Binding of plastoquinone B (QB) within D1 protein of Photosystem II

330-54-1

XMTQQYYKAHVGBJ-UHFFFAOYSA-N

Diuron

Urea, N'-(3,4-dichlorophenyl)-N,N-dimethyl- (3-(3,4-DICHLOROPHENYL)-1,1-DIMETHYL UREA 1-(3,4-Dichlorophenyl)-3,3-dimethylurea 1,1-DIMETHYL-3-(3,4-DICHLOROPHENYL)UREA 3-(3,4-DICHLOROPHENYL)-1,1-DIMETHYL UREA Dironet Dironzol Diuron Nortox DP Hardener 95 Dyhard UR 200 Herbatox HRT Dinron Karmex D Karmex Diuron Herbicide Karmex DW Lucenit N'-(3,4-Dichlorophenyl)-N,N-dimethyl urea N'-(3,4-Dichlorophenyl)-N,N-dimethylurea N-(3,4-Dichlorophenyl)-N',N'-dimethylurea N,N-Dimethyl-N'-(3,4-dichlorophenyl)urea N'-3,4-DICHLOROPHENYL N,N-DIMETHYLUREA NSC 8950 Preventol A 6 Telvar Diuron Weed Killer Urea, 3-(3,4-dichlorophenyl)-1,1-dimethyl- 3-(3,4-Dichlorophenyl)-1,1-dimethylurea

DTXSID0020446

1912-24-9

MXWJVTOOROXGIU-UHFFFAOYSA-N

Atrazine

1,3,5-Triazine-2,4-diamine, 6-chloro-N-ethyl-N'-(1-methylethyl)- 1,3,5-Triazine-2,4-diamine, 6-chloro-N2-ethyl-N4-(1-methylethyl)- 1-Chloro-3-ethylamino-5-isopropylamino-2,4,6-triazine 2-Chloro-4-(ethylamino)-6-(2-propylamino)-s-triazine 2-Chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine 2-Chloro-4-(ethylamino)-6-(isopropylamino)triazine 2-Chloro-4-ethylamineisopropylamine-s-triazine 2-Chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine 2-Chloro-4-ethylamino-6-isopropylamino-s-triazine 2-Ethylamino-4-isopropylamino-6-chloro-s-triazine 6-Chloro-4-(ethylamino)-2-(isopropylamino)-s-triazine 6-Chloro-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine AAtrex Nine-O Akticon Aktikon Aktikon PK Aktinit A Aktinit PK Argezin Atragranz Atranex Atranex 80WP Atraphyt Atrataf Atrazin atrazina Azoprim Chromozin Farmozine Gesamprim Gesaprim Gesaprim 50 Gesaprim 500 Gesaprim L Herbatoxol Hungazin Hungazin PK NSC 163046 Nu-Trazine Oleogesaprim Oleogesaprim 200 Primatol A Primoleo Radazin Radazin T s-Triazine, 2-chloro-4-(ethylamino)-6-(isopropylamino)- Triazine A 1294 Zealin L Zeazine

DTXSID9020112

21087-64-9

FOXFZRUHNHCZPX-UHFFFAOYSA-N

Metribuzin

1,2,4-Triazin-5(4H)-one, 4-amino-6-(1,1-dimethylethyl)-3-(methylthio)- 3-Methylthio-4-amino-6-tert-butyl-1,2,4-triazin-5(4H)-one 3-Methylthio-4-amino-6-tert-butyl-1,2,4-triazin-5-one 4-Amino-6-(1,1-dimethylethyl)-3-(methylthio)-1,2,4-triazin-5(4H)-one 4-Amino-6-tert-butyl-3-(methylthio)-1,2,4-triazin-5(4H)-one 4-Amino-6-tert-butyl-3-(methylthio)-1,2,4-triazin-5-one 4-Amino-6-tert-butyl-3-(methylthio)-1,2,4-triazine-5(4H)-one 4-Amino-6-tert-butyl-3-(methylthio)-4,5-dihydro-1,2,4-triazin-5-one 4-Amino-6-tert-butyl-3-(methylthio)-as-triazin-5(4H)-one 4-Amino-6-tert-butyl-4,5-dihydro-3-methylthio-1,2,4-triazin-5-one as-Triazin-5(4H)-one, 4-amino-6-tert-butyl-3-(methylthio)- Lexone DF Metribuzine Sencor 75DF Sencorex Sencorex L.F. Zontran

DTXSID6024204

15545-48-9

JXCGFZXSOMJFOA-UHFFFAOYSA-N

Chlorotoluron

3-(3-Chloro-p-tolyl)-1,1-dimethylurea Urea, N'-(3-chloro-4-methylphenyl)-N,N-dimethyl- 1-(3-Chloro-4-methyl)-3,3-dimethylurea 1-(3-Chloro-4-methylphenyl)-3,3-dimethylurea 1,1-Dimethyl-3-(3-chloro-4-methylphenyl)urea Chlortoluron clorotoluron Dicuran Dicuran 500FL Lentipur Flo Lentipur Forte N'-(3-Chloro-4-methylphenyl)-N,N-dimethylurea N-(3-Chloro-4-methylphenyl)-N',N'-dimethylurea N-(3-Chloro-4-tolyl)-N',N'-dimethylurea N,N-Dimethyl-N'-(3-chloro-4-methylphenyl)urea Syncuran Syncuran 80DP Tolurex Urea, 3-(3-chloro-p-tolyl)-1,1-dimethyl-

DTXSID8052853

51-28-5

UFBJCMHMOXMLKC-UHFFFAOYSA-N

2,4-Dinitrophenol

DNP 1,3-Dinitro-4-hydroxybenzene 1-Hydroxy-2,4-dinitrobenzene 2,4-dinitrofenol Aldifen Dinitrophenol DINITROPHENOL, 2,4- Dinofan Fenoxyl Carbon N NSC 1532 Phenol, α-dinitro- UN 1320 UN 1599 α-Dinitrophenol Phenol, 2,4-dinitro-

DTXSID0020523

87-86-5

IZUPBVBPLAPZRR-UHFFFAOYSA-N

Pentachlorophenol

PCP Phenol, pentachloro- 1-Hydroxy-2,3,4,5,6-pentachlorobenzene 1-Hydroxypentachlorobenzene Chlorophenasic acid CHLOROPHENATE Dowicide EC 7 Dura Treet II Fungifen Grundier Arbezol Lauxtol Liroprem NSC 263497 Penchlorol Pentachlorphenol Perchlorophenol Permasan Phenol, 2,3,4,5,6-pentachloro- Pole topper Pole topper fluid Preventol P Santophen 20 Satophen UN 3155 Witophen P Woodtreat A 2,3,4,5,6-Pentachlorophenol

DTXSID7021106

3380-34-5

XEFQLINVKFYRCS-UHFFFAOYSA-N

Triclosan

5-Chloro-2-(2,4-dichlorophenoxy)phenol Phenol, 5-chloro-2-(2,4-dichlorophenoxy)- 2, 4, 4'-Trichloro-2'-hydroxydiphenylether 2,2'-Oxybis(1',5'-dichlorophenyl-5-chlorophenol) 2,4,4'-TRICHLORO-2'-HYDROXY DIPHENYLETHER 2',4',4-Trichloro-2-hydroxydiphenyl ether 2',4,4'-Trichloro-2-hydroxydiphenyl ether 2,4,4'-Trichloro-2'-hydroxydiphenyl ether 2'-Hydroxy-2,4,4'-trichlorodiphenyl ether 2-Hydroxy-2',4,4'-trichlorodiphenyl ether 3-Chloro-6-(2,4-dichlorophenoxy)phenol 4-Chloro-2-hydroxyphenyl 2,4-dichlorophenyl ether 5-Chloro-2-(2', 4'-dichlorophenoxy) phenol Aquasept Bacti-Stat soap Cansan TCH DIPHENYL ETHER, 2,4,4'-TRICHLORO-2'-HYDROXY- Irgacare MP Irgacide LP 10 Irgaguard B 1000 Irgaguard B 1325 Irgasan Irgasan CH 3565 Irgasan DP 30 Irgasan DP 300 Irgasan DP 3000 Irgasan DP 400 Irgasan PE 30 Irgasan PG 60 Microban Additive B Microban B Oletron Phenol, 5-chloro-2-(2,4-dichlorophenoxy) Phenol, 5-chloro-2-(2,4-dichlorophenoxy)-, dihydrogen phosphate Sanitized XTX Sapoderm SterZac Tinosan AM 100 Tinosan AM 110 TRICLOSAM Ultra Fresh NM 100 Ultrafresh NM-V 2 Vinyzene DP 7000 Yujiexin Zilesan UW

DTXSID5032498

518-82-1

RHMXXJGYXNZAPX-UHFFFAOYSA-N

Emodin

9,10-Anthracenedione, 1,3,8-trihydroxy-6-methyl- 1,3,8-trihidroxi-6-metilantraquinona 1,3,8-Trihydroxy-6-methyl-9,10-anthraquinone 1,3,8-Trihydroxy-6-methylanthrachinon 1,3,8-trihydroxy-6-methylanthraquinone 1,6,8-Trihydroxy-3-methylanthraquinone 3-Methyl-1,6,8-trihydroxyanthraquinone 4,5,7-Trihydroxy-2-methylanthraquinone Anthraquinone, 1,3,8-trihydroxy-6-methyl- Frangula emodin Frangulic acid NSC 408120 NSC 622947 Rheum emodin Schuttgelb

DTXSID5025231

10537-47-0

MZOPWQKISXCCTP-UHFFFAOYSA-N

Malonoben

DTXSID1042106

CHEBI:15422

CHEBI ATP

PCO:0000001

PCO population of organisms

UBERON:0000468

UBERON multicellular organism

GO:0006754

GO ATP biosynthetic process

PCO:0000008

PCO population growth rate

GO:0040007

GO growth

WIKI decreased

Diuron

2018-05-24T15:29:12

atrazine

2016-11-29T18:42:27

Metribuzin

2026-02-23T08:51:24

Chlorotoluron

2026-02-23T08:52:43

2,4-Dinitrophenol

2016-11-29T18:42:27

Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone

2020-11-12T17:59:28

Carbonyl cyanide m-chlorophenyl hydrazone

2020-11-12T17:59:47

Pentachlorophenol

2020-11-12T17:59:12

Triclosan

2020-11-12T18:00:07

Emodin

2020-11-20T13:48:58

Malonoben

2020-11-27T14:43:47

WCS_4472

common ecological species Lemna minor

41457

NCBI Skeletonema pseudocostatum

208873

NCBI Myriophyllum spicatum

906914

NCBI Chlamydomonas reinhardtii CC3269

2850

NCBI Phaeodactylum tricornutum

WCS_3702

common ecological species Arabidopsis thaliana

WCS_4530

common ecological species Oryza sativa

WCS_35525

common ecological species Daphnia magna

3055

NCBI Chlamydomonas reinhardtii

WikiUser_22

all species

WCS_9606

common toxicological species human

10116

NCBI rat

10090

NCBI mouse

WCS_7955

common ecological species zebrafish

WCS_90988

common ecological species fathead minnow

3349

NCBI Pinus sylvestris

10116

NCBI Rattus norvegicus

10090

NCBI Mus musculus

3888

NCBI Pisum sativum

Binding of plastoquinone B (QB) within D1 protein of Photosystem II

Binding to the QB site D1 protein

Molecular

This Key Event describes the competitive displacement of the endogenous electron carrier plastoquinone B (QB) from its binding site in the D1 protein (encoded by psbA) by an exogenous stressor, typically a Photosystem II (PSII) inhibitor. Under normal conditions, a plastoquinone molecule enters the QB site, accepts two electrons from the primary quinone QA and two protons from the stroma, becomes plastoquinol PQH2, and then leaves the site to continue the electron transport chain. Stressor molecules share structural similarities with the quinone ring of plastoquinone. They enter the QB pocket and form stable hydrogen bonds with specific amino acid residues, most critically Serine 264 and Histidine 215. Because the stressor binds with higher affinity or slower dissociation than the natural quinone. This physically blocks the transfer of electrons from QA to QB, and thus reduced photosynthetic efficiency..

The binding of PSII inhibitors to the QB site in the D1 protein can be detected and quantified using a combination of computational, biochemical, biophysical, and functional approaches, each providing complementary lines of evidence. In silico approaches, including quantitative structure–activity relationship (QSAR) modelling and molecular docking, are widely used to predict the affinity and binding orientation of PSII inhibitors within the QB niche of the D1 protein. These methods support chemical screening and mechanistic interpretation of structure–binding relationships (Arnaud et al., 1994; Battaglino, Grinzato and Pagliano, 2021). Radioligand binding assays represent a classical and highly specific experimental approach, in which radiolabelled herbicides are used to directly quantify competitive displacement of plastoquinone or reference inhibitors from isolated thylakoid membranes or PSII preparations (Tischer and Strotmann, 1977; Vermaas, Renger and Arntzen, 1984). These assays provide quantitative binding constants and competitive inhibition profiles. Fluorescence-based techniques, including chlorophyll a fluorescence measurements, are frequently applied as indirect but sensitive indicators of QB-site occupation. Inhibitor binding disrupts electron transfer from QA to QB, leading to characteristic changes in fluorescence yield and PSII photochemical efficiency (Sundby, Chow and Anderson, 1993; Maxwell and Johnson, 2000). These methods are particularly useful for whole-cell or intact-tissue assessments. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) provide label-free, real-time measurements of binding kinetics and thermodynamics between PSII inhibitors and PSII protein complexes or isolated D1 fragments. These approaches allow direct determination of binding affinity, stoichiometry, and energetics (Piletska, Piletsky and Rouillon, 2006; Zimmermann et al., 2006). Resistance mutant analyses, based on naturally occurring or experimentally induced mutations in the psbA gene encoding the D1 protein, offer strong mechanistic evidence for QB-site binding. Altered sensitivity to PSII inhibitors in mutants affecting key QB-site residues (e.g. Ser-264/268) directly links chemical binding to functional inhibition and photosynthetic impairment (Sundby, Chow and Anderson, 1993; Alfonso et al., 1996; Oettmeier, 1999). Finally, structural biology approaches, including X-ray crystallography of PSII complexes, have provided direct visualization of herbicide occupancy within the QB site, confirming binding modes and key amino-acid interactions at atomic resolution (Broser et al., 2011; Zimmermann et al., 2006).

This KE is applicable to oxygenic photosynthetic organisms that possess a functional PSII complex containing the D1 protein encoded by psbA and a conserved QB binding site. The scientific basis for this domain of applicability is the high structural and functional conservation of the QB niche within the D1 protein across cyanobacteria, algae, and higher plants, which underpins both endogenous plastoquinone binding and competitive binding by PSII-inhibiting chemicals. The Weight of Evidence supporting this KE is high, based on: <ul> <li> Direct evidence from radioligand binding, structural studies, and biophysical measurements demonstrating inhibitor occupancy of the QB site; </li> <li> Indirect functional evidence from chlorophyll fluorescence and electron transport inhibition assays; and </li> <li> Biological plausibility and consistency demonstrated by resistance mutations and cross-species conservation of the binding site. </li> </ul> The strong mechanistic understanding and consistency across experimental systems support high confidence in both the domain of applicability and the causal role of this KE within PSII inhibition-related adverse outcome pathways.

