” tests with increased biomass concentrations were indistinguishable from those in current tests and that reference persistent chemicals would not pass the test i.e. no false positives (Martin et al., 2017a, 2017b). While previous research only validated enhancements for activated sludge RBTs (Martin et al., 2017b), this study focusses on evaluating increased cell numbers and extended test durations for seawater in so-termed “environmentally relevant biodegradation screening tests” (erBSTs). A marine erBST with increased biomass concentrations (here 100-fold by tangential flow filtration) was compared to a marine BST in a modified OECD 301B test with radiolabeled reference chemicals to assess the accuracy and reliability of screening for non-persistent chemicals. 2. Methods This study was conducted in compliance to Good Laboratory Practice (GLP) in an accredited laboratory. 2.1. Sampling and seawater preparation Seawater samples were collected during spring/summer from dailyreplenished on-site storage tanks, prior to on-site treatment, in Brixham, Devon, UK. Seawater was filtered through a 10 μm screen to remove coarse particles for the reference marine BST (referred to as BST). As the chosen test apparatus ensured aerobic incubation conditions over the complete test period, seawater ageing to reduce background respiration was not conducted for either test. For the erBST, biomass from 10 μm pre-screened seawater was increased by tangential flow filtration (TFF) using a 0.22 μm nominal pore size filter (Millipore, Billerica, MA, USA) (Martin et al., 2018). TFF was chosen as it ranked

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highest in a previous comparison of different methods for increasing biomass concentrations in BSTs by accurately representing the microbial community of the initial sampled environment while allowing for a high sample throughput (Martin et al., 2018). During the TFF process, the recirculating seawater increased in biomass as it decreased in volume with the removal of permeate through the TFF unit. When the recirculating retentate was approximately 500 mL, the TFF unit was backwashed with permeate to resuspend any material deposited within the unit and on the filter. 1 L of 100-fold nominally concentrated bacteria in seawater was thus prepared from 100 L of environmental sample. Total cell counts were measured by epifluorescence microscopy with 4′,6-diamidino-2-phenylindole staining (DAPI, Sigma-Aldrich, St. Louis, MO, USA).

2.2. Test chemicals Five test chemicals were selected covering a range of biodegradation potentials to evaluate the limits of the BST and erBST. A positive (aniline: ANI, rapidly biodegradable – non persistent) and negative (pentachlorophenol: PCP, potentially persistent) reference chemical was chosen together with three chemicals previously having shown variable degradation in biodegradation tests: 4-fluorophenol (4-FP, rapidly biodegradable – non persistent), 4-nitrophenol (4-NP, inherently biodegradable – non persistent) and 4-chloroaniline (4-CA, inherently biodegradable – non persistent) (Table 1). The assigned biodegradation classifications were based on environmental fate information from the ECHA database and a review document to develop “a set of reference chemicals for use in biodegradability tests for assessing the persistency of chemicals” (Comber and Holt, 2010), together with relevant Concise International Chemical Assessment Documents (CICADs) (WHO, 2003, 2000) and data from the Finnish Environment Institute (SYKE, 2018). It should be noted that the assigned biodegradation classifications are not definitive as they are restricted by the quality and scope of the evaluated data. For instance in the ECHA database, the amount, reliability and relevance of studies assessed for biodegradation conclusions can vary greatly (Ingre-Khans et al., 2019). The adequacy of these studies is scored on a four point scale depending on the nature of the study (key study, supporting study, weight of evidence, others e.g. disregarded or unspecified type of study), with GLP regulatory standardized tests scored as most reliable (ECHA, 2011). However, the transparency and systematic approach of this framework has recently been questioned (Ingre-Khans et al., 2019). Additionally, registrants summarize the biodegradability conclusions in the ECHA database without review or authorization by the Agency (ECHA, 2018). While acknowledging these limitations, biodegradation results from databases

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are crucial in establishing expected biodegradation behaviors for reference chemicals to validate new test methods (Comber and Holt, 2010). A mixture of stock solutions of radiolabeled and non-radiolabeled test chemicals were applied in the BST and erBST to increase the sensitivity of the analysis, while also reaching the high test chemical concentrations required in OECD regulatory studies. For this, concentrated stock solutions (1 g C L−1) of each chemical (Sigma-Aldrich, St. Louis, MO, USA) were prepared in OECD 301B mineral media (OECD, 1992a). These were then combined with radiolabeled stock solutions of the test chemicals (universally labeled 14C: ARC Inc., St. Louis, MO, USA) and radioactivity was measured via liquid scintillation counting (LSC) (Tri-Carb 2800-TR, Perkin Elmer, Waltham, MA, USA). The chemical solution was added to each system at a ratio of 10 mL to 1 L of seawater to give a final test concentration of 10 mg C L−1. The level of radioactivity applied allowed b1% degradation to be resolved in the sampled NaOH, with an average dose of 20 Bq mL−1 (ranging from 15 to 24 Bq mL−1).

