Evaluation of a Questionnaire-Based Method for the Estimation of Methylmercury Exposure of Recreational Anglers in the James Bay Territory (Québec,Canada)

The impoundment of reservoirs temporarily increases the methylation of mercury bound to flooded soils and vegetation and the transfer of methylmercury (MeHg) to fish. MeHg levels in various fish species of hydroelectric reservoirs located in the James Bay territory increased by factors of 3 to 7, then gradually declined toward initial concentrations 10 to 20 years after flooding, depending on reservoir characteristics. The potential risk of increased MeHg exposure for recreational anglers who consume fish from these reservoirs had not been assessed previously. A less invasive method than systematic measurement of Hg levels in hair was developed to determine MeHg exposure of recreational anglers. A fish consumption questionnaire-based approach was combined with a toxicokinetic model to estimate the corresponding hair MeHg concentrations. The results were compared with actual analytical determinations of hair Hg levels for the 94 recreational anglers recruited for the study. The values predicted by the model based on self-reporting consumption overestimated actual hair Hg levels by an average factor greater than 6. The mean hair level predicted for the most recent period (September-October) was 23.3?µg.g^?1 compared to 3.6 µg.g^?1 for the measured value. Although the questionnaire protocol may certainly be improved to increase the precision of estimations, direct hair Hg measurement remains the more effective means to assess Hg exposure.     

Effect of Bacopa monniera Extract on Methylmercury-Induced Behavioral and Histopathological Changes in Rats

Methylmercury (MeHg) is a well-recognized environmental contaminant with established health risk to human beings by fish and marine mammal consumption. Bacopa monniera (BM) is a perennial herb and is used as a nerve tonic in Ayurveda, a traditional medicine system in India. This study was aimed to evaluate the effect of B. monniera extract (BME) on MeHg-induced toxicity in rat cerebellum. Male Wistar rats were administered with MeHg orally at a dose of 5 mg/kg b.w. for 21 days. Experimental rats were given MeHg and also administered with BME (40 mg/kg, orally) 1 h prior to the administration of MeHg for 21 days. After treatment period, MeHg exposure significantly decreases the body weight and also caused the following behavioral changes. Decrease tail flick response, longer immobility time, significant decrease in motor activity, and spatial short-term memory. BME pretreatment reverted the behavioral changes to normal. MeHg exposure decreases the DNA and RNA content in cerebellum and also caused some pathological changes in cerebellum. Pretreatment with BME restored all the changes to near normal. These findings suggest that BME has a potent efficacy to alleviate MeHg-induced toxicity in rat cerebellum.     

A systems‐based approach to investigate dose‐ and time‐dependent methylmercury‐induced gene expression response in C57BL/6 mouse embryos undergoing neurulation

BACKGROUND: Aberrations during neurulation due to genetic and/or environmental factors underlie a variety of adverse developmental outcomes, including neural tube defects (NTDs). Methylmercury (MeHg) is a developmental neurotoxicant and teratogen that perturbs a wide range of biological processes/pathways in animal models, including those involved in early gestation (e.g., cell cycle, cell differentiation). Yet, the relationship between these MeHg‐linked effects and changes in gestational development remains unresolved. Specifically, current information lacks mechanistic comparisons across dose or time for MeHg exposure during neurulation. These detailed investigations are crucial for identifying sensitive indicators of toxicity and for risk assessment applications. METHODS: Using a systems‐based toxicogenomic approach, we examined dose‐ and time‐dependent effects of MeHg on gene expression in C57BL/6 mouse embryos during cranial neural tube closure, assessing for significantly altered genes and associated Gene Ontology (GO) biological processes. Using the GO‐based application GO‐Quant, we quantitatively assessed dose‐ and time‐dependent effects on gene expression within enriched GO biological processes impacted by MeHg. RESULTS: We observed MeHg to significantly alter expression of 883 genes, including several genes (e.g., Vangl2, Celsr1, Ptk7, Twist, Tcf7) previously characterized to be crucial for neural tube development. Significantly altered genes were associated with development cell adhesion, cell cycle, and cell differentiation–related GO biological processes. CONCLUSIONS: Our results suggest that MeHg‐induced impacts within these biological processes during gestational development may underlie MeHg‐induced teratogenic and neurodevelopmental toxicity outcomes. Birth Defects Res (Part B) 89:188–200, 2010. © 2010 Wiley‐Liss, Inc.     

