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Laccase mediator systems do not detectably degrade PFOA or PFOS; the reported loss is sorption to the enzyme

Can a laccase mediator system be optimised to degrade perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), as two earlier reports of modest losses suggested?

Source: An Artifact of Perfluoroalkyl Acid (PFAA) Removal Attributed to Sorption Processes in a Laccase Mediator System (doi:10.1021/acs.estlett.3c00173). Path record: acs-laccase-path.json. Headline outcome negative-not-replicated. While unable to replicate the oxidation of PFAAs, we show that interactions of the PFAA compounds with laccase and laccase mediator mixtures could cause an artifact that mimics transformation (<=60 %) of PFAAs. the LMS appears to be unable to detectably degrade the perfluorinated compounds PFOA and PFOS ... it is particularly important for researchers to use rigorous controls when analyzing treatment and removal strategies with novel bioinspired technologies All graphs · Back to the pilot

How to read these graphs

A discourse graph breaks a piece of research into typed one-sentence nodes and draws how they bear on one another. The grammar is that of Discourse Graphs (Chan and Akamatsu). Read each graph left to right: a question, the experiments that addressed it, the results they produced, and the hypothesis every result is drawn against.

QUE Question: What the work set out to answer.
HYP Hypothesis: The answer the authors expected. Every result earns one edge against it.
EXP Experiment: One attempt at the question: a trial arm, an assay, a survey, an analysis.
RES Result: One finding from an experiment, with where in the source it comes from.
ISS Issue: An open question or untried branch the work leaves for someone else.

addresses: An experiment addresses the question.
produces: An experiment produces a result.
supports: A positive result that backs the hypothesis.
opposes: A negative result that counts as evidence against the hypothesis, or a refutation of an earlier claim.
informs: A partial or mixed result that bears on the hypothesis without settling it.
consistent with: A negative or inconclusive result that does not count either way: no positive control passed, or there is no sensitivity or power statement, so the absence of an effect cannot be told from a measurement that failed.
relation: A thin dotted line: a link the source itself states between two experiments, such as one being derived from another.

A dashed box marks a result the rule above counts as uninformative, or an issue. In the drawing, click a node to jump to its text below.

The graph

Read left to right. The question (QUE) is addressed by 4 experiments (EXP), each of which produces results (RES), and each result bears on the hypothesis (HYP) with one of four edges. 6 support it, 3 oppose it, 0 inform it, and 2 are merely consistent with it because the informativeness rule would not count them as evidence either way. Click a node to jump to its full text below.

supports positive finding
opposes informative negative, or refutation
informs partial finding
consistent with inconclusive or uninformative: no positive control passed, or no sensitivity statement
thin grey a relation the record itself states between two attempts
dashed box a result the informativeness rule marks uninformative, or an issue

The grammar is that of Discourse Graphs (Chan and Akamatsu): QUE, HYP, EXP, RES and ISS nodes with supports, opposes and informs edges. Every node and edge is derived from fields already in the Negative-Results Record; the edge a finding earns is decided by its outcomeClass and informativeness. Issues (ISS) hang below the graph as a strip: the open questions and untried branches the path leaves for someone else to pick up.

Nodes

Question and hypothesis

QUE: Can a laccase mediator system be optimised to degrade perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), as two earlier reports of modest losses suggested?
HYP: Improve the scope and efficacy of laccase mediator system (LMS) treatment of perfluoroalkyl acids by a mechanistic investigation of each component, starting from literature reports of modest PFOA and PFOS losses over extended periods.

Experiments (4 attempts)

Node	Attempt	Type	Headline	JSON
EXP	Two commercial laccases with five nitroxyl mediators do not remove PFOS over 28 days we observed no significant differences among the enzyme/mediator treatment conditions and the enzyme-only control (Figure S2) p = 0.58 (white rot) and 0.24 (Agaricus bisporus).	`wet-lab`	`inconclusive-no-positive-control`	record
EXP	TvL with HBT does not remove PFOA over two weeks; the 64 to 67 % concentration fall at 0.1 uM is recovered in full on extraction monitoring PFOA treatment with six doses of 1 unit/mL TvL and 1 mM HBT over the course of 2 weeks did not indicate significant removal compared to an untreated control (p = 0.29; two-tailed t test) At 0.1 uM, a 64 % decrease in PFOA concentration and a 67 % decrease in PFOS concentration at the 24 hour time point after two doses, but 99 +/- 14 % of the PFOA mass and 111 +/- 11 % of the PFOS mass were recovered in the extracted solutions, suggesting that the decrease in concentration was likely due to physical phenomena (e.g., sorption)	`wet-lab`	`negative-not-replicated`	record
EXP	The apparent PFOA and PFOS loss is sorption to the laccase, 18 +/- 2 % and 34 +/- 4 %, with a complete mass balance apparent PFOA loss in the TvL/HBT-treated reactors of 18 +/- 2 % compared to the total PFOA mass extracted; for PFOS 34 +/- 4 %; in the reactors containing only the TvL enzyme, similar losses for PFOS of 35 +/- 3 %; for PFOA a substantial mass loss was not observed sorption of PFOS to the reactor was greater in the enzyme-free control (40 +/- 12 % of total mass) relative to the TvL/HBT (14 +/- 2 %) and TvL (12 +/- 1 %) treatments, suggesting that sorption to the protein was more favorable PFOA sorption appeared substantial only where the mediator was present, consistent with side-chain exposure or charge changes on oxidation of TvL; PFOS sorption was unaltered by the mediator, consistent with a structurally intact side chain and PFOS hydrophobicity	`wet-lab`	`positive`	record
EXP	The TvL/HBT system is active: carbamazepine is transformed, the BTNO radical forms, and enzyme activity is retained in the PFAA matrix About 35 % CBZ removal (p < 0.001); BTNO radical detected by EPR in the laccase system; TvL activity retained in CuSO4 and acetate.	`wet-lab`	`positive`	record

