Binding to plastoquinone B site leading to decreased population growth rate via photosystem II inhibition

• Li Xie
• Knut Erik Tollefsen

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Taxonomic Applicability

Since the MIE (binding of an inhibitor to the QB site of the D1 protein) targets one of the most highly conserved components of the photosynthetic apparatus across taxa, AOP 567 is broadly applicable to all oxygenic photosynthetic organisms that contain Photosystem II (PSII), which included photosynthetic primary producers of many different taxonomic groups. The underlying mechanism, PSII inhibitor binding to D1 QB site, is conserved across these groups. Sensitivity differences reflect physiological traits (e.g., pigment composition, repair rates, metabolic flexibility) rather than differences in molecular target presence. Heterotrophic organisms lacking PSII are not directly affected, though they may experience indirect impacts through reduced primary productivity. For regulatory purposes, a representative set of test species (e.g., green alga, cyanobacterium, diatom, aquatic plant) can adequately capture the taxonomic domain of applicability for this AOP.

Higher plants (aquatic angiosperms). Aquatic vascular plants such as seagrasses (Halophila ovalis, Zostera muelleri) and freshwater macrophytes (e.g., Lemna gibba, Myriophyllum spicatum) rely on PSII for photosynthesis and are highly sensitive to PSII herbicides. In seagrasses, diuron or atrazine at 1–10 µg/L can cause ~50% inhibition of PSII yield within 72 h, with chronic exposures leading to growth and survival impacts. Laboratory tests with Lemna show frond multiplication inhibited at low tens of µg/L. The D1 protein structure in higher plants is essentially identical to that in algae, and resistance mutations (e.g., psbA Ser264→Gly in triazine-resistant weeds) confirm cross-taxa conservation of the binding site. While terrestrial plants are the intended targets of these herbicides, aquatic plants are affected via environmental exposure.

Diatoms and algae groups. Diatoms can be amongst the most sensitive taxa; Chaetoceros muelleri has a 72h reduced growth under 1.5 ug/l diuron exposure. There is clear PSII inhibition in cryptophytes( e.g., Rhodomonas salina; 72-h growth EC50 with 6 µg/L diuron). The structure of PSII is the same across rhodophytes and other macroalgae, except that the composition of other pigments can affect the scaling of repair and the sensitivity of repair. 

Cyanobacteria. These prokaryotic phototrophs have PSII functionally equivalent to that in eukaryotes and are susceptible to PSII herbicides. Laboratory studies show inhibition of PSII activity and growth in species such as Nostoc, Aphanocapsa, and Aulosira (96-h atrazine EC₅₀ ~147 µg/L). Some cyanobacteria exhibit greater tolerance or can exploit ecological shifts (e.g., nitrogen fixation) to gain competitive advantage when co-occurring algae are suppressed, but direct effects occur at sufficiently high or prolonged exposures.

Other photosynthetic protists that contains PSII,  Euglenophytes, dinoflagellates, and coral symbionts (Symbiodiniaceae) are also affected. For example, Symbiodinium spp. show significant declines in Fv/Fm at 1–3 µg/L diuron, contributing to coral bleaching events.

Sex and life stage

Sex is not a relevant domain of applicability for this AOP, as the key events are rooted in the universally conserved physiology of PSII and cellular energy metabolism.

All life stages that rely on functional photosynthesis are within the domain of applicability. Early developmental stages, such as germinating seeds, algal spores, or propagules of aquatic plants, may be particularly vulnerable due to their high energy demands and limited capacity for metabolic compensation (Amthor & Baldocchi, 2001).

Biological context:

The AOP is applicable under both laboratory and environmentally realistic exposure conditions. While higher doses are often used in controlled studies to establish mechanistic linkages, environmentally relevant, sublethal concentrations have been shown to impair PSII efficiency, photosynthesis, and growth in aquatic systems (Wilkinson et al., 2015; Hanson et al., 2023). Given the persistence and mobility of PSII inhibitors in surface and ground waters, chronic exposures in aquatic environments are particularly relevant.