High Unspecific

High

All life stages

High Alfonso, M., Pueyo, J.J., Gaddour, K., Etienne, A.-L., Kirilovsky, D. and Picorel, R. (1996). Induced new mutation of D1 serine-268 in soybean photosynthetic cell cultures produced atrazine resistance, increased stability of S2QB- and S3QB-states, and increased sensitivity to light stress. Plant Physiology, 112(4), 1499–1508. Arnaud, L., Taillandier, G., Kaouadji, M., Ravanel, P. and Tissut, M. (1994). Photosynthesis inhibition by phenylureas: A QSAR approach. Ecotoxicology and Environmental Safety, 28(2), 121–133. Battaglino, B., Grinzato, A. and Pagliano, C. (2021). Binding properties of photosynthetic herbicides with the QB site of the D1 protein in plant photosystem II: A combined functional and molecular docking study. Plants, 10(8). Broser, M., Glöckner, C., Gabdulkhakov, A., Guskov, A., Buchta, J., Kern, J., Müh, F., Dau, H., Saenger, W. and Zouni, A. (2011). Structural basis of cyanobacterial photosystem II inhibition by the herbicide terbutryn. Journal of Biological Chemistry, 286(18), 15964–15972. Giardi, M.T. and Pace, E. (2006). Photosynthetic proteins for technological applications. In: Giardi, M.T. and Piletska, E.V. (eds), Biotechnological Applications of Photosynthetic Proteins: Biochips, Biosensors and Biodevices. Springer, Boston, MA, pp. 147–154. Oettmeier, W. (1999). Herbicide resistance and supersensitivity in photosystem II. Cellular and Molecular Life Sciences, 55(10), 1255–1277. Piletska, E.V., Piletsky, S.A. and Rouillon, R. (2006). Sensor systems for photosystem II inhibitors. In: Giardi, M.T. and Piletska, E.V. (eds), Biotechnological Applications of Photosynthetic Proteins: Biochips, Biosensors and Biodevices. Springer, Boston, MA, pp. 130–146. Sundby, C., Chow, W.S. and Anderson, J.M. (1993). Effects on photosystem II function, photoinhibition, and plant performance of the spontaneous mutation of serine-264 in the photosystem II reaction center D1 protein in triazine-resistant Brassica napus L. Plant Physiology, 103(1), 105–113. Tischer, W. and Strotmann, H. (1977). Relationship between inhibitor binding by chloroplasts and inhibition of photosynthetic electron transport. Biochimica et Biophysica Acta (Bioenergetics), 460(1), 113–125. Vermaas, W.F., Renger, G. and Arntzen, C.J. (1984). Herbicide/quinone binding interactions in photosystem II. Zeitschrift für Naturforschung C, 39(5), 368–373. Zimmermann, K., Heck, M., Frank, J., Kern, J., Vass, I. and Zouni, A. (2006). Herbicide binding and thermal stability of photosystem II isolated from Thermosynechococcus elongatus. Biochimica et Biophysica Acta – Bioenergetics, 1757(2), 106–114. AOPWiki 2025-01-14T04:58:47

2026-02-04T06:58:50

Decrease, Photosystem II efficiency

Cellular

The decreased Photosystem II efficiency describes a reduction in the capacity of Photosystem II to convert absorbed light energy into chemical energy, typically quantified as a decline in the maximum or effective quantum yield of PSII (e.g. Fv/Fm or ΦPSII). This impairment reflects disturbances in PSII reaction centers and/or associated electron transport components, resulting in reduced linear electron flow and a consequent limitation in the production of ATP and NADPH required for downstream photosynthetic processes (Maxwell & Johnson, 2000; Baker, 2008).

Decreased Photosystem II (PSII) efficiency can be quantified using a suite of complementary physiological and biochemical methods that assess the functional status of PSII reaction centers and associated electron transport processes. The most widely applied approach is chlorophyll fluorescence analysis, particularly the measurement of the maximum quantum yield of PSII (Fv/Fm), which provides a sensitive, non-invasive indicator of PSII photochemical efficiency and photoinhibitory damage (Maxwell and Johnson, 2000; Xia et al., 2023). Pulse-amplitude-modulated (PAM) fluorometry and related modulated fluorescence techniques allow both dark-adapted and light-adapted measurements, enabling assessment of effective PSII quantum yield and dynamic responses to stressors. PSII efficiency can also be evaluated through measurements of photosynthetic oxygen evolution rates, typically using polarographic oxygen electrodes with leaf discs, algal cultures, or isolated chloroplasts. These measurements directly reflect the functional integrity of the PSII water-splitting complex and downstream electron transport capacity (DELIEU and WALKER, 1981). Reductions in oxygen evolution are indicative of impaired PSII activity and reduced photochemical performance. At the molecular and mechanistic level, degradation or modification of the D1 protein of PSII can be assessed to support evidence of PSII damage. The D1 protein is a primary target of photodamage and herbicide interaction, and increased D1 turnover or instability is closely linked to declines in PSII efficiency (Alfonso et al., 1996). Together, chlorophyll fluorescence parameters, oxygen evolution measurements, and D1 protein degradation assays provide robust and mechanistically informative lines of evidence for identifying and quantifying decreases in Photosystem II efficiency.

The key event “decreased Photosystem II efficiency” is broadly applicable across oxygenic photosynthetic organisms that rely on PSII-mediated light reactions, including higher plants, macroalgae, microalgae, and cyanobacteria. The underlying structure and function of PSII, as well as the photochemical principles captured by chlorophyll fluorescence parameters (e.g. Fv/Fm) and oxygen evolution, are highly conserved across these taxa, supporting cross-species relevance of this KE. This KE is particularly applicable in studies assessing the effects of stressors that directly or indirectly interfere with PSII function, such as photosystem II–inhibiting herbicides, compounds targeting the D1 protein or the QB binding site, excess light, UV radiation, nutrient limitation, and other environmental stressors that induce photoinhibition or disrupt electron transport. It is most reliably measured under controlled laboratory or semi-controlled conditions where light history, acclimation status, and physiological state of the test organism can be standardized. The domain of applicability is strongest for acute to sub-chronic exposures where changes in PSII efficiency precede downstream effects on carbon fixation, growth, and biomass production. While the KE is highly sensitive and diagnostically informative at the cellular and organelle level, its interpretation at higher levels of biological organization (e.g. whole-plant productivity or ecosystem-level responses) requires integration with additional endpoints to account for compensatory mechanisms such as energy dissipation, PSII repair, and alternative electron transport pathways.

2026-01-16T04:26:14

Decrease, Photosynthesis

Cellular

refers to a reduction in the efficiency and/or capacity of photosynthetic organisms to convert light energy into chemical energy stored in organic carbon compounds. This key event encompasses impairments in the light-dependent reactions, the carbon fixation reactions, or both, resulting in diminished overall photosynthetic performance. At the mechanistic level, this KE can be caused by damage or inhibition of photosystems, particularly photosystem II (PSII), or interference with the photosynthetic electron transport chain (ETC). Such perturbations reduce the generation of ATP and NADPH, which are required to drive carbon fixation in the Calvin–Benson cycle. decrease in photosynthesis leads to reduced primary productivity, lower carbohydrate synthesis, and impaired energy availability for growth, reproduction, and maintenance. In aquatic and terrestrial primary producers, this KE represents a critical point of vulnerability linking molecular or cellular stressors (e.g., chemical inhibitors, oxidative stress, nutrient imbalance, or physical stressors such as light limitation) to higher-level adverse outcomes, including reduced biomass accumulation, altered community structure, and ecosystem-level impacts.

A decrease in photosynthesis can be quantified using a suite of complementary physiological and biochemical measurements that capture both light-driven energy conversion and carbon assimilation processes. Carbon fixation rates are most directly assessed using ¹⁴C-bicarbonate uptake assays, which quantify the incorporation of inorganic carbon into organic compounds during photosynthesis. This approach provides an integrative measure of photosynthetic carbon assimilation and is widely applied in algal, phytoplankton, and plant systems, with careful interpretation required to distinguish gross versus net fixation under different experimental conditions (Grant and Howard, 1980; Milligan, Halsey and Behrenfeld, 2015). Oxygen evolution measurements offer a direct proxy for the activity of the photosynthetic light reactions, particularly Photosystem II. Using Clark-type oxygen electrodes or polarographic methods, the rate of O₂ production under illumination can be quantified in intact tissues, leaf discs, or isolated chloroplasts, providing sensitive detection of functional impairment in the photosynthetic electron transport chain (DELIEU and WALKER, 1981; van Gorkom and Gast, 1996). At the whole-organism or leaf level, infrared gas analysis (IRGA) is commonly employed to measure net CO₂ uptake. This non-invasive technique allows continuous monitoring of photosynthetic performance under controlled environmental conditions and integrates stomatal conductance, biochemical capacity, and photochemical efficiency into a single functional endpoint (Amthor and Baldocchi, 2001; Xie et al., 2019). To resolve downstream biochemical constraints, Rubisco activity assays are used to quantify the catalytic capacity of ribulose-1,5-bisphosphate carboxylase/oxygenase. Both ¹⁴C-based assays and NADH-linked spectrophotometric or microtiter plate methods enable discrimination between limitations arising from carbon fixation enzymes versus upstream photochemical processes (Lilley and Walker, 1974; Sales, da Silva and Carmo-Silva, 2020). Together, these measurement approaches provide mechanistically informative and quantitatively robust indicators of decreased photosynthesis, supporting their use as key event measurements in Adverse Outcome Pathway development and ecotoxicological hazard characterization (van Gorkom and Gast, 1996)

This KE applies broadly to organisms that perform oxygenic photosynthesis, including terrestrial higher plants, freshwater and marine macrophytes, macroalgae, and phytoplankton, because all of these taxa utilize PSII–mediated light reactions and downstream carbon fixation to convert light energy into chemical energy. Photosystem II is a multisubunit pigment–protein complex present across cyanobacteria, algae, and plants that catalyzes light-driven water oxidation and initiates electron transport, providing the reducing power required for CO₂ assimilation and organic carbon synthesis. Disruption of PSII electron transport or carbon fixation directly results in decreased photochemical efficiency and reduced primary productivity in these diverse taxa (Sundby et al., 1993; Broser et al., 2011). For example, herbicides and other stressors that target PSII competitively bind to quinone acceptor sites in the D1 protein, blocking electron transport and diminishing carbon fixation efficiency, with effects documented across several photosynthetic groups (Broser et al., 2011; King et al., 2021). Environmental stressors such as high light intensity and other abiotic pressures further exacerbate impairment of PSII function through photoinhibition, in which damage to PSII and imbalances in repair processes reduce photosynthetic rates in both aquatic and terrestrial organisms (Murata et al., 2007). Because the PSII reaction center and associated processes are highly conserved among oxygenic photosynthetic organisms, decreases in photosynthetic performance induced by chemical or physical stressors are broadly applicable across taxa that contribute to ecosystem primary productivity. This KE is relevant across life stages where photosynthetic activity supports growth and energy capture, from juvenile algal cells to mature plant leaves, and under a range of environmental contexts, including natural light variation and anthropogenic pollutant exposure (Sundby et al., 1993; Murata et al., 2007). The universal importance of PSII integrity for carbon fixation and energy transduction supports the domain of applicability of Decrease, Photosynthesis as a mechanistically grounded indicator of photochemical and autotrophic dysfunction across photosynthetic lineages.

High Unspecific

High

Not Otherwise Specified

High High High High High High High Broser, M., Glöckner, C., Gabdulkhakov, A., Guskov, A., Buchta, J., Kern, J., Müh, F., Dau, H., Saenger, W. and Zouni, A. 2011. Structural basis of cyanobacterial photosystem II inhibition by the herbicide terbutryn. J Biol Chem 286(18), 15964–15972. DELIEU, T. and WALKER, D.A. 1981. Polarographic measurement of photosynthetic oxygen evolution by leaf discs. New Phytologist 89(2), 165–178. Grant, B.R. and Howard, R.J. 1980. Kinetics of C distribution during photosynthesis by chloroplast preparations isolated from the siphonous alga Caulerpa simpliciuscula. Plant Physiol 66(1), 29–33. Lilley, R.M. and Walker, D.A. 1974. An improved spectrophotometric assay for ribulosebisphosphate carboxylase. Biochimica et Biophysica Acta (BBA) – Enzymology 358(1), 226–229. Milligan, A.J., Halsey, K.H. and Behrenfeld, M.J. 2015. Advancing interpretations of 14C-uptake measurements in the context of phytoplankton physiology and ecology. Journal of Plankton Research 37(4), 692–698. Murata, N., Takahashi, S., Nishiyama, Y. and Allakhverdiev, S.I. 2007. Photoinhibition of photosystem II under environmental stress. Biochimica et Biophysica Acta (review). Photosystem II. 2025. Wikipedia, The Free Encyclopedia. Sales, C.R.G., da Silva, A.B. and Carmo-Silva, E. 2020. Measuring Rubisco activity: challenges and opportunities of NADH-linked microtiter plate-based and 14C-based assays. Journal of Experimental Botany 71(18), 5302–5312. Sundby, C., Chow, W.S. and Anderson, J.M. 1993. Effects on Photosystem II function, photoinhibition, and plant performance of the spontaneous mutation of serine-264 in the Photosystem II reaction center D1 protein in triazine-resistant Brassica napus L. Plant Physiol 103(1), 105–113. van Gorkom, H.J. and Gast, P. 1996. Measurement of photosynthetic oxygen evolution. Biophysical Techniques in Photosynthesis 3, 391–405. Xie, L., Solhaug, K.A., Song, Y., Brede, D.A., Lind, O.C., Salbu, B. and Tollefsen, K.E. 2019. Modes of action and adverse effects of gamma radiation in an aquatic macrophyte Lemna minor. Science of the Total Environment 680, 23–34. AOPWiki 2017-10-10T07:51:34

2026-01-20T03:44:57

Decreased, mitochondrial oxidative phosphorylation

Decrease in mitochondrial oxidative phosphorylation

Cellular

Mitochondrial oxidative phosphorylation (OXPHOS) is a fundamental cellular process to generate most of ATP to supplies energy for essential cellular functions.  The decrease of OXPHOS can be due to the inhibition or impairment of one or more components of the ETC or ATP synthase, loss of membrane integrity leading to uncoupling of electron transport from proton translocation, limited availability of electron donors (e.g., NADH, FADH₂), reduced metabolic substrate supply due to diminished photosynthetic carbon fixation and associated energy imbalance in photosynthetic cells, or oxygen limitation, or structural damage to mitochondrial membranes. As a consequence, the ability of mitochondria to maintain proton motive force and to synthesize ATP is reduced.