2.3. Screening test preparation Within the OECD testing regime, biodegradability in seawater (OECD 306) can be measured with two methods, which are both variants of existing OECD 301 methods: the closed bottle test (based on OECD 301D) and the shake flask test (based on OECD 301E) (ECHA, 2017b). Both of these tests come with their own advantages and restrictions such as oxygen limitations occurring past 28 days in the former and only test chemicals with restrictive characteristics being suitable for the later (Fiebig et al., 2017; OECD, 1992a, 1992b). This study used a modified OECD 301B method (Courtes et al., 1995; Martin et al., 2017b) for measuring the biodegradability of radioactive labeled chemicals in seawater (Fig. 1). Each test was performed in triplicate. Test vessels (2000 mL) contained processed seawater (986 mL; 10 μm filtered for BST, 10 μm filtered and TFF processed for erBST), OECD mineral media (1 mL each of solutions a, b, c and d (OECD, 1992b)) and the test chemical (10 mL). Air was drawn into the vessel via an influent CO2 scrubber (50 mL of 2 M NaOH, Sigma-Aldrich, St. Louis, MO, USA) and humidifier (50 mL of H2O). Evolved 14CO2 was captured in traps containing 50 mL of 2 M NaOH, positioned after the main test vessel with empty traps positioned on either side of the NaOH traps. Orbo tubes (Orbo 32 and 91, Sigma-Aldrich, St. Louis, MO, USA) were positioned between the test vessel and first empty trap to capture volatilized test compounds and degradation products. Test systems were kept aerobic at 20 °C (± 2 °C) in the dark for at least 60 days. Where extensive lag phases were suspected or degradation had started but not reached a plateau yet, test batches were extended beyond 60 days to a maximum of 110 days.

Table 1 Assigned biodegradation classification for the five reference chemicals based on environmental fate information from the ECHA database, bin assignments from Comber and Holt (2010), Concise International Chemical Assessment Documents (CICADs), the Finnish Environment Institute (SYKE) database and test guidelines. Aniline

4-Fluorophenol

ECHA database: Study results for biodegradation in water screening tests

Readily biodegradable (ECHA, 2019a)

Readily biodegradable (ECHA, 2019b)

Bins assigned by Comber and Holt (2010) based on expected biodegradation behaviour in BSTs and enhanced BSTsa Further relevant information

Bin 1: Would normally pass a BST and enhanced BST Reference chemical for BSTs (e.g. OECD 301, OECD 306)

Assigned biodegradation classification for this study

Rapidly biodegradable – non persistent

4-Nitrophenol

4-Chloroaniline

Under test conditions, no biodegradation observed (ECHA, 2019c) Bin 2: Would normally fail a current BST, but pass an enhanced BST

/

Rapidly biodegradable – non persistent

Inherently biodegradable (ECHA, 2019e)

CICAD conclusion: Inherently biodegradable under WHO (2000) Inherently biodegradable – non persistent

CICAD conclusion: Inherently biodegradable under WHO (2003) Inherently biodegradable – non persistent

Pentachlorophenol No information available (ECHA, 2019d)

Bin 3: Should normally fail a BST and enhanced BST SYKE, 2018: OECD screening test 5% in 28 days; two MITI test results, one negative and one 1% in 28 days. Potentially persistent

a Comber and Holt (2010) define enhanced BSTs as BSTs incorporating following test approaches: increased test durations/testing in larger vessels/increasing the biomass concentration/low-level pre-adaptation test systems/semi-continuous biodegradability tests.