Evidence for essentiality in an AOP refers to demonstrating that if an upstream key event is prevented or reversed, the subsequent downstream events do not occur (or normal recovery).  In AOP 567, the essentiality of events were determined based on exposure-recovery experiments, the inhibition-rescue experiments as well as the mechanistic studies of aquatic plants and algae. The essentiality of the MIE (Event 2307: Binding of plastoquinone B to the D1 protein) is considered high, since this is the well characterised target site of most PSII-inhibitor herbicides. A number of reports show that the herbicide can be artificially removed or that replacement of the PSII acceptors by oxidants with alternative electron acceptors (e.g., decyl-plastoquinone) will allow restoration of the activity of PSII and avoid downstream consequences on photosynthesis and subsequent growth. Similarly, KE1 (Event 1862: Decrease in PSII efficiency) is also considered high in essentiality. Inhibition of PSII activity directly results in reduced quantum yield and electron transport, and restoration of PSII activity has been consistently associated with recovery of photosynthetic performance. There is strong empirical evidence showing that PSII efficiency (e.g., Fv/Fm, ΦPSII) strongly correlates with primary productivity in photosynthetic organisms. And recovery study on seagrass after exoposure to diuron indicated the ΔF/Fm′ and growth rate returned over few weeks after removal to clean water. The KE2 (Event 1472: Decrease in photosynthesis) is assigned to high essentiality as well. Studies in both algae and aquatic plants have shown that when photosynthesis is suppressed, growth rapidly declines, and normal function resumes when photosynthetic activity is restored. In contrast, KE3 (Event 1545: Decrease in mitochondrial oxidative phosphorylation) is considered to have moderate essentiality. While evidence indicates that mitochondrial ATP production is reduced following photosynthetic inhibition due to limited carbohydrate substrate availability, the capacity for partial compensation through glycolysis or alternative pathways reduces the strength of causal inference. Finally, KE4 (Event 1472: Decrease in ATP production) also holds moderate essentiality. Growth inhibition is mechanistically connected to a lack of ATP. However, few direct rescue studies, using ATP precursors or supply of energy, provide uncertainty. 

1. Biological Plausibility

Biological plausibility means the known structural, or functional correlations between upstream key event and downstream key event in unperturbed biology or normal conditions (OECD, 2018). Such basic knowledge forms what can be used to extrapolate or make a hypothesis of the probable effects of a biological perturbation induced by a stressor. 

The biological plausibility of KER3556 (Binding of plastoquinone B leads to Decrease in Photosystem II efficiency) is considered high. Under normal photosynthetic conditions, plastoquinone at the Q_B site of Photosystem II (PSII) accepts electrons from QA and enables the continuation of electron flow through the thylakoid membrane. Binding PSII-inhibitor to the Q_B site blocks this electron transfer, directly impairing photochemical efficiency (Battaglino, Grinzato and Pagliano, 2021). Such a structure-function relationship is known well within plant and algal systems and forms the basis MoA of many commercial herbicides. 

The biological plausibility of KER2333 (Decrease in PSII efficiency leads to Decrease in Photosynthesis) has been rated high as well. The effectiveness of PSII determines the ability of the plant to utilize the light available to generate biochemical energy by the process of ATP and NADPH synthesis and subsequently carbon fixation. Decreased energy transmission causes a downstream decrease in CO₂ assimilation, and hence this relationship is well supported by both mechanistic knowledge and experimentation. 

The biological plausibility of KER3557 (Decrease in Photosynthesis leads to Decrease in Mitochondrial OXPHOS) is considered high. Mitochondrial respiration in photosynthetic organisms relies on photosynthetically produced oxygen-dependent oxidative phosphorylation with carbohydrates. In normal physiological states, the functions of chloroplast and mitochondria are in highly coordinated patterns. A drop in photosynthetic carbon fixation diminishes the availability of substrate to the mitochondrial respiration, therefore restricting the OXPHOS capability. The interdependence in this functional nature is clearly proven in plots and algae systems and this offers a good mechanism to this relationship. 

The biological plausibility is also high in the case of KER3558 (Decrease in Mitochondrial OXPHOS leads to Decrease in ATP production). In eukaryotes mitochondrial oxidative phosphorylation is the primary route of cellular ATP production and any impairment of the electron transport chain or proton gradient will directly reduce the efficiency of ATP synthesis. The structural and functional reliance of generation of ATP on OXPHOS is evolutionarily preserved, and as a result this is an extremely probable causative relationship. 

The biological plausibility of KER3559 (Decrease in ATP production leads to Decrease in Population growth rate) is similarly high. ATP at the cellular level is important in facilitating energy-demanding functions of DNA replication, protein synthesis and cell division all of which are preparatory to the expansion of the population. Photoautotrophic organisms suffer with ATP deficits maintained, which suppresses the cell proliferation and lowers biomass gain. Mathematical prediction of ATP limitation of cellular and population level growth has been borne out by numerous experiments making this KE relationship quite convincing. 

2. Empirical Support

Empirical support is moderate to high.