At environmental doses, dietary methylmercury inhibits mitochondrial energy metabolism in skeletal muscles of the zebra fish (Danio rerio)

The neurotoxic compound methylmercury (MeHg) is a commonly encountered pollutant in the environment, and constitutes a hazard for human health through fish eating. To study the impact of MeHg on mitochondrial structure and function, we contaminated the model fish species Danio rerio with food containing 13 microg of MeHg per gram, an environmentally relevant dose. Mitochondria from contaminated zebrafish muscles presented structural abnormalities under electron microscopy observation. In permeabilized muscle fibers, we observed, a strong inhibition of both state 3 mitochondrial respiration and functionally isolated maximal cytochrome c oxidase (COX) activity after 49 days of MeHg exposure. However, the state 4 respiratory rate remained essentially unchanged. This suggested a defect at the level of ATP synthesis. Accordingly, we measured a dramatic decrease in the rate of ATP release by skinned muscle fibers using either pyruvate and malate or succinate as respiratory substrates. However, the amount and the assembly of the ATP synthase were identical in both control and contaminated muscle mitochondrial fractions. This suggests that MeHg induced a decoupling of mitochondrial oxidative phosphorylation in the skeletal muscle of zebrafish. Western blot analysis showed a 30% decrease of COX subunit IV levels, a 50% increase of ATP synthase subunit alpha, and a 40% increase of the succinate dehydrogenase Fe/S protein subunit in the contaminated muscles. This was confirmed by the analysis of gene expression levels, using RT-PCR. Our study provides a basis for further analysis of the deleterious effect of MeHg on fish health via mitochondrial impairment.     

Selenium Health Benefit Values as Seafood Safety Criteria

Selenium (Se) is absolutely required for activity of 25–30 genetically unique enzymes (selenoenzymes). All forms of life that have nervous systems possess selenoenzymes to protect their brains from oxidative damage. Homeostatic mechanisms normally maintain optimal selenoenzyme activities in brain tissues, but high methylmercury (MeHg) exposures sequester Se and irreversibly inhibit selenoenzyme activities. However, nutritionally relevant amounts of Se can replace the Se sequestered by MeHg and maintain normal selenoenzyme activities, thus preventing oxidative brain damage and other adverse consequences of MeHg toxicity. Findings of studies that seem contradictory from MeHg exposure perspectives are entirely consistent from MeHg:Se molar ratio perspectives. Studies that have reported dose-dependent consequences of maternal MeHg exposures on child development uniformly involved seafoods that contained much more Hg than Se. Meanwhile more typical varieties of ocean fish contain much more Se than Hg. This may explain why maternal MeHg exposure from eating ocean fish is associated with major IQ benefits in children instead of harm. Therefore, instead of being avoided, ocean fish consumption should be encouraged during pregnancy. However, the safety of freshwater fish consumption is less certain. In freshwater fish, MeHg bioaccumulation and toxicity are both inversely related to Se bioavailability. Their Se can be far lower than their MeHg contents, potentially making them more dangerous than pilot whale meats. Therefore, to provide accurate and appropriate regulatory advice regarding maternal consumption of seafoods and freshwater fish, Hg:Se molar ratios need to be incorporated in food safety criteria.     

Linking fish consumption patterns and health risk assessment of mercury exposure in a coastal community of NW Mexico

We aimed at characterizing the methylmercury (MeHg) exposure through fish consumption in two populations (common general and fishing-related population (FRP)) using a probabilistic health risk assessment in children, women of childbearing age, and adults in Mazatlán. The hazard quotients (HQs) were obtained from fish consumption, defined through a survey, and the levels of mercury in fishery products, obtained from published information. The average fish ingestion rate (IR_food) was higher in the FRP (167.85 g d^?1) than in the general population (GP) (140.9 g d^?1). However, HQs were significantly (p < 0.05) higher in the GP (ranging from 0.18 to 10.91) compared to the FRP (0.20 to 2.48); significant differences were also found among groups of both populations. Remarkably, children in both populations exhibited the highest proportions of risk, reaching up to 97% in GP. For all populations, fish consumption was the most important variable influencing MeHg exposure. Overall, for MeHg exposure, there is no safe level of fish consumption without risk, and actions should be taken to mitigate possible risk; further research with current data is needed to assess potential health risks associated with MeHg exposure, particularly in children.     