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were mixed with Gold Star scintillation cocktail and analyzed via LSC. For solids, the filter papers were combusted (A307 sample oxidizer: PerkinElmer, Waltham, MA, USA), mixed with Gold star scintillation cocktail and analyzed via LSC. To quantify chemical loss by volatilization, Orbo tubes were rinsed with 10 mL of methanol, which was subsequently mixed with Gold Star scintillation cocktail and analyzed via LSC. To assess chemical sorption to internal surfaces, glassware and tubing were rinsed with methanol. Methanol was subsequently mixed with Gold Star scintillation cocktail and analyzed via LSC. 3. Results and discussion Fig. 1. Modified OECD 301B experimental apparatus with radiolabeled chemicals for the marine BST and erBST (adapted from Brixham Environmental Laboratories, 2013).

2.4. Biodegradation determination and interpretation Ultimate biodegradation (mineralization) was determined by capturing evolved 14CO2 in 2M NaOH traps (Fig. 1). During periodic sampling (day 0, 2, 4, 7, 10, 14, 21, 28, 35, 42, 49, 56, 60, 67, 74, 81, 90, 110), NaOH was collected in pre-weighed Nalgene bottles (ThermoFisher Scientific, Waltham, MA, USA), which were subsequently reweighed. Triplicate 5 mL NaOH subsamples were transferred to 20 mL glass LSC vials (Perkin Elmer, Waltham, MA, USA), mixed with Gold Star scintillation cocktail (Meridian Biotechnologies Ltd., Tadworth, UK) and analyzed via LSC. The accumulated radioactivity was converted to a percentage of the originally applied radioactivity and used as an indicator of ultimate biodegradation. A further 5 mL sample was taken from the test vessels on each sampling day for other analyses not presented here. The mass balance accounts for the removal of radioactivity by these samples. Biodegradation outcome was assessed based on REACH guidance on interpreting enhanced screening tests for non-persistence (ECHA, 2017a). Reference chemicals with ultimate biodegradation (mineralization) rates of ≥60% over 60 days were classified as non-persistent while values b60% were indicative of potential persistence. Non-persistence assignments based on the BST and erBST results were subsequently compared to the expected biodegradation classifications (Table 1). Additional biodegradation descriptors were applied to compare the BST and erBST. For each chemical, the time to reach 50% degradation, t50 (this descriptor is different to the t50 descriptor mentioned in the OECD 306 – see below), time to reach 10% degradation, i.e. lag time, tL, and dt50 = t50 − tL (this descriptor is equivalent to t50 as mentioned in OECD 306) were determined based on visual assessments of the biodegradation curves. The distinction between t50 and dt50 was made to assess the effect of lag phases. The OECD 301B validity criterion to assess variability between replicates was applied where a test is considered valid if the difference of extremes of replicate values at the end of the test does not exceed 20% (OECD, 1992a). For each time point, the mean degradation and standard deviation was calculated from the triplicate test set-ups, from which the coefficient of variation (CV) was determined. The average of these coefficients was then calculated for each chemical. To compare the BST and erBST variability, only chemicals where degradation was observed were considered for the CV. Data analysis and visualization was performed using R (R Core Team, 2015).

3.1. Reliability of chemical classification The erBST was more accurate and significantly less variable (ANOVA, P b 0.05) than the BST in assigning the reference chemicals to their expected biodegradation classification (non-persistent for ANI, 4-FP, 4NP, 4-CA and potentially persistent for PCP; Tables 1 and 2; Fig. 2). According to the REACH screening criterion of ≥60% biodegradation over 60 days, the erBST correctly classified 60% of the reference chemical replicates to their respective biodegradation category and had a CV of 21%. The BST correctly classified 40% of test compound replicates and had a CV of 36%. Neither, the BST or erBST showed any false positives (Table 2). The false negative rate was reduced in the erBST (50%; BST 75%), but higher than that found in a previous activated sludge (AS) erBST study where no false negatives were detected (Martin et al., 2017b). These differences in test performance between the marine and AS erBST could, amongst others, be associated with higher cell counts (Martin et al., 2017b) and, or an increased microbial diversity in the AS erBST (ECHA, 2017b; Martin, 2014). Indeed, the total cell counts of the sampled seawater were relatively low (104–105 cells mL−1) compared to reported marine cell density ranges of 104– 107 cells mL−1 with average concentrations of 5 × 105 cells mL−1 for the continental shelf and upper 200 m of the open ocean (Whitman et al., 1998). This may have limited the impact seen by increasing biomass concentrations in the erBST. Rather than a nominal-fold concentration, a defined bacterial cell concentration range may be preferred and may assist in standardizing environmental samples between different locations. 3.2. Mass balance Mean recovery rates at test termination were 88% to 109% except for 4-CA with reduced values of 69% and 71% for the BST and erBST, respectively (Fig. 3). The test compound 4-CA was volatile and partly removed through the headspace as indicated by its detection in the Orbo tubes (BST: 41%; erBST: 39%). The missing percentages from the 4-CA radioactivity mass balance could be the result of incomplete Orbo tube extractions as the 14C measurements were still relatively high at the last out of four extractions. Previously reported sorption of volatile chemicals to internal plastic surfaces (Brown et al., 2018) is probably not relevant for this study as the modified OECD 301B test set-up was composed of glass and rubber tubing (Fig. 1). The variation between replicates for mass balance recovery was low with a mean standard deviation of 3.8%, ranging from 0.6% to 6.9%. 3.3. Rapidly biodegradable chemicals