A decrease in mitochondrial oxidative phosphorylation (OXPHOS) can be measured using a combination of functional, biochemical, and structural approaches that collectively assess mitochondrial respiratory capacity, ATP production, and integrity of the electron transport system. Functional impairment of OXPHOS is most commonly evaluated by respirometry, where oxygen consumption rates are measured in intact or permeabilized cells, tissues, or isolated mitochondria under controlled substrate and inhibitor conditions. High-resolution respirometry allows quantification of basal respiration, ADP-stimulated (ATP-linked) respiration, maximal electron transport system capacity, and coupling efficiency. A reduction in ADP-stimulated respiration or maximal respiratory capacity provides direct evidence for decreased mitochondrial oxidative phosphorylation (Djafarzadeh and Jakob, 2017; Coulson, Duffy and Staples, 2024). Complementary evidence for reduced OXPHOS can be obtained by quantifying ATP synthesis rates. Luciferase-based assays enable sensitive measurement of ATP production in real time or at defined endpoints and are widely used to assess mitochondrial ATP output. Reduced ATP synthesis, particularly when observed alongside diminished oxygen consumption, indicates impaired coupling between electron transport and phosphorylation (Lundin, Rickardsson and Thore, 1976; Coulson, Duffy and Staples, 2024). In addition, in vivo ³¹P nuclear magnetic resonance (NMR) spectroscopy provides a non-invasive assessment of high-energy phosphate metabolites, allowing evaluation of cellular energetic status and supporting identification of reduced mitochondrial ATP generation (Hitchins, Cieslar and Dobson, 2001). Biochemical characterization of OXPHOS impairment can be further refined through enzyme activity assays of individual respiratory chain complexes (I–IV). These assays measure the catalytic activity of specific complexes using defined substrates and inhibitors and can identify whether reduced oxidative phosphorylation is associated with inhibition or dysfunction of discrete components of the electron transport chain (Coulson, Duffy and Staples, 2024). Changes in mitochondrial membrane potential (ΔΨm), assessed using potentiometric fluorescent probes, provide additional functional insight, as loss or reduction of ΔΨm reflects impaired proton motive force and compromised capacity for ATP synthesis (Xie et al., 2019). Finally, blue-native polyacrylamide gel electrophoresis (BN-PAGE) is used to examine the structural organization, abundance, and stability of intact OXPHOS complexes and supercomplexes within the inner mitochondrial membrane. Alterations in complex assembly or loss of specific respiratory complexes detected by BN-PAGE support a mechanistic interpretation of reduced oxidative phosphorylation at the protein organization level (Yan and Forster, 2009). Together, these measurement approaches provide robust and complementary lines of evidence for identifying and characterizing decreases in mitochondrial oxidative phosphorylation.

The Key Event Decrease in mitochondrial oxidative phosphorylation is broadly applicable across biological systems that rely on mitochondria for aerobic energy production. This KE is relevant to eukaryotic organisms, including animals, plants, algae, and fungi, as the core molecular machinery of mitochondrial oxidative phosphorylation—comprising the electron transport chain (Complexes I–IV), ATP synthase (Complex V), and the proton motive force across the inner mitochondrial membrane—is highly conserved across taxa. Consequently, the KE can be applied to a wide range of model and non-model species used in ecotoxicology, physiology, and environmental risk assessment. The KE is applicable across life stages, but sensitivity may vary. Early developmental stages, growth phases, reproduction, and energetically demanding physiological processes (e.g. molting, metamorphosis, active growth, and stress responses) are expected to be particularly vulnerable to perturbations in mitochondrial ATP production. In photosynthetic organisms, this KE is also applicable under both light and dark conditions, reflecting the metabolic integration of mitochondrial respiration with photosynthetic carbon metabolism. In terms of stressors, the domain of applicability includes chemical, physical, and environmental factors that impair mitochondrial function. These include direct inhibitors of respiratory chain complexes or ATP synthase, uncouplers of oxidative phosphorylation, agents that damage mitochondrial membranes or proteins, stressors that disrupt cellular redox balance, and conditions that limit substrate or oxygen availability. In photosynthetic organisms, stressors that reduce photosynthesis can indirectly contribute to this KE by decreasing the supply of carbon substrates and reducing equivalents that support mitochondrial respiration, thereby altering cellular energy balance.

High Unspecific

High

Not Otherwise Specified

High High Coulson, S.Z., Duffy, B.M. and Staples, J.F. 2024. Mitochondrial techniques for physiologists. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 271, 110947. Djafarzadeh, S. and Jakob, S.M. 2017. High-resolution respirometry to assess mitochondrial function in permeabilized and intact cells. Journal of Visualized Experiments (120). Hitchins, S., Cieslar, J.M. and Dobson, G.P. 2001. ³¹P NMR quantitation of phosphorus metabolites in rat heart and skeletal muscle in vivo. American Journal of Physiology-Heart and Circulatory Physiology 281(2), H882–H887. Lundin, A., Rickardsson, A. and Thore, A. 1976. Continuous monitoring of ATP-converting reactions by purified firefly luciferase. Analytical Biochemistry 75(2), 611–620. Xie, L., Solhaug, K.A., Song, Y., Brede, D.A., Lind, O.C., Salbu, B. and Tollefsen, K.E. 2019. Modes of action and adverse effects of gamma radiation in an aquatic macrophyte Lemna minor. Science of the Total Environment 680, 23–34. Yan, L.J. and Forster, M.J. 2009. Resolving mitochondrial protein complexes using nongradient blue native polyacrylamide gel electrophoresis. Analytical Biochemistry 389(2), 143–149. AOPWiki 2018-12-19T09:36:59

2026-02-04T03:52:42

Decrease, ATP production

Cellular

Decrease in ATP production refers to a reduced ability of cells to generate adenosine triphosphate (ATP), the main energy source for cellular processes. ATP is mainly produced in mitochondria, and its reduction typically reflects impaired energy metabolism. Lower ATP levels limit energy-dependent functions such as active transport, biosynthesis, and cell maintenance. If ATP production remains reduced, normal cellular function cannot be sustained, leading to cellular dysfunction or failure.

A decrease in ATP production is commonly measured by quantifying cellular ATP levels or by assessing the activity of ATP-producing pathways. Luciferase-based ATP assays are widely used to directly measure ATP concentrations, relying on the light emitted during the luciferase–luciferin reaction, which is proportional to ATP content (Lundin et al., 1976). Enzymatic assays targeting ATP synthase activity provide complementary information on the functional capacity of the mitochondrial phosphorylation machinery and have been applied to link electron transport efficiency with ATP synthesis rates (Allakhverdiev et al., 2005; Coulson et al., 2024). ATP and other high-energy phosphates can also be quantified using ^31P nuclear magnetic resonance (NMR) spectroscopy, which allows non-destructive measurement of intracellular phosphorus metabolites and energy status (Hitchins et al., 2001). In addition, high-performance liquid chromatography (HPLC) methods enable sensitive and accurate separation and quantification of adenosine phosphates (ATP, ADP, AMP) in cell extracts, providing detailed information on cellular energy balance (Juarez-Facio et al., 2021)

Taxonomic applicability domain This key event is generally considered applicable to all organisms that rely on ATP as the universal cellular energy currency, including both prokaryotes and eukaryotes, as ATP production is a fundamental and conserved biological process. Life stage applicability domain This key event is considered applicable to all life stages, as continuous ATP production is required to support cellular maintenance, growth, development, and normal physiological function throughout the life cycle. Sex applicability domain This key event is considered sex-unspecific, as ATP production and utilization are essential cellular processes in both males and females and are not inherently dependent on sex-specific biology.

CL:0000000

CL cell

High Unspecific

High

All life stages

High High High

Allakhverdiev, S.I., Nishiyama, Y., Takahashi, S., Miyairi, S., Suzuki, I. and Murata, N. (2005). Systematic analysis of the relation of electron transport and ATP synthesis to the photodamage and repair of photosystem II in Synechocystis. Plant Physiology, 137(1), 263–273. Coulson, S.Z., Duffy, B.M. and Staples, J.F. (2024). Mitochondrial techniques for physiologists. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 271, 110947. Hitchins, S., Cieslar, J.M. and Dobson, G.P. (2001). ^31P NMR quantitation of phosphorus metabolites in rat heart and skeletal muscle in vivo. American Journal of Physiology – Heart and Circulatory Physiology, 281(2), H882–H887. Juarez-Facio, A.T., Martin de Lagarde, V., Monteil, C., Vaugeois, J.M., Corbiere, C. and Rogez-Florent, T. (2021). Validation of a fast and simple HPLC-UV method for the quantification of adenosine phosphates in human bronchial epithelial cells. Molecules, 26(20). Lundin, A., Rickardsson, A. and Thore, A. (1976). Continuous monitoring of ATP-converting reactions by purified firefly luciferase. Analytical Biochemistry, 75(2), 611–620. AOPWiki 2017-10-10T07:49:23

2026-02-04T05:16:50

Decrease, Population growth rate

Population

A population can be defined as a group of interbreeding organisms, all of the same species, occupying a specific space during a specific time (Vandermeer and Goldberg 2003, Gotelli 2008).  As the population is the biological level of organization that is often the focus of ecological risk assessments, population growth rate (and hence population size over time) is important to consider within the context of applied conservation practices. If N is the size of the population and t is time, then the population growth rate (dN/dt) is proportional to the instantaneous rate of increase, r, which measures the per capita rate of population increase over a short time interval. Therefore, r, is a difference between the instantaneous birth rate (number of births per individual per unit of time; b) and the instantaneous death rate (number of deaths per individual per unit of time; d) [Equation 1]. Because  r is an instantaneous rate, its units can be changed via division.  For example, as there are 24 hours in a day, an r of 24 individuals/(individual x day) is equal to an r of 1 individual/(individual/hour) (Caswell 2001, Vandermeer and Goldberg 2003, Gotelli 2008, Murray and Sandercock 2020).  Equation 1:  r = b - d This key event refers to scenarios where r < 0 (instantaneous death rate exceeds instantaneous birth rate). Examining r in the context of population growth rate: ● A population will decrease to extinction when the instantaneous death rate exceeds the instantaneous birth rate (r < 0).              ● The smaller the value of r below 1, the faster the population will decrease to zero.   ● A population will increase when resources are available and the instantaneous birth rate exceeds the instantaneous death rate (r > 0)            ● The larger the value that r exceeds 1, the faster the population can increase over time       ● A population will neither increase or decrease when the population growth rate equals 0 (either due to N = 0, or if the per capita birth and death rates are exactly balanced).  For example, the per capita birth and death rates could become exactly balanced due to density dependence and/or to the effect of a stressor that reduces survival and/or reproduction (Caswell 2001, Vandermeer and Goldberg 2003, Gotelli 2008, Murray and Sandercock 2020).      Effects incurred on a population from a chemical or non-chemical stressor could have an impact directly upon birth rate (reproduction) and/or death rate (survival), thereby causing a decline in population growth rate.   ● Example of direct effect on r:  Exposure to 17b-trenbolone reduced reproduction (i.e., reduced b) in the fathead minnow over 21 days at water concentrations ranging from 0.0015 to about 41 mg/L (Ankley et al. 2001; Miller and Ankley 2004).              Alternatively, a stressor could indirectly impact survival and/or reproduction.   ● Example of indirect effect on r:  Exposure of non-sexually differentiated early life stage fathead minnow to the fungicide prochloraz has been shown to produce male-biased sex ratios based on gonad differentiation, and resulted in projected change in population growth rate (decrease in reproduction due to a decrease in females and thus recruitment) using a population model. (Holbech et al., 2012; Miller et al. 2022) Density dependence can be an important consideration: ● The effect of density dependence depends upon the quantity of resources present within a landscape.  A change in available resources could increase or decrease the effect of density dependence and therefore cause a change in population growth rate via indirectly impacting survival and/or reproduction.   ● This concept could be thought of in terms of community level interactions whereby one species is not impacted but a competitor species is impacted by a chemical stressor resulting in a greater availability of resources for the unimpacted species.  In this scenario, the impacted species would experience a decline in population growth rate. The unimpacted species would experience an increase in population growth rate (due to a smaller density dependent effect upon population growth rate for that species).        Closed versus open systems: ● The above discussion relates to closed systems (there is no movement of individuals between population sites) and thus a declining population growth rate cannot be augmented by immigration.   ● When individuals depart (emigrate out of a population) the loss will diminish population growth rate.   Population growth rate applies to all organisms, both sexes, and all life stages.

Population growth rate (instantaneous growth rate) can be measured by sampling a population over an interval of time (i.e. from time t = 0 to time t = 1).  The interval of time should be selected to correspond to the life history of the species of interest (i.e. will be different for rapidly growing versus slow growing populations). The population growth rate, r, can be determined by taking the difference (subtracting) between the initial population size, Nt=0 (population size at time t=0), and the population size at the end of the interval, Nt=1 (population size at time t = 1), and then subsequently dividing by the initial population size.  Equation 2:  r = (Nt=1 - Nt=0) / Nt=0 The diversity of forms, sizes, and life histories among species has led to the development of a vast number of field techniques for estimation of population size and thus population growth over time (Bookhout 1994, McComb et al. 2021).   ● For stationary species an observational strategy may involve dividing a habitat into units. After setting up the units, samples are performed throughout the habitat at a select number of units (determined using a statistical sampling design) over a time interval (at time t = 0 and again at time t = 1), and the total number of organisms within each unit are counted. The numbers recorded are assumed to be representative for the habitat overall, and can be used to estimate the population growth rate within the entire habitat over the time interval.   ● For species that are mobile throughout a large range, a strategy such as using a mark-recapture method may be employed (i.e. tags, bands, transmitters) to determine a count over a time interval (at time = 0 and again at time =1).    Population growth rate can also be estimated using mathematical model constructs (for example, ranging from simple differential equations to complex age or stage structured matrix projection models and individual based modeling approaches), and may assume a linear or nonlinear population increase over time (Caswell 2001, Vandermeer and Goldberg 2003, Gotelli 2008, Murray and Sandercock 2020). The AOP framework can be used to support the translation of pathway-specific mechanistic data into responses relevant to population models and output from the population models, such as changing (declining) population growth rate, can be used to assess and manage risks of chemicals (Kramer et al. 2011). As such, this translational capability can increase the capacity and efficiency of safety assessments both for single chemicals and chemical mixtures (Kramer et al. 2011).   Some examples of modeling constructs used to investigate population growth rate: ● A modeling construct could be based upon laboratory toxicity tests to determine effect(s) that are then linked to the population model and used to estimate decline in population growth rate.  Miller et al. (2007) used concentration–response data from short term reproductive assays with fathead minnow (Pimephales promelas) exposed to endocrine disrupting chemicals in combination with a population model to examine projected alterations in population growth rate.   ● A model construct could be based upon a combination of effects-based monitoring at field sites (informed by an AOP) and a population model.  Miller et al. (2015) applied a population model informed by an AOP to project declines in population growth rate for white suckers (Catostomus commersoni) using observed changes in sex steroid synthesis in fish exposed to a complex pulp and paper mill effluent in Jackfish Bay, Ontario, Canada. Furthermore, a model construct could be comprised of a series of quantitative models using KERs that culminates in the estimation of change (decline) in population growth rate.   ● A quantitative adverse outcome pathway (qAOP) has been defined as a mathematical construct that models the dose–response or response–response relationships of all KERs described in an AOP (Conolly et al. 2017, Perkins et al. 2019). Conolly et al. (2017) developed a qAOP using data generated with the aromatase inhibitor fadrozole as a stressor and then used it to predict potential population‐level impacts (including decline in population growth rate). The qAOP modeled aromatase inhibition (the molecular initiating event) leading to reproductive dysfunction in fathead minnow (Pimephales promelas) using 3 computational models: a hypothalamus–pituitary–gonadal axis model (based on ordinary differential equations) of aromatase inhibition leading to decreased vitellogenin production (Cheng et al. 2016), a stochastic model of oocyte growth dynamics relating vitellogenin levels to clutch size and spawning intervals (Watanabe et al. 2016), and a population model (Miller et al. 2007). ● Dynamic energy budget (DEB) models offer a methodology that reverse engineers stressor effects on growth, reproduction, and/or survival into modular characterizations related to the acquisition and processing of energy resources (Nisbet et al. 2000, Nisbet et al. 2011).  Murphy et al. (2018) developed a conceptual model to link DEB and AOP models by interpreting AOP key events as measures of damage-inducing processes affecting DEB variables and rates. ● Endogenous Lifecycle Models (ELMs), capture the endogenous lifecycle processes of growth, development, survival, and reproduction and integrate these to estimate and predict expected fitness (Etterson and Ankley, 2021).  AOPs can be used to inform ELMs of effects of chemical stressors on the vital rates that determine fitness, and to decide what hierarchical models of endogenous systems should be included within an ELM (Etterson and Ankley, 2021).