A Probabilistic Risk Assessment of the Effects of Methylmercury on Great Egrets and Bald Eagles Foraging at a Constructed Wetland in South Florida Relative to the Everglades

This case study summarizes an assessment of risk of methylmercury (MeHg) exposure to fish–eating birds foraging at Stormwater Treatment Area–2 (STA–2). This assessment was required as a special condition for a permit modification authorizing flow–through operation of STA–2 Cell 1 without it first satisfying formal mercury start–up criteria. The assessment estimates the risks posed by MeHg to the great egret (Ardea albus) and the bald eagle (Haliaeetus leucocephalus). Exposure models were based on literature–derived life history parameters combined with site–specific MeHg concentrations in water, sediment, and fish. To assess risk, daily MeHg intake by females and cumulative MeHg consumed by nestlings were compared to literature–derived effects thresholds. The results indicated the likelihood was low that MeHg exposures to birds foraging throughout STA–2 would exceed effects thresholds at the time of this assessment. Birds foraging exclusively from Cell 1 or the small discharge canal were predicted to experience greater exposures and could be at potential risk. However, this worst–case risk was comparable or lower than risk levels encountered in nearby water conservation areas or the Everglades National Park.     

Importance of dose‐response model form in probabilistic risk assessment: A case study of health effects from methylmercury in fish

We compared the effect of uncertainty in dose‐response model form on health risk estimates to the effect of uncertainty and variability in exposure. We used three different dose‐response models to characterize neurological effects in children exposed in utero to methylmercury, and applied these models to calculate risks to a native population exposed to potentially contaminated fish from a reservoir in British Columbia. Uncertainty in model form was explicitly incorporated into the risk estimates. The selection of dose‐response model strongly influenced both mean risk estimates and distributions of risk, and had a much greater impact than altering exposure distributions. We conclude that incorporating uncertainty in dose‐response model form is at least as important as accounting for variability and uncertainty in exposure parameters in probabilistic risk assessment.     

Metabolic and oxidative impairments in human salivary gland cells line exposed to MeHg

Background/aimThe ingestion of contaminated seafood by MeHg is considered the main route of human exposure, turning the salivary gland one important target organ. The salivary glands play critical roles in maintaining oral health homeostasis, producing saliva that maintains the oral microbiota, initiation of the digestion of macromolecules, and being essential in maintaining the integrity of the adjacent soft tissues and teeth. Thus, this study aimed to investigate the effects of MeHg exposure on human salivary gland cells line.MethodsCells were exposed to 1–6 μM of MeHg for 24 h, and analysis of toxicity was performed. Based on these results, the LC50 was calculated and two concentrations were chosen (0.25 and 2.5 μM MeHg) to evaluate intracellular mercury (Hg) accumulation (THg), metabolic viability and oxidative stress parameters (GSH:GSSG ratio, lipid peroxidation, protein oxidation and DNA damage).ResultsThe results demonstrated accumulation of THg as we increased the MeHg concentrations in the exposure and, the higher the dose, the lower is the cell metabolic response. In addition, the 2.5 μM MeHg concentration also triggered oxidative stress in human salivary gland cells by depleting the antioxidant competence of GSH:GSSG ratio and increasing lipid peroxidation and proteins carbonyl levels, but no damages to DNA integrity.ConclusionIn conclusion, although these two elected doses did not show lethal effects, the highest dose triggered oxidative stress and new questionings about long-term exposure models are raised to investigate furthers cellular damages to human salivary gland cells caused by MeHg exposure to extrapolate in a translational perspective.     

Comparison of dietary mercury exposure in two sympatric top predator fishes, largemouth bass and northern pike: a bioenergetics modeling approach

Physical and ecologicalfactors, including lake temperature, fishphysiology, and diet, influence methylmercury(MeHg) exposure in fish. We employedbioenergetics modeling to compare dietary MeHgexposure in sympatric top predators, largemouthbass (Micropterus salmoides) and northernpike (Esox lucius). We comparedsimulations using field data to hypotheticalsimulations with (1) ± 25% change in meandaily lake temperature for juvenile and adultbass and pike; (2) ± 25% change inlong-term growth rate of pike; (3) adult bassdiet shift from generalist predator to strictpiscivore. Bass and pike MeHg exposures weresimilar in baseline simulations and reflectedpatterns in field tissue concentrations. Thisoccurred despite the fact that bass consumedhighly contaminated benthic invertebrates,while pike exclusively consumed lesscontaminated fish prey. Higher temperaturesincreased adult bass and pike MeHg exposures by35% and 27%, respectively. Shifting adultbass diets to 100% fish resulted in a 54%decrease in exposure, while increasing pikegrowth rates resulted in a 24% decrease. Bioenergetics modeling proved useful inunderstanding the influence of temperature,prey-base, and predator growth on differencesin Hg exposure across fish species.     