2.5. Mass balance calculation At the end of the test period, recovery of the initially applied radioactivity was used to determine the fate for each reference chemical (Martin et al., 2017b). Evolved 14CO2 radioactivity was determined as previously described. After test termination, test vessel contents were filtered through Whatman grade 4 filter paper (Sigma-Aldrich, St. Louis, MO, USA). The filtrate was stored in a pre-weighed Nalgene bottle, which was subsequently re-weighed and triplicate 5 mL subsamples

The positive control aniline showed degradation in both test set-ups (Fig. 2). Aniline was correctly assigned as non-persistent in all three erBST replicates with an average biodegradation of 66 ± 3% on day 60 (Table 2). Under BST conditions however, it failed to reach 60% degradation over 60 days in one replicate (Table 2). The failure of positive reference chemicals in marine biodegradation tests has been reported not only in screening tests but also in simulation studies such as the OECD 309 (ECETOC, 2013), with long lag phases typically being identified as

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Table 2 Biodegradation results for the five reference chemicals in the marine BST and erBST. For descriptor data in days, mean values are reported with ranges in parentheses. For degradation data in percentages, mean values are shown with standard deviations. Chemical

Test

CV (%)

Descriptor (days)

Degradation (%) on

tL

t50

dt50

day 28

day 60

na (23–b60) 23 (19–26) b110

na (17–b60) 16 (13–18) b110

45 ± 10

60 ± 11

55 ± 4

66 ± 3

19 ± 5

31 ± 4

69 (67–70) 62 (50–83) 44 (39–55) b110 b110

63 (62–63) 13 (10–18) 8 (8–9) b110 b110

27 ± 2

46 ± 2

1±0

45 ± 37

2±1

81 ± 10

1±0 1±0

2±0 2±0

b90 b90

b90 b90

0±1 0±0

0±1 0±0

Aniline; CAS 62-53-3 Purity: 99%

BST

37

erBST

21

4-Fluorophenol;CAS 371-41-5 Purity: 99%

BST

50

erBST

31

4-Nitrophenol;CAS 100-02-7 Purity: ≥99%

BST

20

erBST

11

4-Chloraniline;CAS 106-47-8P urity: 98% Pentachlorophenol;CAS 87-86-5 Purity: 97%

BST erBST

14 22

6 (4–6) 7 (7–8) 13 (9–16) 6 (4–9) 49 (39–67) 36 (30–48) b110 b110

BST erBST

50 67

b90 b90

CV: coefficient of variation; dt50 = t50 − tL; na: not applicable, indicates that mean could not be calculated as not all three replicates showed sufficient degradation to determine the descriptor; t50: time to reach 50% degradation; tL: lag phase, time to reach 10% degradation.

the cause of false negative outcomes (Comber and Holt, 2010). This has resulted in a call to review reference chemicals currently used in marine testing and the consideration of more appropriate reference chemicals (ECETOC, 2017). 4-FP degraded in the BST and erBST. Slow, gradual removal of 4-FP was observed with no distinct lag phase (Fig. 2; Table 2). After 110 days 55 ± 1% and 44 ± 7% average removal were observed in the erBST and BST, respectively. Higher bacterial concentrations in the erBST increased 4-FP degradation compared to the BST, however, it failed to pass the 60% over 60 days REACH threshold for nonpersistence. Comber and Holt (2010) reported mixed results for 4-FP in BSTs and indicated that 4-FP may occasionally fail even enhanced screening tests. Increasing the volume of erBST data, testing

Fig. 2. 14CO2 evolution over time for the reference chemicals selected to validate the marine erBST against the BST. Rapidly degradable, inherently degradable and potentially persistent classifications of the five reference chemicals according to Table 1.

environmental samples from multiple locations, could increase confidence in the characterization of 4-FP biodegradability.