According to OECD AOP guidance, the empirical support of a key event relationship (KER) is determined based on evidence to illustrate dose-response consistency (the upstream KE is impacted at a concentration or dose equal to or lower than that which impacts the downstream KE), temporal concordance (the upstream KE occurs before the downstream KE is observed) and incidence concordance (the upstream KE is observed in an equal or greater proportion of the test population compared to the downstream KE). For AOP 567, empirical support for the KERs ranges from moderate to high, with particularly strong evidence for the early, photochemical stages.

The empirical evidence in support of KER3556 (Binding of plastoquinone B results in Decrease in photosystem II efficiency) is high, as  many experiments on photosystem II (PSII) inhibitors (including the herbicides diuron, atrazine, and etc.) show instantaneous, dose-dependent reductions in PSII efficiency when the Q_B-binding site is occupied by an inhibitor. For instance, in isolated pea thylakoid membranes, Q_B site herbicides with higher inferred Q_B affinity (diuron, terbuthylazine, metribuzin) inhibited PSII electron transfer and fluorescence-based PSII performance at substantially lower concentrations than lower-affinity inhibitors (e.g., bentazon, metobromuron). Reported I50​ values were around 7–8 × 10⁻⁸M for diuron and 1–2 × 10⁻⁷ M for terbuthylazine/metribuzin, derived from both DPIP photoreduction (PSII activity) and OJIP fluorescence (1–V_j, reflecting QA reduction and Q_B site interference). (Battaglino, Grinzato and Pagliano, 2021).

The empirical evidence in support of KER2333 (Decrease in PSII efficiency leads to Decrease in Photosynthesis) is high, as multiple studies demonstrate linear or proportional correlations between PSII inhibition (e.g., reduced Fv/Fm or ΔF/Fm′) and declines in net photosynthetic rate, CO2 fixation, or overall photosynthetic efficiency. Dose-dependent reductions in photosynthesis follow PSII blockage, with EC50 values often aligning closely with those for PSII efficiency (e.g., diuron causing ~50% Pn reduction at ~1 µM in algae; terbuthylazine EC50 ≈0.3 µM in sugar beet). Temporal evidence shows photosynthetic decline occurring shortly after PSII inhibition, typically within 2–4 hours, with chronic exposure amplifying effects through photodamage and ROS accumulation (Morin et al., 2018). In freshwater biofilms, diuron induced rapid photosynthesis inhibition (t1/2 for 50% inhibition ranging from <30 s at high doses to ~7 min at lower doses), directly linking PSII blockage to downstream photosynthetic output (Morin et al., 2018). This linkage holds robustly in microalgae (e.g., Chlorella, Scenedesmus), seagrasses, and higher plants via OJIP kinetics and gas exchange data.

The empirical evidence in support of KER3557 (Decrease in Photosynthesis leads to Decrease in Mitochondrial OXPHOS) is moderate, primarily inferred from aquatic models with limited direct data in terrestrial plants. Photosynthetic inhibition restricts carbohydrate substrate supply, reducing NADH/FADH2 availability for mitochondrial respiration and leading to decreased oxygen consumption rates (e.g., 30–50% reduction at 1 µM diuron in Chlamydomonas reinhardtii). Temporal patterns indicate mitochondrial effects follow photosynthetic decline, measurable within 6–12 hours and intensifying under chronic exposure (days–weeks) due to persistent energy deficits (Seloto et al., 2024). Bioenergetic principles support this cascade in algae, with analogous inferences for terrestrial species (e.g., Arabidopsis thaliana), though direct terrestrial measurements remain sparse.

The empirical evidence in support of KER3558 (Decrease in Mitochondrial OXPHOS leads to Decrease in ATP production) is moderate, based on studies showing direct proportionality between reduced OXPHOS and ATP depletion. Dose-response data indicate ATP levels decline with OXPHOS impairment (e.g., ~40% reduction at 1 µM diuron in Chlorella; EC50 ≈0.5 µM for diuron in algae), as diuron inhibits photochemical (but not oxidative) ATP synthesis, while related compounds affect both pathways (John, 1971). Temporal dynamics reveal ATP depletion following OXPHOS reduction within 12–24 hours in aquatic models, with sustained exposure causing severe cellular energy shortages. This linkage is conserved across mitochondrial bioenergetics in algae and inferred for terrestrial plants (e.g., Pisum sativum, Arabidopsis thaliana), though direct evidence in plants is limited (Seloto et al., 2024).