Consideration of population size and changes in population size over time is potentially relevant to all living organisms.

Not Specified Unspecific

Not Specified

All life stages

High

<ul> <li>Ankley GT, Jensen KM, Makynen EA, Kahl MD, Korte JJ, Hornung MW, Henry TR, Denny JS, Leino RL, Wilson VS, Cardon MD, Hartig PC, Gray LE. 2003. Effects of the androgenic growth promoter 17b-trenbolone on fecundity and reproductive endocrinology of the fathead minnow. Environ. Toxicol. Chem. 22: 1350–1360.</li> <li>Bookhout TA. 1994. Research and management techniques for wildlife and habitats. The Wildlife Society, Bethesda, Maryland. 740 pp.</li> <li>Caswell H. 2001. Matrix Population Models. Sinauer Associates, Inc., Sunderland, MA, USA</li> <li>Cheng WY, Zhang Q, Schroeder A, Villeneuve DL, Ankley GT, Conolly R.  2016.  Computational modeling of plasma vitellogenin alterations in response to aromatase inhibition in fathead minnows. Toxicol Sci 154: 78–89.</li> <li>Conolly RB, Ankley GT, Cheng W-Y, Mayo ML, Miller DH, Perkins EJ, Villeneuve DL, Watanabe KH. 2017. Quantitative adverse outcome pathways and their application to predictive toxicology. Environ. Sci. Technol. 51:  4661-4672.</li> <li>Etterson MA, Ankley GT.  2021.  Endogenous Lifecycle Models for Chemical Risk Assessment. Environ. Sci. Technol. 55:  15596-15608. </li> <li>Gotelli NJ, 2008. A Primer of Ecology. Sinauer Associates, Inc., Sunderland, MA, USA.</li> <li>Holbech H, Kinnberg KL, Brande-Lavridsen N, Bjerregaard P, Petersen GI, Norrgren L, Orn S, Braunbeck T, Baumann L, Bomke C, Dorgerloh M, Bruns E, Ruehl-Fehlert C, Green JW, Springer TA, Gourmelon A. 2012 Comparison of zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) as test species in the Fish Sexual Development Test (FSDT). Comp. Biochem. Physiol. C Toxicol. Pharmacol. 155:  407–415.</li> <li>Kramer VJ, Etterson MA, Hecker M, Murphy CA, Roesijadi G, Spade DJ, Stromberg JA, Wang M, Ankley GT.  2011.  Adverse outcome pathways and risk assessment: Bridging to population level effects.  Environ. Toxicol. Chem. 30, 64-76.</li> <li>McComb B, Zuckerberg B, Vesely D, Jordan C.  2021.  Monitoring Animal Populations and their Habitats: A Practitioner's Guide.  Pressbooks, Oregon State University, Corvallis, OR Version 1.13, 296 pp. </li> <li>Miller DH, Villeneuve DL, Santana Rodriguez KJ, Ankley GT. 2022.  A multidimensional matrix model for predicting the effect of male biased sex ratios on fish populations. Environmental Toxicology and Chemistry 41(4): 1066-1077.</li> <li>Miller DH, Tietge JE, McMaster ME, Munkittrick KR, Xia X, Griesmer DA, Ankley GT. 2015. Linking mechanistic toxicology to population models in forecasting recovery from chemical stress: A case study from Jackfish Bay, Ontario, Canada. Environmental Toxicology and Chemistry 34(7):  1623-1633.</li> <li>Miller DH, Jensen KM, Villeneuve DE, Kahl MD, Makynen EA, Durhan EJ, Ankley GT. 2007. Linkage of biochemical responses to population-level effects: A case study with vitellogenin in the fathead minnow (Pimephales promelas). Environ Toxicol Chem 26:  521–527.</li> <li>Miller DH, Ankley GT. 2004. Modeling impacts on populations: Fathead minnow (Pimephales promelas) exposure to the endocrine disruptor 17b-trenbolone as a case study. Ecotox Environ Saf 59: 1–9.</li> <li>Murphy CA, Nisbet RM, Antczak P, Garcia-Reyero N, Gergs A, Lika K, Mathews T, Muller EB, Nacci D, Peace A, Remien CH, Schultz IR, Stevenson LM, Watanabe KH.  2018.  Incorporating suborganismal processes into dynamic energy budget models for ecological risk assessment.  Integrated Environmental Assessment and Management 14(5):  615–624.</li> <li>Murray DL, Sandercock BK (editors).  2020.  Population ecology in practice.  Wiley-Blackwell, Oxford UK, 448 pp.</li> <li>Nisbet RM, Jusup M, Klanjscek T, Pecquerie L.  2011.  Integrating dynamic energy budget (DEB) theory with traditional bioenergetic models.  The Journal of Experimental Biology 215: 892-902.</li> <li>Nisbet RM, Muller EB, Lika K, Kooijman SALM. 2000. From molecules to ecosystems through dynamic energy budgets. J Anim Ecol 69:  913–926.</li> <li>Perkins EJ,  Ashauer R, Burgoon L, Conolly R, Landesmann B,, Mackay C, Murphy CA, Pollesch N, Wheeler JR, Zupanic A, Scholzk S.  2019.  Building and applying quantitative adverse outcome pathway models for chemical hazard and risk assessment.  Environmental Toxicology and Chemistry 38(9): 1850–1865. </li> <li>Vandermeer JH, Goldberg DE. 2003.  Population ecology: first principles.  Princeton University Press, Princeton NJ, 304 pp.</li> <li>Villeneuve DL, Crump D, Garcia-Reyero N, Hecker M, Hutchinson TH, LaLone CA, Landesmann B, Lattieri T, Munn S, Nepelska M, Ottinger MA, Vergauwen L, Whelan M. Adverse outcome pathway (AOP) development 1: Strategies and principles. Toxicol Sci. 2014: 142:312–320</li> <li>Watanabe KH, Mayo M, Jensen KM, Villeneuve DL, Ankley GT, Perkins EJ.  2016.  Predicting fecundity of fathead minnows (Pimephales promelas) exposed to endocrine‐disrupting chemicals using a MATLAB(R)‐based model of oocyte growth dynamics. PLoS One 11:  e0146594.</li> </ul> AOPWiki 2016-11-29T18:41:24

2023-01-03T09:09:06

Decrease, Growth

Individual

Decreased growth refers to a reduction in size and/or weight of a tissue, organ or individual organism. Growth is normally controlled by growth factors and mainly achieved through cell proliferation (Conlon 1999).

Growth can be indicated by measuring weight, length, total volume, and/or total area of a tissue, organ or individual organism.

Taxonomic applicability domain This key event is in general applicable to all eukaryotes.   Life stage applicability domain This key event is applicable to early life stages such as embryo and juvenile.   Sex applicability domain This key event is sex-unspecific.

High Unspecific

High

Embryo

High

Juvenile

Moderate Moderate Moderate High High High Moderate

Conlon I, Raff M. 1999. Size control in animal development. Cell 96:235-244. DOI: 10.1016/s0092-8674(00)80563-2.  AOPWiki 2018-05-24T15:24:11

2022-07-06T07:36:50

<upstream-id>d47c1ff5-31a3-46a0-a7d4-2dc133806b24</upstream-id> <downstream-id>3f0d9d03-fc32-4f89-b0c0-c7d240933741</downstream-id> Binding of a stressor to the QB site of the D1 protein directly interferes with electron transfer within Photosystem II by blocking plastoquinone binding and reduction. This disruption slows or prevents electron flow from QA to QB, leading to impaired charge separation efficiency and increased recombination reactions. As a result, the functional efficiency of Photosystem II is reduced, which is commonly observed as decreases in parameters such as quantum yield and maximum photochemical efficiency.

<big>The literature was systematically filtered through a tiered process, where the AOP-helpFinder (v3.0) was applied to screening the initial candidate literature that related to the stressor-event and event-event relationships, followed by the Swift-Reviewer (Version 1.43.1063) to refine the corpus of evidence. </big>

The Key Event Relationship (KER) between binding at the Q_B site of the D1 protein and decreased Photosystem II (PSII) efficiency is strongly supported by structural, biochemical, and physiological evidence. Crystallographic analyses demonstrate that PSII-inhibiting herbicides bind specifically to the Q_B niche within the D1 protein, competitively blocking plastoquinone exchange and electron transfer from Q_A to Q_B (Broser et al., 2011). This disruption rapidly reduces photochemical efficiency, reflected by declines in Fv/Fm and impaired oxygen evolution (Maxwell & Johnson, 2000; Delieu & Walker, 1981). Mutational studies of D1 further confirm that alterations at the Q_B site modify herbicide sensitivity and PSII performance (Alfonso et al., 1996), establishing a direct mechanistic linkage.

The biological plausibility of this key event relationship is high. The QB binding site of the D1 protein is a well-defined and essential component of Photosystem II, responsible for the binding and reduction of plastoquinone during photosynthetic electron transport (Lambreva et al., 2014; Velthuys, 1981). Binding of chemicals to this site is known to competitively inhibit plastoquinone exchange, thereby directly disrupting electron transfer from QA to QB (Battaglino et al., 2021; Zobnina et al., 2017). This mechanism leads to a rapid accumulation of reduced QA, increased charge recombination, and impaired stabilization of photochemical charge separation (Ermakova-Gerdes & Vermaas, 1998; Ohad & Hirschberg, 1992). Consequently, the efficiency of Photosystem II is reduced, which is consistently reflected by decreases in established photosynthetic performance metrics (Jansen et al., 1993). The direct structural and functional role of the QB site in electron transport provides a clear and mechanistically supported causal link between QB binding in the D1 protein and decreased Photosystem II efficiency (Nain-Perez et al., 2017; Zobnina et al., 2017).

The empirical support of this KER is considered high. Rationale: A large body of experimental evidence demonstrates strong incidence, dose, and temporal concordance between chemical binding or interference at the QB site of the D1 protein and reduced Photosystem II (PSII) efficiency across cyanobacteria, algae, aquatic plants, and higher plants. These effects are consistently observed for well-characterized PSII inhibitors, with relatively few inconsistencies. Evidence: Dose concordance: In isolated pea thylakoid membranes, QB site herbicides with higher inferred QB 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–Vj, reflecting QA reduction and QB site interference) (Battaglino, Grinzato and Pagliano, 2021).  Incidence concordance: Direct inhibition of plastoquinone binding at the QB site leads to accumulation of reduced QA and impaired QA to QB electron transfer, which is consistently associated with reduced PSII efficiency in functional assays, including fluorescence-based measurements (Vermaas et al., 1984; Sundby et al., 1993; Broser et al., 2011). Structural–functional concordance: Crystallographic and molecular studies have shown that PSII inhibitors bind within the QB_BB pocket of the D1 protein, physically blocking plastoquinone exchange. This structural interference provides direct mechanistic evidence linking QB_BB binding to impaired PSII electron transport and decreased PSII efficiency (Broser et al., 2011; Zimmermann et al., 2006). Genetic concordance: Mutations in the D1 protein affecting the QB_BB region alter herbicide binding and are accompanied by changes in PSII electron transport efficiency and photochemical performance, supporting a causal relationship between perturbation at the QB_BB site and decreased PSII efficiency (Ohad and Hirschberg, 1992; Sundby et al., 1993). Temporal concordance: In aquatic plants and seagrasses, inhibition of PSII efficiency occurs rapidly following exposure to PSII herbicides, while partial or full recovery of PSII function is observed after removal of the stressor, consistent with a direct and reversible inhibition of QB_BB-mediated electron transport (Macinnis-Ng and Ralph, 2003; Wilkinson et al., 2015).

Uncertainties remain regarding species-specific sensitivity and compensatory responses following Q_B-site binding. Structural evidence is strong in model organisms, but quantitative linkage between binding affinity and magnitude of PSII efficiency decline varies across taxa and exposure conditions. Additionally, secondary oxidative damage may confound the direct attribution solely to primary Q_B-site inhibition.

<div> <table class="table table-bordered table-fullwidth"> <thead> <tr> <th>Modulating Factor (MF)</th> <th>MF Specification</th> <th>Effect(s) on the KER</th> <th>Reference(s)</th> </tr> </thead> <tbody> <tr> <td> D1 protein genotype (psbA mutations) </td> <td> Amino acid substitutions in the D1 protein affecting the QB binding niche (e.g. Ser264, Ser268 mutations) </td> <td>Alters herbicide binding affinity at the QB site, leading to reduced or enhanced inhibition of QA→QB electron transfer and corresponding changes in the magnitude of PSII efficiency reduction</td> <td>Ohad and Hirschberg (1992); Sundby et al. (1993); Alfonso et al. (1996); Oettmeier (1999)</td> </tr> <tr> <td>Chemical binding affinity to QB</td> <td>Structural differences among PSII inhibitors (e.g. diuron, atrazine, terbutryn, metribuzin) influencing QB site affinity</td> <td>Higher QB binding affinity results in stronger and more rapid inhibition of PSII efficiency at lower concentrations, while lower-affinity compounds require higher doses</td> <td>Tischer and Strotmann (1977); Fuerst and Michael (1991); Vermaas et al. (1984); Broser et al. (2011)</td> </tr> <tr> <td>Photosystem II repair capacity</td> <td>Ability to replace damaged D1 protein via de novo synthesis and PSII repair cycle</td> <td>High repair capacity reduces duration and magnitude of PSII efficiency loss following QB site inhibition; limited repair capacity prolongs inhibition</td> <td>Allakhverdiev et al. (2005); Wilhelm and Selmar (2011)</td> </tr> <tr> <td>Species / taxonomic group</td> <td>Cyanobacteria, algae, aquatic macrophytes, terrestrial plants</td> <td>Sensitivity of PSII efficiency to QB-site inhibition varies among taxa due to differences in PSII structure, herbicide uptake, and repair dynamics</td> <td>Broser et al. (2011); Wilkinson et al. (2015); Macinnis-Ng and Ralph (2003)</td> </tr> <tr> <td>Exposure duration </td> <td>Short-term pulse exposure versus sustained exposure</td> <td>Short exposures often cause reversible PSII inhibition, while continuous exposure increases probability and magnitude of sustained PSII efficiency loss</td> <td>Macinnis-Ng and Ralph (2003); Wilkinson et al. (2015)</td> </tr> <tr> <td>Light intensity</td> <td>High irradiance versus low or moderate light conditions</td> <td>High light exacerbates PSII efficiency loss following QB site binding due to increased excitation pressure and photodamage; low light reduces severity of functional inhibition</td> <td>Sundby et al. (1993); Wilhelm and Selmar (2011)</td> </tr> </tbody> </table> </div>

The quantitative understanding of the linkage between Q_B-site binding in the D1 protein and decreased Photosystem II (PSII) efficiency is currently considered low. Although inhibition constants and structural binding interactions have been characterized for several PSII inhibitors (Broser et al., 2011), these molecular parameters are rarely quantitatively coupled with functional endpoints such as Fv/Fm decline or oxygen evolution rates (Maxwell & Johnson, 2000; Delieu & Walker, 1981). Most studies report concentration–response relationships at the whole-PSII level rather than linking fractional Q_B-site occupancy to photochemical impairment. Furthermore, species-specific repair capacity of the D1 protein and light-dependent damage processes introduce additional variability (Alfonso et al., 1996). Consequently, the mathematical response function necessary for qAOP parameterization remains insufficiently defined.