3.4. Inherently biodegradable chemicals 4-NP degradation was observed in the BST and erBST after extensive average lag phases of 49 and 36 days, respectively (Table 2). Following these lag phases, degradation occurred fairly rapidly, indicated by average dt50 values of 13 and 8 days compared to t50 values of 62 and 44 days for the BST and erBST, respectively (Table 2). These long lag phases have been previously reported by Nyholm and Kristensen (1992) amongst others (Comber and Holt, 2010). The lag phases may be a batch artefact due to the low cell numbers incorporated in marine screening tests and BST conditions. They probably represent either the time taken for slow growing competent degraders, initially present at very low abundance in the sampled environment, to establish themselves and build a population density within the community, or the time taken to activate metabolic pathways required for degradation (Nyholm et al., 1984). Extending test duration beyond the currently accepted 28 days in some marine RBTs allows the capture of adaptation.

Fig. 3. Mass balance at study end (ranging from day 60 to day 110 depending on chemical, see Fig. 2) indicating mean recovery of initially applied radioactivity as evolved 14CO2 (pink), in the aqueous phase following filtration (blue), recovered from solids following combustion (yellow), volatile organics from Orbo tubes (orange), vessel rinse (purple) and removed from test vessels for additional analysis (green). 4-CA: 4-chloroaniline; 4FP: 4-fluorophenol; 4-NP: 4-nitrophenol; ANI: aniline; PCP: pentachlorophenol. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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The erBST correctly assigned 4-NP as non-persistent in all three replicates based on ≥60% biodegradation over 60 days, while in the BST, only one replicate passed this threshold. No 4-CA degradation was observed in either test set-up (Fig. 2). 14C labeling showed that a significant amount of initially applied radioactivity was lost for 4-CA by volatilization in both tests (Fig. 3). While the Henry's law constant of 0.1 Pa m3/mol suggests low volatility of 4-CA from aqueous solutions (Lyman et al., 1990; WHO, 2003), Schauerte et al. (1982) reported high initial volatility losses of 4-CA from an experimental pond. 4-CA also showed some volatile characteristics in the AS BST and erBST, however, here rates did not exceed 5% which could have been due to degradation starting relatively early in the incubation period (Martin et al., 2017a, 2017b). Variable degradation has been observed for 4-CA for BSTs and inherent tests (WHO, 2003) with extended lag phases documented (Ahtiainen et al., 2003; Ingerslev and Nyholm, 2000). In this study, 4CA potentially did not degrade due to the mentioned bioavailability issues, lag phases extending beyond the test duration of 110 days or no potential degraders being present or encaptured from the sampled environment. 3.5. Potentially persistent chemical PCP was correctly assigned as potentially persistent in all BST and erBST replicates (Tables 1 and 2, Fig. 2). Consequently, in this study, the erBST was not too powerful and increased cell concentrations did not result in a false positive chemical assessment. 3.6. Validity and suitability of the modified OECD 301B for measuring biodegradability in seawater Following the validity criteria of the OECD 301B, biodegradation values of replicates at test termination did not vary by more than 20% (OECD, 1992a). To adapt the OECD 301B for seawater, phosphate was added at lower concentrations following the nutrient additions described in the OECD 306 protocol (1 mL of solution a) instead of OECD 301B protocol (10 mL of solution a). In OECD screening tests, phosphate is not only added as a nutrient to provide optimal bacterial growth conditions but can also serve as a buffer (Painter, 1995). Whereas the OECD 301B test relies on inoculating deionized or distilled water with a source of microorganisms (e.g. from activated sludge), the OECD 306 test directly uses seawater (OECD, 1992a, 1992b). While it is not explicitly stated in the OECD guidelines, the natural buffering capacity of seawater is most likely the reason for the lower phosphate additions in the OECD 306 test (Painter, 1995). On the other side, higher phosphate concentrations have not been shown to interfere or increase biodegradation in BSTs (Courtes et al., 1995; Painter, 1995). For future research, it is recommended to prepare the test chemical stock solutions in seawater instead of in OECD 301B mineral medium (as described here). Despite test chemical solutions only contributing marginally to the overall test volume (here 1%), this improvement can help to better represent the environment by capturing more bacteria in the test vessel (Ott et al., 2019), while altering the salinity of the test medium less (OECD, 1992b). A well-reported disadvantage of the standard OECD 301B method is that inorganic carbon can accumulate in the test medium resulting in an underestimation of biodegradation based on evolved CO2 captured in NaOH traps (OECD, 2014; Weytjens et al., 1994). An unpublished study exploring alternative test designs for marine BSTs found the OECD 301B method not suitable for seawater due to an underestimation of biodegradation, possibly linked to the buffering capacity of seawater (Fiebig et al., 2017). While these limitations do not appear to have occurred in this study, further research should investigate the use of other systems to monitor biodegradation of non-radiolabeled chemicals in marine erBST (for instance, modifying the OECD 301F manometric