Mercury and selenium interaction in vivo: effects on thioredoxin reductase and glutathione peroxidase

Mercury compounds exert toxic effects via interaction with many vital enzymes involved in antioxidant regulation, such as selenoenzymes thioredoxin reductase (TrxR) and glutathione peroxidase (GPx). Selenium supplementation can reactivate the mercury-inhibited TrxR and recover the cell viability in vitro. To gain an insight on how selenium supplementation affects mercury toxicity in vertebrates, we investigated the effects of selenium on the mercury accumulation and TrxR and GPx activities in a fish model. Juvenile zebra-seabreams were exposed either to methylmercury (MeHg) or inorganic mercury (Hg(2+)) in the presence or absence of sodium selenite (Se) for 28 days followed by 14 days of depuration. Mercury accumulation was found to be 10-fold higher under MeHg exposure than under Hg(2+) exposure. Selenium supplementation caused a half decrease of the accumulation of MeHg but did not influence Hg(2+) accumulation. Exposure to both mercurials led to a decrease of the activity of TrxR (<50% of control) in all organs. Se supplementation coincident with Hg(2+) exposure protected the thioredoxin system in fish liver. However, supplementation of Se during the depuration phase had no effects. The activity of GPx was only affected in the brain of fishes upon the exposure to MeHg and coexposure to MeHg and Se. Selenium supplementation has a limited capacity to prevent mercury effects in brain and kidney. These results demonstrate that Se supplementation plays a protective role in a tissue-specific manner and also highlight the importance of TrxR as a main target for mercurials in vivo.     

Effects of dietary methylmercury on the zebrafish brain: histological, mitochondrial, and gene transcription analyses

The neurotoxic compound methylmercury (MeHg) is a commonly encountered pollutant in the environment, and constitutes a hazard for wildlife and human health through fish consumption. To study the neurotoxic impact of MeHg on piscivorous fish, we contaminated the model fish species Danio rerio for 25 and 50 days with food containing 13.5 μg/g dry weight (dw) of MeHg (0.6 μg MeHg/fish/day), an environmentally relevant dose leading to brain mercury concentrations of 30 ± 4 μg of Hg g⁻¹ (dw) after 25 days of exposure and 46 ± 7 μg of Hg g⁻¹ (dw) after 50 days. Brain mitochondrial respiration was not modified by exposure to MeHg, contrary to what happens in skeletal muscles. A 6-fold increase in the expression of the sdh gene encoding the succinate dehydrogenase Fe/S protein subunit was detected in the contaminated brain after 50 days of exposure. An up regulation of 3 genes, atp2b3a, atp2b3b, and slc8a2b, encoding for calcium transporters was noticed after 25 days of exposure but the atp2b3a and atp2b3b were repressed and the slc8a2b gene expression returned to its basal level after 50 days, suggesting a perturbation of calcium homeostasis. After 50 days, we detected the up regulation of glial fibrillary acidic protein and glutathione S-transferase genes (gfap and gst), along with a repression of the glutathione peroxidase gene gpx1. These results match well with a MeHg-induced onset of oxidative stress and inflammation. A transmission electron microscopic observation confirmed an impairment of the optical tectum integrity, with a decrease of the nucleal area in contaminated granular cells compared to control cells, and a lower density of cells in the contaminated tissue. A potential functional significance of such changes observed in optical tectum when considering wild fish contaminated in their natural habitat might be an impaired vision and therefore a lowered adaptability to their environment.     

Methylmercury and brain development: A review of recent literature

Methylmercury (MeHg) is a potent environmental pollutant, which elicits significant toxicity in humans. The central nervous system (CNS) is the primary target of toxicity, and is particularly vulnerable during development. Maternal exposure to MeHg via consumption of fish and seafood can have irreversible effects on the neurobehavioral development of children, even in the absence of symptoms in the mother. It is well documented that developmental MeHg exposure may lead to neurological alterations, including cognitive and motor dysfunction. The neurotoxic effects of MeHg on the developing brain have been extensively studied. The mechanism of toxicity, however, is not fully understood. No single process can explain the multitude of effects observed in MeHg-induced neurotoxicity. This review summarizes the most current knowledge on the effects of MeHg during nervous system development considering both, in vitro and in vivo experimental models. Considerable attention was directed towards the role of glutamate and calcium dyshomeostasis, mitochondrial dysfunction, as well as the effects of MeHg on cytoskeletal components/regulators.     