The empirical evidence in support of KER3559 (Decrease in ATP production leads to Decrease in Population growth rate) is high, as ATP depletion limits energy for cell division, growth, and reproduction, resulting in clear dose-dependent reductions in population growth rates across primary producers. Dose-response curves show consistent EC50 values for growth inhibition (e.g., diuron 50% inhibition in Chlorella vulgaris at ≈0.5 µM; terbuthylazine ≈0.4 µM in diatoms; diuron EC50 3.1–206 µg/L in marine microalgae like Tisochrysis lutea and Tetraselmis sp.) (Flores et al., 2024). Temporal evidence indicates growth declines lag ATP reduction, becoming significant after 24–48 hours in microalgae and weeks in terrestrial/macrophyte systems (e.g., reduced biomass, seed germination). Chronic exposures (weeks) severely restrict population expansion in aquatic primary producers (e.g., seagrasses, microalgae), with field-relevant diuron levels (e.g., 0.3 µg/L) causing sublethal PSII inhibition and growth suppression (Magnusson et al., 2008; Thomas et al., 2020).

Overall WoE Considerations 

The AOP is biologically plausible and mechanistically conserved across primary producers. Dose-response and temporal concordance are strong for early KEs (MIE to photosynthesis) and moderate-to-strong for downstream linkages, supported by consistent patterns in dose- and time-resolved experiments (Morin et al., 2018; Battaglino et al., 2021). Evidence gaps remain for direct terrestrial plant data on mitochondrial/ATP KEs, warranting further targeted studies. This AOP supports predictive risk assessment for PSII herbicides in aquatic and terrestrial ecosystems.

Uncertainties, inconsistencies, and critical gaps

Overall, AOP 567 still constrains several uncertainties and knowledge gaps that need to be addressed to strengthen the reliability. First, for KER3557, the direct causal relationship between decreased photosynthesis (KE1472) and subsequent reduction in mitochondrial oxidative phosphorylation (OXPHOS; KE1545) remains not well-established. While it is logical that loss of chloroplast-derived ATP/NADPH due to PSII inhibition will lead to cellular energy deficits, empirical evidence for a direct impairment of mitochondrial function is limited. Studies on PSII-inhibiting herbicides (e.g., atrazine) have primarily documented chloroplast effects (reduced CO₂ fixation and chloroplast ATP synthesis) and oxidative stress in the chloroplast, with much less focus on mitochondria. Any mitochondrial impacts may occur only under prolonged or high exposures, if at all, introducing uncertainty in temporal and dose concordance for KER3557. In other words, mitochondrial dysfunction might lag behind the initial photosynthesis decline or require higher doses, rather than occurring in parallel at lower exposure levels. This gap in evidence leaves the Photosynthesis→OXPHOS linkage supported more by biological rationale than by consistent experimental data.. Additionally, Some studies on PSII inhibitors show no significant mitochondrial disruption, attributing growth declines solely to photosynthetic energy loss. For example, in certain algal species, ATP deficits are predominantly linked to chloroplastic ATP synthase inhibition rather than mitochondrial OXPHOS, creating inconsistencies with the proposed pathway. This may reflect species-specific differences or assay sensitivities, but it challenges the universality of the KER between photosynthesis decrease and mitochondrial OXPHOS impairment. Empirical data from population growth assays (e.g., in Lemna or Chlamydomonas) occasionally show non-monotonic responses, where low doses stimulate growth (hormesis) despite PSII efficiency drops, inconsistent with linear KER predictions. Such inconsistencies could arise from compensatory mechanisms like enhanced respiration, which are not fully accounted for in the AOP. The AOP is primarily applicable to photosynthetic eukaryotes (e.g., algae, aquatic plants), but gaps exist for non-photosynthetic taxa or prokaryotes, where plastoquinone analogs (e.g., ubiquinone in bacteria) might alter the MIE. Life stage-specific data are limited; juvenile stages may be more sensitive due to higher metabolic demands, but few studies address this. Sex applicability is generally not relevant for hermaphroditic or asexual species but requires exploration in dioecious plants. Important data gaps include the paucity of long-term studies linking the early key events (e.g., decreases in cellular ATP production) to the final adverse outcome (population growth decline) under realistic environmental conditions. Most evidence supporting this AOP comes from short-term laboratory exposures with constant conditions, whereas in nature, organisms experience fluctuating light, nutrient levels, and intermittent exposures. We lack chronic or multi-generation experiments that demonstrate a direct, quantitative progression from sustained ATP/OXPHOS deficits to measurable population-level effects (such as reduced reproduction or population growth rates) in photosynthetic organisms. Additionally, the AOP in its current form does not incorporate alternative or parallel pathways that could also lead to growth impairment. For instance, severe energy stress from PSII inhibition can trigger oxidative damage leading to cell death (e.g., via programmed cell death/apoptotic-like pathways in plants) or could impair other physiological functions like nutrient uptake and assimilation. These outcomes could contribute to growth and survival impacts independently of—or alongside—ATP shortage, but they are not accounted for in the linear sequence of key events. This represents a gap in the network completeness of the AOP. Addressing these uncertainties will require targeted research. Future studies should focus on experiments that probe the interaction between chloroplast and mitochondrial function under stress (to clarify the causality and timing in KER3557) and include a broader array of species (including different algal/plant taxa and life stages) to test the AOP’s generality. Conducting longer-duration exposures that monitor recovery or compounding effects over time would help establish whether short-term energy deficits indeed translate to long-term population declines. Moreover, incorporating emerging omics data is a promising avenue: transcriptomic and proteomic analyses under PSII-inhibitor exposure can reveal early biomarkers and compensatory changes (e.g., upregulation of respiratory genes or stress-response pathways), thereby refining our understanding of each key event and its quantitative relationship. Integrating such omics insights in future AOP updates could improve the weight of evidence for the KERs and support the development of predictive models for regulatory use.