The response–response relationship between Q_B-site binding and decreased Photosystem II (PSII) efficiency remains insufficiently quantified. Empirically, increasing inhibitor concentration produces a sigmoidal decline in Fv/Fm and oxygen evolution rates (Maxwell & Johnson, 2000; Delieu & Walker, 1981). However, these functional responses are measured at the system level and are not directly parameterized against fractional Q_B-site occupancy (Broser et al., 2011). Variability in D1 turnover and photorepair dynamics further modifies the slope and threshold of the response curve (Alfonso et al., 1996), limiting precise quantitative extrapolation.

The time scale of this KER is rapid at the functional level. Q_B-site binding occurs within minutes of exposure, leading to immediate inhibition of electron transport and measurable declines in Fv/Fm and oxygen evolution (Delieu & Walker, 1981; Maxwell & Johnson, 2000). Longer-term effects depend on D1 repair dynamics and sustained exposure.

Inhibition of electron transfer at the Q_B site promotes over-reduction of Q_A and enhances charge recombination, which increases reactive oxygen species (ROS) formation under illumination (Broser et al., 2011). Elevated ROS can further damage the D1 protein, reinforcing PSII impairment through a positive feedforward loop. Conversely, photosynthetic organisms possess a repair cycle involving proteolytic removal and resynthesis of damaged D1, partially restoring PSII efficiency (Alfonso et al., 1996). The balance between ROS-mediated damage and D1 repair capacity determines the persistence and magnitude of PSII inhibition under sustained exposure.

High Unspecific

High

All life stages

High High High

axonomic applicability: This KER applies to all oxygenic photosynthetic taxa possessing Photosystem II with a conserved D1 protein and functional Q_B binding site, including cyanobacteria, chlorophyte algae (e.g., Chlamydomonas reinhardtii), macrophytes (e.g., Lemna minor), and higher plants. The Q_B niche and its role in plastoquinone reduction are structurally conserved across these groups (Broser et al., 2011), supporting broad cross-taxon relevance. Sex applicability: Not sex-specific. PSII structure and function are conserved in both male and female individuals of dioecious plants, and in clonal or unicellular species sex differentiation is not relevant. Life-stage applicability: Applicable across all photosynthetically active life stages (e.g., vegetative cells, seedlings, mature leaves). Sensitivity may vary with developmental stage due to differences in D1 turnover, pigment composition, and photorepair capacity (Maxwell & Johnson, 2000), but the mechanistic linkage remains conserved. Chemical Applicability: This KER is specifically applicable to chemicals that bind to the Q_B site of the D1 protein and inhibit plastoquinone exchange. It is strongly supported for classical PSII inhibitors, including triazines (e.g., atrazine), phenylureas (e.g., diuron), triazinones (e.g., metribuzin), and substituted ureas, which competitively occupy the Q_B niche and block electron transport (Broser et al., 2011; Alfonso et al., 1996). Applicability depends on structural compatibility with the hydrophobic Q_B binding pocket. Chemicals that impair photosynthesis via alternative mechanisms (e.g., PSI inhibitors, uncouplers, pigment synthesis inhibitors) fall outside this KER’s mechanistic domain, although they may produce similar downstream reductions in PSII efficiency through indirect pathways.

2026-02-23T08:10:58

<upstream-id>3f0d9d03-fc32-4f89-b0c0-c7d240933741</upstream-id> <downstream-id>5c7d7a53-fec1-4683-a1cd-9543dc16ddce</downstream-id> The decrease in photosystem II (PSII) efficiency impairs the effectiveness of light-driven electron transport in thylakoid membranes, thereby limiting the reduction of plastoquinone, weakening proton gradient formation, and consequently reducing ATP and NADPH production. Since these energy carriers are crucial for carbon fixation, diminished PSII efficiency directly leads to reduced photosynthetic rates. This relationship exhibits direct mechanistic causality and has been consistently validated across all photosynthetic oxygen-producing organisms.

A systematic literature search will be conducted in Web of Science, Scopus, and PubMed using combinations of terms related to PSII efficiency (e.g., Fv/Fm, ΦPSII, ETR, chlorophyll fluorescence) and photosynthesis (e.g., oxygen evolution, CO₂ fixation, ¹⁴C uptake). Studies will be included if they report paired measurements of PSII performance and photosynthetic rate in oxygenic phototrophs under controlled exposure conditions. Priority will be given to experiments demonstrating temporal or dose-response concordance. Extracted data will include species, exposure details, endpoints, effect magnitude, and methodological quality to support WoE evaluation and potential quantitative linkage.

Strong empirical evidence supports the relationship between decreased PSII efficiency and reduced photosynthesis. Declines in chlorophyll fluorescence parameters (e.g., Fv/Fm) are consistently associated with reduced oxygen evolution and carbon fixation rates in plants and algae (Maxwell and Johnson, 2000; DELIEU and WALKER, 1981). PSII-inhibiting herbicides such as atrazine and terbutryn directly impair electron transport at the D1 protein QB site, leading to decreased photosynthetic performance (Alfonso et al., 1996; Broser et al., 2011). These studies demonstrate mechanistic causality, dose-response concordance, and cross-taxa consistency, providing strong support for this KER.

Biological plausibility was considered as high. Photosystem II (PSII) is the primary site of light-driven charge separation and water oxidation in oxygenic photosynthesis, and its photochemical efficiency governs electron entry into the photosynthetic electron transport chain (Maxwell and Johnson, 2000). Reduced PSII efficiency limits electron transfer from QA to QB, decreasing plastoquinone reduction and proton gradient formation across the thylakoid membrane, thereby constraining ATP and NADPH production (Broser et al., 2011). Because ATP and NADPH drive the Calvin–Benson cycle, impaired PSII function mechanistically results in decreased carbon fixation and overall photosynthetic rate.

The empirical support of this KER is considered high. Rationale: A substantial body of experimental evidence demonstrates strong incidence, dose, and temporal concordance between decreased Photosystem II (PSII) efficiency and reduced photosynthetic performance across cyanobacteria, algae, aquatic macrophytes, and higher plants. Reductions in chlorophyll fluorescence parameters (e.g., Fv/Fm, ΦPSII) are consistently accompanied by declines in oxygen evolution and carbon fixation, with few inconsistencies reported. Evidence: Dose concordance: Increasing concentrations of PSII inhibitors (e.g., diuron, atrazine, terbutryn) cause progressive declines in Fv/Fm and ΦPSII, which are quantitatively paralleled by reductions in oxygen evolution and CO₂ assimilation rates. In controlled experiments, decreases in fluorescence-based PSII efficiency strongly correlate with inhibition of electron transport and reduced photosynthetic activity (Maxwell and Johnson, 2000; Delieu and Walker, 1981). Incidence concordance: Impaired PSII photochemistry, reflected by altered QA reoxidation kinetics and reduced quantum yield, is consistently associated with diminished photosynthetic oxygen evolution in functional assays across taxa (Sundby et al., 1993; Broser et al., 2011). Temporal concordance: Reductions in PSII efficiency occur rapidly (minutes to hours) after herbicide exposure or photoinhibitory stress, followed by measurable decreases in carbon fixation and net photosynthesis, supporting upstream–downstream sequence consistency (Macinnis-Ng and Ralph, 2003; Wilkinson et al., 2015). Genetic/functional concordance: Mutations or damage affecting PSII reaction center components reduce photochemical efficiency and are directly accompanied by decreased photosynthetic capacity, supporting causal linkage between PSII impairment and reduced photosynthesis (Ohad and Hirschberg, 1992; Alfonso et al., 1996).

Some variability exists across species, light regimes, and exposure conditions. PSII efficiency reductions do not always translate proportionally to decreased whole-organism photosynthesis due to compensatory mechanisms, alternative electron pathways, or short-term acclimation. Differences in measurement methods and recovery dynamics may also introduce variability in observed effect magnitude.

<table> <thead> <tr> <th>Modulating Factor (MF)</th> <th>MF Specification</th> <th>Effect(s) on the KER</th> <th>Reference(s)</th> </tr> </thead> <tbody> <tr> <td>Light intensity</td> <td>Low vs. high irradiance; fluctuating light</td> <td>High light amplifies PSII photoinhibition and accelerates decline in photosynthesis; low light may partially mask PSII impairment</td> <td>Maxwell and Johnson, 2000</td> </tr> <tr> <td>Temperature</td> <td>Suboptimal vs. optimal thermal range</td> <td>Alters membrane fluidity and enzyme kinetics, modifying electron transport and recovery capacity</td> <td>Sundby et al., 1993</td> </tr> <tr> <td>Nutrient status</td> <td>Nitrogen or iron limitation</td> <td>Reduces chlorophyll content and D1 repair capacity, strengthening coupling between PSII decline and photosynthesis reduction</td> <td>Maxwell and Johnson, 2000</td> </tr> <tr> <td>Species-specific traits</td> <td>NPQ capacity, cyclic electron flow, antenna size</td> <td>Enhanced photoprotective mechanisms buffer translation of PSII impairment into reduced photosynthesis</td> <td>Maxwell and Johnson, 2000</td> </tr> <tr> <td>Exposure duration</td> <td>Acute vs. chronic exposure</td> <td>Short-term inhibition may be reversible; prolonged exposure leads to sustained reduction in photosynthesis</td> <td>Macinnis-Ng and Ralph, 2003</td> </tr> </tbody> </table>

The quantitative understanding is high. PSII efficiency endpoints consistently show equal or greater sensitivity than whole-photosynthesis measurements, reflecting upstream positioning in the pathway. In Rhodomonas salina, the EC₅₀ for PSII photoinhibition (ΔF/Fm′) was 1.71 µg/L diuron, compared to 6.27 µg/L for growth (Thomas et al., 2020). Oxygen evolution in Synechococcus elongatus was inhibited by 50% at 20 µg/L diuron and 65 µg/L atrazine (Jones et al., 2003). Regulatory assessments report similar EC₅₀ values for atrazine based on fluorescence (232 µg/L) and oxygen evolution (222 µg/L), demonstrating quantitative concordance (USEPA, n.d.).

A strong response–response relationship exists between PSII efficiency and photosynthetic. Declines in Fv/Fm or ΦPSII correlate proportionally with reductions in electron transport, oxygen evolution, and CO₂ fixation. This quantitative alignment supports a mechanistically consistent, predictive linkage between upstream photochemical impairment and downstream photosynthetic performance.

The time-scale of this KER is rapid and sequential. Decreases in PSII efficiency typically occur within minutes to hours following exposure to PSII inhibitors or photoinhibitory stress. Reductions in oxygen evolution and carbon fixation follow shortly thereafter, while longer-term decreases in growth emerge under sustained exposure conditions.

PSII impairment activates feedback mechanisms such as increased non-photochemical quenching (NPQ) and cyclic electron flow, which transiently compensate for reduced photochemical efficiency (Maxwell and Johnson, 2000). However, sustained PSII inhibition enhances reactive oxygen species (ROS) formation, leading to oxidative damage of the D1 protein and amplifying photosynthetic decline through feedforward stress pathways (Sundby, Chow and Anderson, 1993; Alfonso et al., 1996).

High Unspecific

High

All life stages

High High High High

Taxonomic applicability: This KER applies to all oxygenic photosynthetic taxa possessing Photosystem II (PSII), including cyanobacteria, chlorophytes (green algae), diatoms, macrophytes (e.g., Lemna spp.), seagrasses, and higher terrestrial plants. The mechanistic linkage is conserved because PSII structure and electron transport function are highly evolutionarily conserved across these groups. Sex applicability: Not sex-specific. Photosynthesis is not sexually dimorphic; therefore, the KER is applicable to both male and female individuals in dioecious plant species, as well as hermaphroditic or clonal organisms. Life-stage applicability: Applicable across all photosynthetically active life stages, including vegetative growth stages, juvenile and adult plants, and algal exponential growth phases. Sensitivity may vary with developmental stage due to differences in chloroplast density, metabolic demand, and repair capacity, but the mechanistic relationship remains consistent. Chemical applicability: Most relevant for PSII-targeting chemicals (e.g., triazines, phenylureas, triazinones) and stressors causing photoinhibition, oxidative damage to D1 protein, or disruption of thylakoid electron transport. Environmental applicability: Relevant under light-exposed conditions in freshwater, marine, and terrestrial ecosystems. The linkage may be modulated by irradiance, temperature, and nutrient status.

Alfonso, M., Pueyo, J.J., Gaddour, K., Etienne, A.-L., Kirilovsky, D. and Picorel, R., 1996. Induced new mutation of D1 serine-268 in soybean photosynthetic cell cultures produced atrazine resistance, increased stability of S2QB⁻ and S3QB⁻ states, and increased sensitivity to light stress. Plant Physiology, 112(4), pp.1499–1508. Broser, M., Glöckner, C., Gabdulkhakov, A., Guskov, A., Buchta, J., Kern, J., Müh, F., Dau, H., Saenger, W. and Zouni, A., 2011. Structural basis of cyanobacterial Photosystem II inhibition by the herbicide terbutryn. Journal of Biological Chemistry, 286(18), pp.15964–15972. Delieu, T. and Walker, D.A., 1981. Polarographic measurement of photosynthetic oxygen evolution by leaf discs. New Phytologist, 89(2), pp.165–178. Macinnis-Ng, C.M.O. and Ralph, P.J., 2003. Short-term response and recovery of the seagrass Zostera capricorni to the herbicide diuron. Marine Environmental Research, 55(2), pp.153–166. Maxwell, K. and Johnson, G.N., 2000. Chlorophyll fluorescence—A practical guide. Journal of Experimental Botany, 51(345), pp.659–668. Ohad, N. and Hirschberg, J., 1992. Mutations in the D1 protein of photosystem II affect herbicide binding and electron transport properties. Plant Cell, 4(3), pp.273–282. Sundby, C., Chow, W.S. and Anderson, J.M., 1993. Effects on Photosystem II function, photoinhibition, and herbicide binding caused by mutation of the D1 protein. Photosynthesis Research, 36(2), pp.123–135. Wilkinson, A.D., Collier, C.J., Flores, F. and Ralph, P.J., 2015. Assessing the toxicity of herbicides to tropical seagrasses using chlorophyll fluorescence. Marine Pollution Bulletin, 95(2), pp.449–455. Jones, R.J., Muller, J., Haynes, D. and Schreiber, U., 2003. Effects of herbicides diuron and atrazine on corals of the Great Barrier Reef, Australia. Marine Ecology Progress Series, 251, pp.153–167. Thomas, M.C. et al., 2020. Toxicity of ten herbicides to the tropical marine microalgae Rhodomonas salina. Scientific Reports, 10, Article 7521. U.S. Environmental Protection Agency (EPA), n.d. Ambient Aquatic Life Water Quality Criteria for Atrazine AOPWiki 2021-04-11T08:25:30

2026-02-23T07:43:31

<upstream-id>5c7d7a53-fec1-4683-a1cd-9543dc16ddce</upstream-id> <downstream-id>af5e0245-f693-4132-be67-876410e24500</downstream-id> The KER between decreased photosynthesis and decreased mitochondrial oxidative phosphorylation (OXPHOS) reflects the tight metabolic coupling between chloroplast carbon fixation and mitochondrial respiration in photosynthetic eukaryotes. Reduced photosynthetic electron transport lowers ATP and NADPH production and limits CO₂ assimilation, thereby decreasing carbohydrate availability for mitochondrial respiration (Maxwell & Johnson, 2000). As a consequence, reduced supply of pyruvate and tricarboxylic acid (TCA) cycle intermediates constrains electron input into the mitochondrial electron transport chain, diminishing OXPHOS activity and ATP synthesis (Milligan et al., 2015). Furthermore, disruption of photosynthetic redox balance can alter respiratory flux and mitochondrial energy metabolism, reinforcing cross-organelle energetic dependence.