respirometer test or testing oxygen sensors (Brown et al., 2018; Fiebig et al., 2017)). 3.7. Regulatory implications and interpretation of pass criteria This study together with other research (Martin et al., 2018, 2017b) suggests that increasing biomass concentration can be a suitable approach to improve the reliability of screening tests for persistence assessment and challenges the recent exclusion of this option in REACH (ECHA, 2017b). The marine erBST with increased biomass and extended test durations is still a conservative screening test as unrealistically high test chemical concentrations are employed to overcome analytical method constraints in RBTs. This results in a biomass to test chemical ratio that is not environmentally relevant but is improved over the traditional marine OECD 306 test. The improved biomass to test chemical ratio and better representation of microbial diversity in the test system result in decreased variability between replicates and improve the ability to screen for non-persistence. While the concept of increasing biomass concentration was removed in the latest version of REACH guidance, a UBA report published in the same year described this approach to improve BSTs for persistence assessment (Gartiser et al., 2017), citing the PhD thesis (Martin, 2014) linked to this paper. A marine erBST could complement the existing OECD 306 method, which would still be pertinent for identifying rapidly degrading chemicals, but the erBST would offer a cost-effective screening test for non-persistence when the OECD 306 test gives the result “not readily/ rapidly biodegradable”. The erBST could form part of the first tier of non-persistence screening before the more complex, costly and timeconsuming simulation tests. Even if improvements in screening test methods are accepted and successful, there is still a requirement to establish clearer guidance on interpreting the data. The extensive lag phases observed with marine samples are a well-known issue (Elf Akvamiljo, 1996). Guidance on the interpretation of long lag phases is an important consideration that could influence the ability to draw meaningful assessments where long lag phases are observed. Recommendations have been made to extend screening tests to 60 days (ECETOC, 2007; ECHA, 2008). There is evidence presented here and in the literature to suggest that test durations could be more flexible and allow extension beyond this to 120 days, with a 60 day window for degradation to occur beginning once 10% degradation has been reached. The rates of biodegradation following long lag phases are often similar to those observed in freshwater tests, with rapid and high levels of removal observed (ECETOC, 2009). 3.8. Outlook: multi-laboratory validation of the marine erBST in a ring test The recommendations initially made to increase bacterial cell concentration and extend test duration should be seen as the first step in a process to address the known limitations of marine biodegradation testing and interpretation. These recommendations have been shown to improve the reliability of marine BSTs, providing sufficient evidence to merit further investigation. Based on this study and other previously published research (Martin et al., 2018, 2017b), an ECETOC funded workshop was delivered in 2015 with regulatory, industry and academic involvement to discuss improvements to marine biodegradation testing (ECETOC, 2017; Ott et al., 2019). One of the outcomes from this workshop was the agreement to validate a marine erBST with increased biomass concentrations and extended test durations in a ring test at several locations with different seawater. In 2017, the multi-laboratory validation was conducted at 13 laboratories in Europe, Japan and North America, closely following the OECD Guidance Document 34 on the correct validation of new or updated test methods (OECD, 2005). The ring test compared the accuracy and reliability of classifying the biodegradability of a set of five unlabeled reference chemicals in the standard