Results (11 findings)

Node	Finding	Id	Outcome and informativeness	Edge	JSON
RES	Does a native white-rot laccase (Creative Enzymes) with any of five nitroxyl mediators remove PFOS over 28 days? target: PFOS; PFOS concentration relative to the no-mediator control after evaporation correction = no significant difference (n = 3)	`screen-white-rot` in acs-laccase-mediator-screen	`inconclusive-no-positive-control` uninformative no positive control	consistent-with HYP	record
RES	Does Agaricus bisporus laccase (Sigma) with any of five nitroxyl mediators remove PFOS over 28 days? target: PFOS; PFOS concentration relative to the no-mediator control after evaporation correction = no significant difference (n = 3)	`screen-agaricus` in acs-laccase-mediator-screen	`inconclusive-no-positive-control` uninformative no positive control	consistent-with HYP	record
RES	Does TvL with HBT, dosed six times over two weeks, remove 1 uM PFOA? target: PFOA; PFOA concentration relative to untreated control after two weeks = no significant removal (n = 3)	`pfoa-two-weeks` in acs-laccase-pfaa-degradation	`negative-not-replicated` informative designated positive control passed; negative control clean; sensitivity or power stated	opposes HYP	record
RES	Is the fall in measured PFOA concentration after 24 h of TvL/HBT dosing a transformation? target: perfluorooctanoic acid (PFOA); decrease in aqueous concentration at 24 h = 64 % (n = 3)	`pfoa-96h-apparent-loss` in acs-laccase-pfaa-degradation	`refuted` not-applicable an affirmative refutation is judged on the refuting analysis, not on the controls of the run it refutes	opposes HYP	record
RES	Is the fall in measured PFOS concentration after 24 h of TvL/HBT dosing a transformation? target: perfluorooctanesulfonic acid (PFOS); decrease in aqueous concentration at 24 h = 67 % (n = 3)	`pfos-96h-apparent-loss` in acs-laccase-pfaa-degradation	`refuted` not-applicable an affirmative refutation is judged on the refuting analysis, not on the controls of the run it refutes	opposes HYP	record
RES	Where does the PFOA mass that disappears from solution in TvL/HBT reactors go: protein, vessel or transformation? target: perfluorooctanoic acid (PFOA); apparent loss from the 24 h subsample relative to total extracted mass, TvL + HBT = 18 % (n = 3)	`pfoa-partition` in acs-laccase-sorption	`positive` not-applicable rule applies to negative and inconclusive findings only	supports HYP	record
RES	Where does the PFOS mass that disappears from solution in TvL/HBT reactors go: protein, vessel or transformation? target: perfluorooctanesulfonic acid (PFOS); apparent loss from the 24 h subsample relative to total extracted mass, TvL + HBT = 34 % (n = 3)	`pfos-partition` in acs-laccase-sorption	`positive` not-applicable rule applies to negative and inconclusive findings only	supports HYP	record
RES	Does PFOS sorb to the glass reactor, and does the presence of enzyme change how much? target: perfluorooctanesulfonic acid (PFOS); PFOS mass recovered from the emptied reactor, enzyme-free control = 40 % (n = 3)	`pfos-reactor-sorption` in acs-laccase-sorption	`positive` not-applicable rule applies to negative and inconclusive findings only	supports HYP	record
RES	Does the TvL/HBT system transform the surrogate substrate carbamazepine under literature conditions? target: carbamazepine (CBZ); CBZ removal after three doses over 120 h = 35 % (n = 3)	`cbz-transformed` in acs-laccase-system-checks	`positive` not-applicable rule applies to negative and inconclusive findings only	supports HYP	record
RES	Does TvL retain activity in the CuSO4 solution used for PFAA reactors, compared with acetate buffer? target: Trametes versicolor laccase (TvL); laccase activity profile by DMP assay = similar in 5 mM CuSO4 and 0.1 M sodium acetate; none in 5 mM FeSO4	`activity-retained` in acs-laccase-system-checks	`positive` not-applicable rule applies to negative and inconclusive findings only	supports HYP	record
RES	Does the TvL/HBT/CuSO4 system generate the benzotriazole-N-oxyl (BTNO) radical proposed to oxidise contaminants? target: benzotriazole-N-oxyl radical from HBT; EPR detection of BTNO = detected in both routes	`btno-radical` in acs-laccase-system-checks	`positive` not-applicable rule applies to negative and inconclusive findings only	supports HYP	record

Issues (5 open questions and untried branches)

ISS Why did the earlier Pleurotus ostreatus laccase systems report PFOA and PFOS loss: sorption of the kind shown here, a property of that enzyme, or something else? (open-question)
ISS Which side chains of TvL bind PFOA after oxidation by the mediator? (open-question)
ISS Use rigorous controls, in particular full mass balances including vessel and protein extraction, when evaluating bioinspired PFAS treatments. (proposed-by-authors)
ISS Study PFAS-protein binding mechanisms as a route to bioinspired adsorptive materials, noting that the quantity of enzyme needed for adsorptive removal may be prohibitive. (proposed-by-authors)
ISS Repeat the prior reports' conditions with a Pleurotus ostreatus laccase, with full mass balance, if a source of that enzyme can be found. (inferred-by-ingest)