Evidence was collected through targeted literature searches linking photosynthetic inhibition to respiratory and mitochondrial endpoints, including oxygen consumption, ATP synthesis rates, and OXPHOS enzyme activity. Screening incorporated studies measuring both chlorophyll fluorescence (Fv/Fm) and mitochondrial respiration parameters to ensure mechanistic continuity between reduced carbon fixation and diminished oxidative phosphorylation.

Experimental evidence demonstrates that inhibition of photosynthesis reduces mitochondrial respiration due to diminished carbon substrate supply. Studies measuring chlorophyll fluorescence (Fv/Fm) and carbon fixation show that reduced photosynthetic performance directly limits carbohydrate production (Maxwell & Johnson, 2000). In phytoplankton and plants, decreased ¹⁴C assimilation is associated with reduced respiratory oxygen consumption, reflecting constrained substrate flow into the tricarboxylic acid cycle (Milligan et al., 2015). Additionally, stress-induced impairment of photosynthetic electron transport alters cellular redox balance, influencing mitochondrial metabolic flux. Together, these findings support a mechanistic link between decreased photosynthetic carbon assimilation and reduced mitochondrial oxidative phosphorylation activity.

Substantial evidence demonstrates tight functional integration between chloroplast photosynthesis and mitochondrial oxidative phosphorylation in photosynthetic eukaryotes. Photosynthetically derived carbohydrates provide the primary substrates for glycolysis and the tricarboxylic acid (TCA) cycle, which generate NADH and FADH₂ for the mitochondrial electron transport chain (Raghavendra & Padmasree, 2003). When photosynthetic carbon assimilation declines, respiratory substrate availability is reduced, resulting in decreased mitochondrial electron transport and ATP synthesis (Noguchi & Yoshida, 2008). In addition, chloroplast–mitochondria redox shuttles coordinate cellular NAD(P)H balance; disruption of photosynthetic electron flow alters mitochondrial respiratory flux and energy homeostasis (Noctor et al., 2007). Together, these studies support a mechanistic linkage whereby decreased photosynthesis constrains mitochondrial oxidative phosphorylation through substrate limitation and redox coupling.

The empirical support of this KER is considered moderate. Rationale: A substantial body of experimental evidence demonstrates incidence and temporal concordance between reduced photosynthetic carbon assimilation and decreased mitochondrial respiration in photosynthetic organisms. However, fewer studies directly quantify oxidative phosphorylation parameters (e.g., ATP synthesis rate, respiratory control ratio) alongside photosynthetic inhibition, limiting quantitative resolution. <h3>Evidence</h3> Dose concordance: In plant mesophyll cells, inhibition of photosynthesis using PSII inhibitors (e.g., DCMU) reduces carbohydrate production and is accompanied by decreases in mitochondrial respiratory flux and coupled oxidative metabolism (Padmasree et al., 2001; Noguchi & Yoshida, 2008). Incidence concordance: Experimental reductions in carbon fixation consistently correspond with reduced mitochondrial oxygen consumption due to limited substrate flow into glycolysis and the TCA cycle (Raghavendra, 1994; Raghavendra & Padmasree, 2003). Temporal concordance: Respiratory adjustments occur rapidly following changes in photosynthetic performance, particularly under illuminated conditions where chloroplast–mitochondrial coupling is strongest (Noguchi & Yoshida, 2008). Mechanistic concordance: Chloroplast–mitochondria metabolite exchange and redox shuttling provide a mechanistic basis whereby reduced photosynthetic ATP/NADPH production constrains mitochondrial electron transport and oxidative phosphorylation (Noctor et al., 2007; Igamberdiev, 2023).

Uncertainty arises because mitochondrial respiration can be partially maintained through alternative substrates (e.g., stored carbohydrates or photorespiration), buffering short-term declines in photosynthesis. Additionally, some studies report compensatory increases in respiratory activity under stress. Direct measurements of OXPHOS parameters are limited, reducing quantitative precision of this linkage.

<table> <thead> <tr> <th>Modulating Factor (MF)</th> <th>MF Specification</th> <th>Effect(s) on the KER</th> <th>Reference(s)</th> </tr> </thead> <tbody> <tr> <td>Light intensity</td> <td>Low vs. high irradiance; photoinhibitory vs. optimal light</td> <td>High light amplifies chloroplast–mitochondria redox coupling and increases respiratory demand; low light reduces substrate flux, weakening the linkage</td> <td>Noguchi & Yoshida, 2008</td> </tr> <tr> <td>Carbon availability</td> <td>CO₂ concentration; carbohydrate reserves</td> <td>Low CO₂ or depleted carbohydrate pools strengthen substrate limitation, enhancing the impact of reduced photosynthesis on OXPHOS</td> <td>Raghavendra & Padmasree, 2003</td> </tr> <tr> <td>Developmental stage</td> <td>Young vs. mature leaves/cells</td> <td>Younger tissues with higher metabolic turnover show stronger coupling between carbon fixation and respiration</td> <td>Padmasree et al., 2001</td> </tr> <tr> <td>Alternative oxidase (AOX) activity</td> <td>Induction of AOX pathway under stress</td> <td>AOX can partially uncouple electron transport from ATP production, modifying the magnitude of OXPHOS reduction</td> <td>Noctor et al., 2007</td> </tr> <tr> <td>Temperature</td> <td>Suboptimal vs. optimal temperature</td> <td>Temperature affects both photosynthesis and mitochondrial enzyme kinetics, altering respiratory compensation capacity</td> <td>Noguchi & Yoshida, 2008</td> </tr> <tr> <td>Nutrient status</td> <td>Nitrogen or phosphate limitation</td> <td>Nutrient stress modifies carbon allocation and respiratory metabolism, influencing the strength of organelle coupling</td> <td>Raghavendra, 1994</td> </tr> <tr> <td>Stress-induced ROS</td> <td>Elevated ROS levels</td> <td>Oxidative stress can impair both chloroplast and mitochondrial function, potentially amplifying the KER</td> <td>Igamberdiev, 2023</td> </tr> </tbody> </table>

The quantitative understanding of this KER is moderate Rationale: few experimental studies demonstrate that reductions in photosynthetic carbon assimilation (via chemical inhibition or metabolic perturbation) coincide with decreases in mitochondrial respiratory activity in iprimary producers. While dose–response characterization of mitochondrial OXPHOS secondary to photosynthesis inhibition is not common, proportional decreases in respiration align with graded photosynthetic impairment. Dose-reponse concordance: In Lemna minor exposed to gamma radiation for 7 days, CO₂ uptake declined dose-dependently (EDR10 = 2.8 mGy/h; EDR50 = 53.2 mGy/h), while mitochondrial membrane potential (MMP, proxy for OXPHOS) decreased at higher thresholds (EDR10 = 21.8 mGy/h; EDR50 = 144.7 mGy/h). The lower sensitivity threshold for photosynthesis relative to MMP supports upstream–downstream quantitative concordance between decreased carbon assimilation and mitochondrial dysfunction (Xie et al., 2019). Temporal concordance: Both photosynthesis endpoints and mitochondrial membrane potential (MMP) were measured after the same 7-day exposure period. Declines in CO₂ uptake and PSII efficiency co-occurred with significant reductions in MMP at ≥24 mGy/h, demonstrating coordinated impairment of chloroplast and mitochondrial function within the same biological timeframe (Xie et al., 2019).

The biological plausibility of this KER is high due to the fundamental metabolic coupling between chloroplasts and mitochondria in photosynthetic eukaryotes. Photosynthesis generates carbohydrates that serve as substrates for glycolysis and the tricarboxylic acid (TCA) cycle, providing reducing equivalents (NADH, FADH₂) to the mitochondrial electron transport chain (Milligan et al., 2015). Reductions in PSII efficiency and carbon fixation therefore constrain respiratory substrate availability (Maxwell & Johnson, 2000). Furthermore, chloroplast and mitochondrial redox states are interconnected through metabolite exchange and shared energy demands, linking photosynthetic performance with respiratory flux. Disruption of photosynthetic electron transport alters cellular redox balance and impacts downstream mitochondrial ATP production (Broser et al., 2011; Delieu & Walker, 1981).

The time-scale of this KER is rapid under illuminated conditions. Experimental inhibition of photosynthesis leads to measurable decreases in mitochondrial respiratory flux within minutes to hours, reflecting immediate limitation of carbon substrates and redox imbalance (Noguchi & Yoshida, 2008; Padmasree et al., 2001). Sustained inhibition can prolong respiratory suppression.

Reduced photosynthesis limits carbohydrate supply to mitochondria, decreasing OXPHOS and ATP production. Lower ATP availability can further constrain chloroplast metabolism, reinforcing energetic limitation (Raghavendra & Padmasree, 2003). Additionally, redox imbalance promotes reactive oxygen species formation, which can impair both chloroplast and mitochondrial function, amplifying metabolic suppression (Noctor et al., 2007).

High Unspecific

High

All life stages

High High High

Taxonomic applicability: This KER applies broadly to oxygenic photosynthetic eukaryotes in which chloroplast carbon fixation supplies substrates for mitochondrial respiration. It is strongly supported in higher plants, green algae, and photosynthetic protists, where chloroplast–mitochondria metabolic coupling is well documented (Raghavendra & Padmasree, 2003; Noguchi & Yoshida, 2008). Applicability to cyanobacteria is limited because respiration and photosynthesis occur within the same cellular membrane system rather than in distinct organelles. Sex applicability: Not sex-specific. The metabolic linkage between photosynthesis and mitochondrial oxidative phosphorylation is fundamental to cellular bioenergetics and operates similarly in male and female individuals of dioecious plants. In unicellular algae and clonal macrophytes, sex differentiation is generally not relevant. Life-stage applicability: Applicable across all photosynthetically active life stages, including vegetative cells, seedlings, mature leaves, and reproductive tissues that perform photosynthesis. The magnitude of coupling may vary with developmental stage due to differences in metabolic demand, carbohydrate storage capacity, and respiratory flexibility (Padmasree et al., 2001). Chemical domain: Relevant to chemicals that reduce photosynthetic carbon assimilation, including PSII inhibitors (e.g., triazines, phenylureas), PSI inhibitors, electron transport disruptors, pigment synthesis inhibitors, and carbon fixation inhibitors. Chemicals acting exclusively on mitochondria without affecting photosynthesis fall outside this KER. Indirect stressors (e.g., light deprivation, CO₂ limitation) are also within scope if they reduce carbon fixation and secondarily constrain mitochondrial oxidative phosphorylation.

Broser, M., Glöckner, C., Gabdulkhakov, A., Guskov, A., Buchta, J., Kern, J., Müh, F., Dau, H., Saenger, W., & Zouni, A. (2011). Structural basis of cyanobacterial photosystem II inhibition by the herbicide terbutryn. Journal of Biological Chemistry, 286(18), 15964–15972. Delieu, T., & Walker, D.A. (1981). Polarographic measurement of photosynthetic oxygen evolution by leaf discs. New Phytologist, 89(2), 165–178. Igamberdiev, A.U. (2023). Mitochondria in photosynthetic cells: Coordinating redox and energy metabolism. Plant Physiology, 191(4), 2104–2120. Maxwell, K., & Johnson, G.N. (2000). Chlorophyll fluorescence—a practical guide. Journal of Experimental Botany, 51(345), 659–668. Milligan, A.J., Halsey, K.H., & Behrenfeld, M.J. (2015). Advancing interpretations of ¹⁴C-uptake measurements in the context of phytoplankton physiology and ecology. Journal of Plankton Research, 37(4), 692–698. Noctor, G., De Paepe, R., & Foyer, C.H. (2007). Mitochondrial redox biology and homeostasis in plants. Trends in Plant Science, 12(3), 125–134. Noguchi, K., & Yoshida, K. (2008). Interaction between photosynthesis and respiration in illuminated leaves. Mitochondrion, 8(1), 87–99. Padmasree, K., Padmavathi, L., & Raghavendra, A.S. (2001). Essentiality of mitochondrial oxidative metabolism for photosynthetic performance in plant cells. Plant Physiology, 125(2), 617–626. Raghavendra, A.S. (1994). Interdependence of photosynthesis and respiration in plant cells. Photosynthesis Research, 38, 3–14. Raghavendra, A.S., & Padmasree, K. (2003). Beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation. Trends in Plant Science, 8(11), 546–553. Xie, L., Solhaug, K.A., Song, Y., Brede, D.A., Lind, O.C., Salbu, B., & Tollefsen, K.E. (2019). Modes of action and adverse effects of gamma radiation in an aquatic macrophyte Lemna minor. Science of the Total Environment, 680, 23–34. AOPWiki 2025-08-05T07:27:08

2026-02-23T08:46:45

<upstream-id>af5e0245-f693-4132-be67-876410e24500</upstream-id> <downstream-id>84d5f25e-ef7c-40d0-b428-15e5eda9b31e</downstream-id> Oxidative phosphorylation (OXPHOS) is the primary mechanism for ATP synthesis in aerobic eukaryotic cells, coupling electron transport chain (ETC) activity to ATP production via the proton motive force (Mitchell, 1961; Nicholls & Ferguson, 2013). Experimental inhibition of ETC complexes (I–IV) or ATP synthase consistently results in decreased mitochondrial membrane potential and reduced ATP generation (Brand & Nicholls, 2011). Chemical uncouplers and respiratory chain inhibitors (e.g., rotenone, antimycin A) produce concentration-dependent declines in cellular ATP levels (Wallace, 2012). Together, biochemical, pharmacological, and bioenergetic studies provide strong mechanistic and quantitative evidence linking impaired OXPHOS directly to decreased ATP production.

Evidence was gathered through targeted literature searches focusing on mitochondrial bioenergetics, electron transport chain inhibition, and ATP synthesis measurements. Studies were selected that quantified both OXPHOS impairment (e.g., membrane potential, respiratory control ratio, complex activity) and ATP levels to ensure direct mechanistic linkage between decreased mitochondrial oxidative phosphorylation and reduced ATP production.

Experimental evidence demonstrates that suppression of mitochondrial respiratory activity results in measurable declines in cellular ATP content. In isolated mitochondria, inhibition of complex I (rotenone), complex III (antimycin A), or complex IV (cyanide) reduces oxygen consumption rates and is accompanied by proportional decreases in ATP synthesis rates (Chance & Williams, 1955; Hatefi, 1985). Measurements of respiratory control ratios show that impaired electron flow diminishes ADP-stimulated phosphorylation efficiency (Nicholls, 2004). Genetic defects affecting ETC components similarly reduce ATP output and cellular energy charge (Wallace, 1999). Together, biochemical, pharmacological, and genetic evidence consistently supports a direct causal linkage between decreased OXPHOS capacity and reduced ATP production.