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OECD 306 closed bottle test with a new test, which was based on the erBST (Ott et al., 2020). 4. Conclusions The environmentally relevant biodegradation screening test (erBST) described is an improved test to screen for non-persistent chemicals in the marine environment. The marine erBST was more reliable and less variable than the comparator BST in assessing the persistence of a set of five reference compounds. In the erBST, increased biomass aimed to better represent the bacterial diversity inherent in the sampled environment. In addition, prolonged test durations allowed the capture of adaptation during the lag phase followed by growth-linked biodegradation. It is crucial to improve these first tier screening tests to reduce any further potentially unnecessary persistence, bioaccumulation and toxicity testing. Improved persistence assessment can save high numbers of fish lives and reduce costs by reliably screening out non-persistent chemicals earlier in the risk assessment process. Declaration of competing interest The authors declare no competing financial interest. Acknowledgement This work was supported by the European Chemical Industry Council Long-range Initiative (Cefic LRI; project ECO11) with in-kind support from AstraZeneca. Russell Davenport also acknowledges a Challenging Engineering award from the Engineering and Physical Sciences Research Council (EP/I025782/1). References Ahtiainen, J., Aalto, M., Pessala, P., 2003. Biodegradation of chemicals in a standardized test and in environmental conditions. Chemosphere 51, 529–537. https://doi.org/ 10.1016/S0045-6535(02)00861-5. Brixham Environmental Laboratories, 2013. Guidelines for Assessing the Ready Biodegradability of Chemicals: Overview of OECD 301 B Testing Procedures. Brown, D.M., Hughes, C.B., Spence, M., Bonte, M., Whale, G., 2018. Assessing the suitability of a manometric test system for determining the biodegradability of volatile hydrocarbons. Chemosphere 195, 381–389. https://doi.org/10.1016/j. chemosphere.2017.11.169. Comber, M., Holt, M., 2010. Developing a Set of Reference Chemicals for Use in Biodegradability Tests for Assessing the Persistency of Chemicals. Courtes, R., Bahlaoui, A., Rambaud, A., Deschamps, F., Sunde, E., Dutrieux, E., 1995. Ready biodegradability test in seawater: a new methodological approach. Ecotoxicol. Environ. Saf. https://doi.org/10.1006/eesa.1995.1054. ECETOC, 2003. Persistence of Chemicals in the Environment No. 90, Technical Report. (Brussels) ECETOC, 2007. Workshop on Biodegradation and Persistence No. 10, Workshop Report. Brussels ECETOC, 2009. Collation of Existing Marine Biodegradation Data and its Use in Environmental Risk Assessment No. 108, Technical Report. Brussels ECETOC, 2013. Assessing Environmental Persistence No. 24, Workshop Report. Brussels ECETOC, 2017. Improvement of the OECD 306 screening test: Workshop held at CEFAS laboratories, Lowestoft, UK 17-18 February 2015 and subsequent ring test No. 34, Workshop Report. (Brussels) ECHA, 2008. Guidance on information requirements and chemical safety assessment, chapter R . 7b : endpoint specific guidance (version 1.0) ECHA, 2008. Guidance on information requirements and chemical safety assessment, chapter R. 7b : endpoint specific guidance (Version 1.1) ECHA, 2017a. Guidance on information requirements and chemical safety assessment, chapter R.11: PBT/vPvB assessment (version 3.0). doi:10.2823/128621 ECHA, 2017b. Guidance on information requirements and chemical safety assessment, chapter R . 7b : endpoint specific guidance (version 4.0). doi:10.2823/84188 ECHA, 2018. What is a Registered Substance Factsheet? doi:10.2823/42973 ECHA, 2019a. Brief profile aniline [WWW document]. URL. https://echa.europa.eu/fi/ brief-profile/-/briefprofile/100.000.491. ECHA, 2019b. Brief profile 4-fluorophenol [WWW document]. URL. https://www.echa.europa.eu/brief-profile/-/briefprofile/100.006.124. ECHA, 2019c. Brief profile 4-chloroaniline [WWW document]. URL. https://www.echa.europa.eu/brief-profile/-/briefprofile/100.003.093. ECHA, 2019d. Substance information pentachlorophenol [WWW document]. URL. https://echa.europa.eu/fr/substance-information/-/substanceinfo/100.001.617. ECHA, 2018. Brief profile 4-nitrophenol [WWW Document]. URL https://echa.europa.eu/ fr/brief-profile/-/briefprofile/100.002.556 (accessed 5.11.19)

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