Overall, these uncertainties, inconsistencies, and gaps highlight the need for caution when applying AOP 567 and point to specific areas (temporal concordance, dose-response shape, taxonomic scope, and additional pathways) where further research and data generation would greatly enhance the AOP’s robustness and utility in risk assessment.

The quantitative understanding of KER#3556 (Q_B binding → PSII efficiency) is considered low, because although target-site inhibition occurs at nanomolar concentrations (I₅₀ diuron ≈ 7–8 × 10⁻⁸ M in PSII assays; Battaglino et al., 2021), the quantitative relationship between fractional Q_B occupancy and functional decline in Fv/Fm has not been parameterised. The available data demonstrate high potency at the binding site, but there is no established response–response model linking molecular occupancy to photochemical efficiency reduction.

The quantitative understanding of KER#2333 (PSII efficiency → Photosynthesis) is considered high, because concordant EC₅₀ values have been reported between fluorescence-derived PSII endpoints and functional photosynthetic measures. In Rhodomonas salina, diuron produced an EC₅₀ of 1.71 µg/L for ΔF/Fm′ compared to 6.27 µg/L for growth (Thomas et al., 2020). Similarly, atrazine EC₅₀ values for fluorescence (232 µg/L) closely matched those for oxygen evolution (222 µg/L) (USEPA, n.d.). These datasets demonstrate strong quantitative alignment between reduced PSII efficiency and decreased photosynthetic performance, although compensatory non-photochemical quenching introduces variability across taxa.

The quantitative understanding of KER#3557 (Photosynthesis → Mitochondrial OXPHOS) is considered moderate, because limited simultaneous multi-endpoint studies are available. In Lemna minor, the effect dose rate for a 10% reduction in CO₂ uptake (EDR₁₀ = 2.8 mGy/h) was substantially lower than that for mitochondrial membrane potential (EDR₁₀ = 21.8 mGy/h) (Xie et al., 2019), indicating an approximate eightfold separation between upstream functional decline and downstream mitochondrial impairment. While this supports directional linkage, a fully parameterised quantitative response–response model has not yet been established.

The quantitative understanding of KER#3558 (Mitochondrial OXPHOS → ATP production) is considered moderate, because direct respiratory inhibition studies demonstrate clear concentration–response relationships. Rotenone inhibits Complex I with IC₅₀ values of approximately 5–20 nM and produces 40–80% ATP reduction, with nonlinear behaviour observed when OXPHOS inhibition exceeds 70–80% (Hatefi, 1985; Nicholls, 2004). However, within the context of AOP 567, the relevant mechanism is substrate-limited OXPHOS following reduced photosynthesis rather than direct electron transport inhibition, limiting direct quantitative extrapolation.

The quantitative understanding of KER#3559 (ATP production → Population growth rate) is considered moderate, because quantitative concordance between ATP depletion and growth inhibition has been demonstrated but not yet fully parameterised across the entire concentration range. In Chlamydomonas reinhardtii, the EC₅₀ for ATP reduction (0.16 µM) was lower than that for growth inhibition (0.41 µM), representing an approximately 2.6-fold separation (Nestler et al., 2012). Additionally, Bayesian modelling in Lemna minor provides a quantitative framework linking energetic impairment to demographic outcomes (Moe et al., 2021). Nevertheless, a fully integrated quantitative AOP model spanning all key events has not yet been developed.

Table 1. Summary of quantitative understanding for each KER in AOP 567