The biological plausibility of this KER is high because ATP synthesis in aerobic cells is fundamentally dependent on oxidative phosphorylation (OXPHOS). Electron transport through complexes I–IV establishes a proton gradient across the inner mitochondrial membrane, which drives ATP synthase activity (Mitchell, 1961). Any reduction in electron flux decreases proton motive force and directly limits ATP generation (Hatefi, 1985). Experimental measurements of respiratory control demonstrate that impaired coupling efficiency reduces ADP phosphorylation rates (Chance & Williams, 1955). Furthermore, genetic defects in mitochondrial DNA or ETC components consistently result in reduced ATP availability and energetic failure at the cellular level (Wallace, 1999), confirming the mechanistic dependency.

The empirical support of this KER is considered high. Rationale: Extensive biochemical and pharmacological evidence demonstrates strong dose and temporal concordance between inhibition of mitochondrial oxidative phosphorylation (OXPHOS) and reduction in ATP production. Because ATP synthase activity depends directly on the proton motive force generated by the electron transport chain (ETC), disruption at any ETC complex consistently results in decreased ATP synthesis across experimental systems. Dose-response concordance: In isolated mitochondria, inhibition of Complex I by rotenone produces an IC₅₀ of ~5–20 nM, with 40–80% ATP reduction observed at 10–100 nM (Hatefi, 1985; Wallace, 1999). Antimycin A (Complex III inhibitor) shows IC₅₀ values of ~1–10 nM and induces >50% ATP depletion at low nanomolar concentrations (Nicholls, 2004). Oligomycin directly inhibits ATP synthase with IC₅₀ ~10–50 nM, reducing ATP production by >70% (Chance & Williams, 1955). These data demonstrate tight quantitative coupling between respiratory inhibition and ATP decline. Incidence concordance: Across diverse eukaryotic systems, inhibition of ETC complexes I–IV or ATP synthase consistently results in decreased mitochondrial membrane potential and reduced cellular ATP levels, confirming mechanistic dependency (Hatefi, 1985). Temporal concordance: ATP decline occurs rapidly following OXPHOS inhibition. In isolated mitochondria, suppression of ATP synthesis is detectable within seconds to minutes after inhibitor addition (Chance & Williams, 1955). In intact cells, measurable ATP depletion occurs within 5–30 minutes depending on metabolic reserve capacity (Nicholls, 2004), indicating immediate energetic consequences.

because ATP levels may be transiently sustained through glycolysis, phosphocreatine buffering, or light reactions in chloroplasts, partially compensating for reduced mitochondrial OXPHOS (Nicholls, 2004; Raghavendra & Padmasree, 2003). Additionally, cell type–specific metabolic flexibility and mitochondrial reserve capacity can modify the quantitative relationship between respiratory inhibition and ATP depletion (Wallace, 1999).

<table> <thead> <tr> <th>Modulating Factor (MF)</th> <th>MF Specification</th> <th>Effect(s) on the KER</th> <th>Reference(s)</th> </tr> </thead> <tbody> <tr> <td>Glycolytic capacity</td> <td>High vs. low glycolytic flux</td> <td>High glycolytic capacity buffers ATP decline despite partial OXPHOS inhibition, reducing apparent magnitude of the KER</td> <td>Nicholls, 2004</td> </tr> <tr> <td>Phosphocreatine buffering</td> <td>Creatine kinase system activity</td> <td>Phosphocreatine stores transiently maintain ATP levels during acute OXPHOS inhibition, delaying ATP depletion</td> <td>Nicholls, 2004</td> </tr> <tr> <td>Photosynthetic light reactions</td> <td>Chloroplast ATP production under illumination</td> <td>In photosynthetic cells, light-driven ATP synthesis can partially compensate for reduced mitochondrial ATP production</td> <td>Raghavendra & Padmasree, 2003</td> </tr> <tr> <td>Mitochondrial reserve capacity</td> <td>Spare respiratory capacity</td> <td>Cells with high reserve capacity tolerate partial ETC inhibition before ATP levels decline</td> <td>Wallace, 1999</td> </tr> <tr> <td>Oxygen availability</td> <td>Normoxia vs. hypoxia</td> <td>Hypoxia independently limits ETC activity, amplifying ATP decline under OXPHOS impairment</td> <td>Hatefi, 1985</td> </tr> <tr> <td>Substrate availability</td> <td>NADH/FADH₂ supply from TCA cycle</td> <td>Limited reducing equivalents exacerbate ATP reduction during ETC inhibition</td> <td>Hatefi, 1985</td> </tr> <tr> <td>Cell type / tissue metabolic demand</td> <td>High vs. low energy-demand tissues</td> <td>High-demand tissues (e.g., muscle, neurons) exhibit more rapid ATP depletion when OXPHOS declines</td> <td>Wallace, 1999</td> </tr> </tbody> </table>

The quantitative understanding of this KER is high at the mechanistic level. ATP synthesis rate is directly proportional to proton motive force generated by electron transport, and reductions in respiratory flux translate into proportional declines in ATP production (Mitchell, 1961; Nicholls, 2004). Pharmacological inhibition of Complex I–III typically shows IC₅₀ values in the low nanomolar range (≈1–20 nM), with 40–80% ATP reduction observed at concentrations causing ≥50% respiration inhibition (Hatefi, 1985). The response–response relationship is approximately linear under moderate inhibition but becomes nonlinear near complete OXPHOS collapse due to loss of membrane potential and energetic failure (Wallace, 1999).

The response–response relationship between decreased OXPHOS and ATP production is typically proportional under moderate inhibition, as ATP synthesis depends directly on proton motive force generated by electron transport (Mitchell, 1961). When respiratory inhibition exceeds ~70–80%, ATP decline becomes nonlinear due to membrane potential collapse and loss of phosphorylation capacity (Nicholls, 2004).

The time-scale of this KER is rapid. Following acute inhibition of electron transport, ATP synthesis declines within seconds to minutes in isolated mitochondria due to immediate loss of proton motive force (Chance & Williams, 1955). In intact cells, measurable ATP depletion typically occurs within 5–30 minutes (Nicholls, 2004).

Reduced ATP production activates AMP-activated protein kinase (AMPK), which downregulates anabolic pathways and can suppress mitochondrial activity, reinforcing energy limitation (Hardie, 2011). Conversely, ATP depletion stimulates glycolysis as a compensatory feedback mechanism. Severe ATP loss further destabilizes membrane potential, amplifying OXPHOS impairment.

High Unspecific

High

All life stages

High High High High

Taxonomic applicability: This KER applies broadly to aerobic organisms possessing mitochondria with a functional electron transport chain (ETC) and ATP synthase. It is conserved across eukaryotes including animals, plants, fungi, and protists, as oxidative phosphorylation is the principal mechanism for ATP generation in mitochondria (Mitchell, 1961; Hatefi, 1985). In prokaryotes, analogous coupling between membrane-bound electron transport and ATP synthase occurs, but structural organization differs. Sex applicability: Not sex-specific. The biochemical mechanism of oxidative phosphorylation and ATP synthase function is conserved across sexes. However, quantitative sensitivity may vary due to sex-specific mitochondrial density, hormonal regulation, or metabolic demand (Wallace, 1999). Life-stage applicability: Applicable across all life stages that rely on mitochondrial respiration. Rapidly proliferating or high-energy-demand stages (e.g., embryonic, larval, neuronal, muscle tissues) may exhibit greater sensitivity to OXPHOS inhibition due to limited energetic buffering capacity (Nicholls, 2004). Chemical domain: Relevant to chemicals that impair mitochondrial electron transport or ATP synthase activity, including Complex I–IV inhibitors (e.g., rotenone, antimycin A, cyanide), ATP synthase inhibitors (e.g., oligomycin), and uncouplers of oxidative phosphorylation. Agents acting exclusively on glycolysis without affecting mitochondrial respiration fall outside this KER.

Brand, M.D., & Nicholls, D.G. (2011). Assessing mitochondrial dysfunction in cells. Biochemical Journal, 435(2), 297–312. Chance, B., & Williams, G.R. (1955). Respiratory enzymes in oxidative phosphorylation. I. Kinetics of oxygen utilization. Journal of Biological Chemistry, 217, 383–393. Hardie, D.G. (2011). AMP-activated protein kinase: An energy sensor that regulates all aspects of cell function. Genes & Development, 25(18), 1895–1908. Hatefi, Y. (1985). The mitochondrial electron transport and oxidative phosphorylation system. Annual Review of Biochemistry, 54, 1015–1069. Mitchell, P. (1961). Coupling of phosphorylation to electron and hydrogen transfer by a chemiosmotic type of mechanism. Nature, 191, 144–148. Nicholls, D.G. (2004). Mitochondrial membrane potential and aging. Aging Cell, 3(1), 35–40. Nicholls, D.G., & Ferguson, S.J. (2013). Bioenergetics 4. Academic Press. Raghavendra, A.S., & Padmasree, K. (2003). Beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation. Trends in Plant Science, 8(11), 546–553. Wallace, D.C. (1999). Mitochondrial diseases in man and mouse. Science, 283(5407), 1482–1488. Wallace, D.C. (2012). Mitochondria and cancer. Nature Reviews Cancer, 12(10), 685–698. AOPWiki 2025-08-05T07:27:20

2026-02-26T07:50:38

<upstream-id>84d5f25e-ef7c-40d0-b428-15e5eda9b31e</upstream-id> <downstream-id>680b6500-c8fb-4b06-897d-bcd9081e564c</downstream-id>

AOPWiki 2026-04-10T08:16:23

2026-04-10T08:16:23

<upstream-id>680b6500-c8fb-4b06-897d-bcd9081e564c</upstream-id> <downstream-id>0bb4c93f-10f1-4def-abc1-6a6ed2ca4827</downstream-id>

AOPWiki 2020-10-08T08:17:08

2020-10-08T08:17:08

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

Qb protein binding leading to decrease, population growth via PSII inhibition

Li Xie

Li Xiea, Knut Erik Tollefsena a Norwegian Institute for Water Research (NIVA), NO-0349, Oslo, Norway

BY-SA

2.7

Photosystem II (PSII) is essential for photosynthesis in primary producers, facilitating the primary photochemical reaction to oxidize water and drive the production of ATP and NADPH. A critical interaction within PSII occurs at the plastoquinone B (QB) site on the D1 protein, where electron transfer from QA to QB ensures the continuity of photosynthetic electron flow and energy transduction. PSII-inhibitors can competitively bind to this QB site, blocking electron flow and suppressing the production of ATP and NADPH in chloroplasts. This disruption constrains downstream processes at different biological levels and consequently result in growth inhibition. PSII inhibitors are frequently detected in surface and ground waters, especially in agricultural regions. Their prolonged mobility results in chronic exposure of non-target organisms, particularly primary producers such as algae, aquatic macrophytes, and terrestrial plants that share conserved QB binding sites with target weeds. To improve mechanistic understanding and strengthen ecological risk assessment of PSII-inhibitor, such as herbicides, AOP #567 was developed. This AOP delineates a linear cascade of key events, beginning with the binding of PSII inhibitors to the QB site on the D1 protein, leading through successive reductions in PSII efficiency, photosynthesis, mitochondrial OXPHOS, and ATP production, and culminating in decreased growth rates of primary producer populations. By systematically linking molecular interactions to population-level outcomes, AOP #567 provides a transparent and biologically plausible framework to assess the environmental hazards posed by PSII inhibitors. Photosynthesis is the fundamental biological process to convert solar energy into chemical energy and stored as carbohydrates in plants, algae, and cyanobacteria. It aids energy needs of both terrestrial and aquatic ecosystems. Being at the centre of the photosynthesis, photosystem II (PSII) performs the most important photochemical reaction through light capture resulting in oxidation of the water molecules releasing oxygen, protons and electrons (Chen et al., 2007). Such electrons are then passed on through the photosynthetic electron transport pathway, and eventually to the generation of ATP and NADPH. In a situation within the PSII complex, a momentous interaction that is in place is the plastoquinone B (QB) interaction capsule with the D1 protein, a pivotal location which receives electrons at the plastoquinone A (QA) (Ohad and Hirschberg, 1992). This is an essential electron transfer between QA and QB which is essential to the constancy of electron flow and energy transduction in photosynthesis (Vermaas, Renger and Arntzen, 1984). The binding site of the QB is common among herbicidal molecular targets because it is the key protein to electron transport (Fuerst and Michael, 1991).  PSII-inhibiting herbicides exert their phytotoxic effects by competitively binding to the QB site on the D1 protein. This binding disrupts the natural electron flow between QA and QB that stops electron flow in photosynthesis apparatus. This blockade direct suppressed the production of ATP and NADPH in chloroplast, which has serious impact on the followed metabolic process, such as such as carbon fixation and carbohydrate synthesis (Wilkinson et al., 2015). Reduced amount of generated carbohydrates output results in shorter amount of photosynthate to serve as mitochondrial oxidative phosphorylation (OXPHOS) substrate and consequently less ATP formation via the respiratory pathway (Hanson et al., 2023). At the whole-plant scale, about 50 percent of carbon dioxide fixed through photosynthesis is usually respired (Amthor and Baldocchi, 2001). Thus, constriction of carbon assimilation achieved by PSII inhibitors magnifies the cellular energy deficiency by restraining not only photosynthetic but as well as respiratory ATP synthesis. This deconvoluted energy deficiency is the root cause of metabolism malfunction and major reduction of growth in PSII-stressed plants.  Although useful as agricultural compounds, PSII inhibitors have elicited extreme environmental concerns considering how chemically stable they are, moderately water soluble, and resistance to breakdown and movement throughout the ecosystem (Brock, Lahr and Van den Brink, 2000). These chemicals are often found in surface and ground water, especially in agriculture areas as it is introduced through run off and leaching. Their prolonged environmental persistence and mobility mean that they can affect non-target organisms in aquatic and terrestrial habitats long after application. Primary producers, such as algae, aquatic macrophytes and terrestrial plants, are particularly susceptible to PSII herbicides, since they contain highly conserved QB binding sites the same as the targeted weed species. Even low environmentally relevant concentrations can negatively affect photosynthetic efficiency, growth rates and normal primary productivity (Oettmeier, 1999). These effects may cascade through ecosystems and change species composition, community structures, and stability and functioning of whole ecosystems. To improve mechanistic insight and risk assessment of herbicides that inhibit photosystem II, AOP#567 (Binding of plastoquinone B causing a lowered population growth rate through a reduced population photosystem II efficiency) was developed to illustrate how the binding of PSII inhibitor to the QB site of the D1 protein interferes with photosynthetic electron transport and leads to a decreased growth rate of populations on primary producers.

The development of AOP 567 followed the guidance outlined in the OECD Users’ Handbook Supplement to the Guidance Document for Developing and Assessing AOPs (OECD, 2018). The pathway was constructed using a structured, evidence-based, and transparent process integrating systematic literature review, expert consultation, and formal weight-of-evidence (WoE) evaluation consistent with modified Bradford Hill considerations. 3.1 Problem Formulation and AOP Scoping The AOP was initiated based on the well-characterised mode of action of PSII-inhibiting chemicals that interact with the Q_B binding niche of the D1 protein. The molecular initiating event (MIE) was defined as interference at the Q_B site within PSII. Downstream key events (KEs) were sequentially identified based on established biological continuity: decreased PSII efficiency, reduced photosynthesis, impaired mitochondrial oxidative phosphorylation (OXPHOS), decreased ATP production, and reduced population growth rate. These relationships correspond to KER#3556, KER#2333, KER#3557, KER#3558, and KER#3559. 3.2 Systematic Literature Identification and Screening   A tiered and reproducible literature screening pipeline was applied. The AOP-helpFinder (v3.0) was used to screen bibliographic datasets for stressor–event and event–event relationships. Swift-Reviewer (Version 1.43.1063) was subsequently used to refine the evidence corpus through structured tagging, relevance scoring, and confidence ranking. This combined approach ensured traceability and reproducibility in evidence selection while reducing manual bias. 3.3 Weight-of-Evidence Evaluation The data extraction and assessment were done according to a weight-of-evidence (WOE) methodology in resonance to the modified Bradford Hill requirements (Becker et al., 2015; Collier et al., 2016). In particular, each key event relationship (KER) was evaluated by: (3) Essentiality of individual key events (KEs);  (2) biological plausibility in that it is based on known photosynthetic and bioenergetic principles; (3) empirical evidence such as the dose response, temporality, and occurrence of effects.

Growth is a regulatory relevant chronic toxicity endpoint for almost all organisms. Multiple OECD test guidelines have included growth either as a main endpoint of concern, or as an additional endpoint to be considered in the toxicity assessments. Relevant test guidelines include, but not only limited to:   -Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test -Test No. 208: Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test -Test No. 211: Daphnia magna Reproduction Test -Test No. 212: Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages -Test No. 215: Fish, Juvenile Growth Test -Test No. 221: Lemna sp. Growth Inhibition Test -Test No. 228: Determination of Developmental Toxicity to Dipteran Dung Flies (Scathophaga stercoraria L. (Scathophagidae), Musca autumnalis De Geer (Muscidae)) -Test No. 241: The Larval Amphibian Growth and Development Assay (LAGDA) -Test No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents -Test No. 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents -Test No. 416: Two-Generation Reproduction Toxicity -Test No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test -Test No. 443: Extended One-Generation Reproductive Toxicity Study -Test No. 453: Combined Chronic Toxicity/Carcinogenicity Studies

Maintenance of sustainable fish and wildlife populations (i.e., adequate to ensure long-term delivery of valued ecosystem services) is a widely accepted regulatory goal upon which risk assessments and risk management decisions are based.

adjacent

Low

High adjacent

High

High adjacent

Moderate

Moderate adjacent

High

High adjacent

Moderate

High adjacent

Low

High

High Unspecific

High

All life stages

High High High

The present AOPdescribes the potential causal events initiated by the binding of photosystem II (PSII) inhibitors to the plastoquinone B (QB) site of the D1 protein within the PSII complex (MIE, event 1975), leading to a decrease in population growth rate in primary producers (adverse outcome, event 2181) via a cascade of intermediate key events (KEs), including inhibition of PSII electron transport (event 1976), decrease in PSII efficiency (event 1977), and ultimately, decrease in growth rate (event 2031). The Table 1 provides a summary of  the KEs that constitute the AOP and the representative methods for measurement of each KE.  The MIE constitutes the competitive binding of PSII inhibitors to the QB site, which disrupts the transfer of electrons between QA and QB. This blockage has a direct inhibitory impact on the functioning of the PSII complex and causes the first downstream KE: the inhibition of PSII efficiency, which is typically reported as Fv/Fm ratio or ΦPSII. The lower PSII efficiency is an indication of reduced energy conversion in the light-dependent components of photosynthesis. As a logical physiological consequence, this leads to a decrease in carbon fixation and the availability of ATP and NADPH, both of which are essential for cellular growth. here is a consequent decrease in the rate of organismal growth, particularly on the taxa that grows fast like phytoplankton and aquatic plants. This series of KEs represents the minimal essential path of causally linked events required to describe how a molecular-level interaction with a chemical stressor produces an ecologically relevant adverse outcome. Each KE and its connection to the adjacent events are evidenced by the mechanistic and empirical evidence, dose-response relationships, as well as taxonomic concordance, as it is elaborated in the section “Summary of scientific evidence assessment” below. Although the subsequent secondary effects, such as oxidative stress and thylakoid membrane damage, also can happen after the PSII disruption, they do not constitute a necessary part of this AOP but rather play a modifying role. The mechanisms of the reactions discussed are very well conserved and the events are consequently found in very wide range of photosynthetic organisms i.e. algae, cyanobacteria, and aquatic vascular plants. This linear AOP can be included in a network of herbicide-related inhibition of photosynthesis and downstream effects at the population level that can occur across primary producers.     <table cellspacing="0" class="MsoTable15Plain4" style="border-collapse:collapse"> <tbody> <tr> <td style="height:328px; vertical-align:top; width:601px"> Table 1. Summary of key events in AOP #567 and related measurement methods <table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none; width:574px"> <tbody> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:19px; width:89px"> Event ID </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:19px; width:84px"> Description </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:19px; width:208px"> Measurement Methods </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:19px; width:194px"> Reference </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:52px; width:89px"> 2307 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:52px; width:84px"> Binding of PSII inhibitors to plastoquinone B </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:52px; width:208px"> In vitro binding assays (radioligand binding, fluorescence-based assays, surface plasmon resonance, isothermal titration calorimetry) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:52px; width:194px"> (Battaglino, Grinzato and Pagliano, 2021; Broser et al., 2011; Giardi and Pace, 2006; Piletska, Piletsky and Rouillon, 2006; Tischer and Strotmann, 1977; Zimmermann et al., 2006) </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:61px; width:89px"> 1862 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:61px; width:84px"> Decrease, photosystem II efficiency </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:61px; width:208px"> Chlorophyll fluorescence analysis (Fv/Fm ratio), oxygen evolution rates, PAM fluorometry, modulated fluorometry, D1 protein degradation assays </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:61px; width:194px"> (Alfonso et al., 1996; DELIEU and WALKER, 1981; Maxwell and Johnson, 2000; Xia et al., 2023) </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:47px; width:89px"> 1442 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:47px; width:84px"> Decrease, photosynthesis </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:47px; width:208px"> Carbon fixation rates (14C uptake), oxygen production, infrared gas analysis (CO2 uptake), measurement of Rubisco activity,O2 evolution </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:47px; width:194px"> (Grant and Howard, 1980; Lilley and Walker, 1974; Milligan, Halsey and Behrenfeld, 2015; Sales, da Silva and Carmo-Silva, 2020; van Gorkom and Gast, 1996; Xie et al., 2019) </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:52px; width:89px"> 1545 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:52px; width:84px"> Decrease, mitochondrial OXPHOS </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:52px; width:208px"> Respirometry, ATP synthesis rates, enzyme activity assays for complexes I-IV, mitochondrial membrane potential assays, blue-native PAGE </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:52px; width:194px"> (Coulson, Duffy and Staples, 2024; Djafarzadeh and Jakob, 2017; Hitchins, Cieslar and Dobson, 2001; Lundin, Rickardsson and Thore, 1976; Xie et al., 2019; Yan and Forster, 2009) </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:52px; width:89px"> 1472 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:52px; width:84px"> Decrease, ATP production </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:52px; width:208px"> Luciferase-based ATP assays, enzymatic assays for ATP synthase, NMR spectroscopy, high-performance liquid chromatography (HPLC) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:52px; width:194px"> (Allakhverdiev et al., 2005; Coulson, Duffy and Staples, 2024; Hitchins, Cieslar and Dobson, 2001; Juarez-Facio et al., 2021) </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:56px; width:89px"> 360 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:56px; width:84px"> Decrease, population growth rate </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:56px; width:208px"> Growth rate calculations from biomass or cell count, specific growth rate in cultures, flow cytometry for cell cycle analysis, optical density measurements </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:56px; width:194px"> (Peniuk, Schnurr and Allen, 2016; RICHARD, 1971; Xie, Macken and Tollefsen, 2025; Xie et al., 2019). OECD guidelines </td> </tr> </tbody> </table>   </td> </tr> </tbody> </table> <h2>Taxonomic Applicability</h2> 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. <h2>Sex and life stage</h2> 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). <h2>Biological context:</h2> 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.

<h2>1. Biological Plausibility</h2> 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 QB 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 QB 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.  <h2>2. Empirical Support</h2> 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 QB-binding site is occupied by an inhibitor. For instance, in isolated pea thylakoid membranes, QB site herbicides with higher inferred QB 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–Vj, reflecting QA reduction and QB 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). <h2>Overall WoE Considerations </h2> 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. <h2>Uncertainties, inconsistencies, and critical gaps</h2> 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.

<table> <thead> <tr> <th>Modulating Factor (MF)</th> <th>Influence or Outcome</th> <th>KER(s) involved</th> </tr> </thead> <tbody> <tr> <td>Light intensity</td> <td>High irradiance can amplify the impact of PSII inhibition by increasing excitation pressure and ROS formation; low light may partially buffer translation from PSII efficiency decline to measurable reductions in photosynthesis.</td> <td>KER#3556; KER#2333</td> </tr> <tr> <td>Temperature</td> <td>Elevated temperatures can exacerbate PSII instability and D1 turnover, increasing sensitivity to Q_B inhibitors; low temperatures may slow metabolic propagation to mitochondrial processes.</td> <td>KER#3556; KER#2333; KER#3557</td> </tr> <tr> <td>Nutrient availability (N, Fe)</td> <td>Nutrient limitation reduces baseline photosynthetic capacity and mitochondrial activity, potentially increasing susceptibility to energy limitation or masking proportional declines.</td> <td>KER#2333; KER#3557; KER#3558</td> </tr> <tr> <td>Species-specific photoprotective capacity (NPQ, cyclic electron flow)</td> <td>Enhanced non-photochemical quenching or alternative electron sinks can mitigate the functional impact of PSII inhibition on net photosynthesis.</td> <td>KER#3556; KER#2333</td> </tr> <tr> <td>Carbohydrate reserves</td> <td>High intracellular carbohydrate storage may buffer short-term reductions in photosynthesis, delaying downstream OXPHOS and ATP decline.</td> <td>KER#3557; KER#3558</td> </tr> <tr> <td>Mitochondrial respiratory flexibility</td> <td>Alternative oxidase pathways or metabolic reprogramming may partially compensate for reduced substrate supply, moderating ATP decline.</td> <td>KER#3557; KER#3558</td> </tr> <tr> <td>Exposure duration</td> <td>Short exposures may produce transient PSII inhibition without measurable population-level consequences; chronic exposure increases likelihood of ATP-mediated growth reduction.</td> <td>KER#3556; KER#2333; KER#3557; KER#3558; KER#3559</td> </tr> <tr> <td>Chemical potency and binding affinity at Q_B site</td> <td>Higher affinity inhibitors produce PSII inhibition at lower concentrations, shifting the quantitative threshold for downstream effects.</td> <td>KER#3556</td> </tr> <tr> <td>Life stage metabolic demand</td> <td>Rapidly growing or reproducing stages have higher ATP demand and may exhibit earlier population-level consequences following ATP depletion.</td> <td>KER#3558; KER#3559</td> </tr> <tr> <td>Environmental stress co-exposure (e.g., oxidative stressors)</td> <td>Additional stressors that impair mitochondrial function can synergistically enhance ATP depletion and growth suppression.</td> <td>KER#3557; KER#3558; KER#3559</td> </tr> </tbody> </table>

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 <table cellspacing="0" style="border-collapse:collapse; width:679px"> <tbody> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:40px; text-align:center; vertical-align:middle; white-space:normal; width:44px">KER</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; text-align:center; vertical-align:middle; white-space:normal; width:184px">KE Upstream → KE Downstream</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; text-align:center; vertical-align:middle; white-space:normal; width:162px">Rating</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; text-align:center; vertical-align:middle; white-space:normal; width:141px">Key Quantitative Data</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; text-align:center; vertical-align:middle; white-space:normal; width:146px">Limiting Factor</td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:76px; vertical-align:middle; white-space:normal; width:44px">3556</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:184px">QB binding → PSII efficiency</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:162px">Low</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:141px">I₅₀ diuron ≈ 7–8 × 10⁻⁸ M (PSII assays; Battaglino et al., 2021)</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:146px">Fractional QB occupancy not coupled to Fv/Fm decline</td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:190px; vertical-align:middle; white-space:normal; width:44px">2333</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:184px">PSII efficiency → Photosynthesis</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:162px">High</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:141px">EC₅₀ diuron: 1.71 µg/L (ΔF/Fm′) vs. 6.27 µg/L (growth) in Rhodomonas salina (Thomas et al., 2020); atrazine EC₅₀ fluorescence 232 µg/L ≈ O₂ evolution 222 µg/L (USEPA, n.d.)</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:146px">Compensatory NPQ varies across taxa</td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:95px; vertical-align:middle; white-space:normal; width:44px">3557</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:184px">Photosynthesis → Mitochondrial OXPHOS</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:162px">Moderate</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:141px">Lemna minor: EDR₁₀ CO₂ uptake = 2.8 mGy/h vs. EDR₁₀ MMP = 21.8 mGy/h (Xie et al., 2019)</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:146px">Limited simultaneous multi-endpoint studies under PSII herbicide exposure</td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:152px; vertical-align:middle; white-space:normal; width:44px">3558</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:184px">Mitochondrial OXPHOS → ATP production</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:162px">Moderate</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:141px">Rotenone IC₅₀ Complex I ≈ 5–20 nM → 40–80% ATP reduction; nonlinear at >70–80% OXPHOS inhibition (Hatefi, 1985; Nicholls, 2004)</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:146px">AOP-relevant mechanism is substrate-limited OXPHOS, not direct ETC inhibition</td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:171px; vertical-align:middle; white-space:normal; width:44px">3559</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:184px">ATP production → Population growth rate</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:162px">Moderate</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:141px">EC₅₀ ATP = 0.16 µM vs. EC₅₀ growth = 0.41 µM (~2.6-fold ratio) in C. reinhardtii (Nestler et al., 2012); Bayesian model for Lemna minor (Moe et al., 2021)</td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:146px">No fully parameterised qAOP model across the full concentration range</td> </tr> </tbody> </table>

This AOP provides a mechanistically anchored framework linking PSII-targeting stressors to ecologically relevant growth outcomes and is particularly suitable for application in environmental hazard assessment of PSII-inhibiting herbicides and related compounds. Structural and functional evidence demonstrating Q_B site inhibition and suppression of PSII electron transport (Broser et al., 2011; Battaglino et al., 2021) supports its relevance for identifying chemicals acting through this conserved molecular target.  For regulatory applications, AOP 567 can inform mode-of-action classification, weight-of-evidence evaluations, and ecological risk assessment of plant protection products and other PSII-active chemicals. The mechanistic continuity from PSII inhibition to growth impairment aligns with population-level risk frameworks linking individual performance to population growth rate (Forbes and Calow, 2002; Kramer et al., 2011). The pathway may also support mixture risk assessment where multiple stressors converge on photosynthetic or energetic processes. Limitations include environmental modulating factors such as light intensity and species-specific photoprotective mechanisms (e.g., NPQ), which may influence translation from PSII impairment to functional photosynthetic decline (Maxwell and Johnson, 2000). Additionally, while quantitative anchors exist for individual KERs, a fully parameterised qAOP model spanning the entire cascade has not yet been developed.

Not Specified

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