Extraction of monoclonal antibodies (IgG1) using anionic and anionic/nonionic reverse micelles

Purification schemes for antibody production based on affinity chromatography are trying to keep pace with increases in cell culture expression levels and many current research initiatives are focused on finding alternatives to chromatography for the purification of Monoclonal antibodies (MAbs). In this article, we have investigated an alternative separation technique based on liquid–liquid extraction called the reverse micellar extraction. We extracted MAb (IgG1) using reverse micelles of an anionic surfactant, sodium bis 2‐ethyl‐hexyl sulfosuccinate (AOT) and a combination of anionic (AOT) and nonionic surfactants (Brij‐30, Tween‐85, Span‐85) using isooctane as the solvent system. The extraction efficiency of IgG1 was studied by varying parameters, such as pH of the aqueous phase, cation concentration, and type and surfactant concentration. Using the AOT/Isooctane reverse micellar system, we could achieve good overall extraction of IgG1 (between 80 and 90%), but only 30% of the bioactivity of IgG1 could be recovered at the end of the extraction by using its binding to affinity chromatography columns as a surrogate measure of activity. As anionic surfactants were suspected as being one of the reasons for the reduced activity, we decided to combine a nonionic surfactant with an anionic surfactant and then study its effect on the extraction efficiency and bioactivity. The best results were obtained using an AOT/Brij‐30/Isooctane reverse micellar system, which gave an overall extraction above 90 and 59% overall activity recovery. An AOT/Tween‐85/Isooctane reverse micellar system gave an overall extraction of between 75 and 80% and overall activity recovery of around 40–45%. The results showed that the activity recovery of IgG1 can be significantly enhanced using different surfactant combination systems, and if the recovery of IgG1 can be further enhanced, the technique shows considerable promise for the downstream purification of MAbs. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Experimental and Natural Warming Elevates Mercury Concentrations in Estuarine Fish

Marine food webs are the most important link between the global contaminant, methylmercury (MeHg), and human exposure through consumption of seafood. Warming temperatures may increase human exposure to MeHg, a potent neurotoxin, by increasing MeHg production as well as bioaccumulation and trophic transfer through marine food webs. Studies of the effects of temperature on MeHg bioaccumulation are rare and no study has specifically related temperature to MeHg fate by linking laboratory experiments with natural field manipulations in coastal ecosystems. We performed laboratory and field experiments on MeHg accumulation under varying temperature regimes using the killifish, Fundulus heteroclitus. Temperature treatments were established in salt pools on a coastal salt marsh using a natural temperature gradient where killifish fed on natural food sources. Temperatures were manipulated across a wider range in laboratory experiments with killifish exposed to MeHg enriched food. In both laboratory microcosms and field mesocosms, MeHg concentrations in killifish significantly increased at elevated temperatures. Moreover, in field experiments, other ancillary variables (salinity, MeHg in sediment, etc.) did not relate to MeHg bioaccumulation. Modeling of laboratory experimental results suggested increases in metabolic rate as a driving factor. The elevated temperatures we tested are consistent with predicted trends in climate warming, and indicate that in the absence of confounding factors, warmer sea surface temperatures could result in greater in bioaccumulation of MeHg in fish, and consequently, increased human exposure.     

Automated Speciation of Mercury in the Hair of Breastfed Infants Exposed to Ethylmercury from Thimerosal-Containing Vaccines

A simplified thiourea-based chromatography method, originally developed for methyl and inorganic mercury, was adapted to separate methylmercury (MeHg), ethylmercury (EtHg), and inorganic mercury (Hg^II) in infants' hair. Samples were weighed and leached with an acidic thiourea solution. Leachates were concentrated on a polymeric resin prior to analysis by Hg-thiourea liquid chromatography/cold vapor atomic fluorescence spectrometry. All but one sample showed small amounts of EtHg, and four of the six analyzed samples had proportionally higher Hg^II as a percent of total Hg. Breastfed infants from riverine Amazonian communities are exposed to mercury in breast milk (from high levels of maternal sources that include both fish consumption and dental amalgam) and to EtHg in vaccines (from thimerosal). The method proved sensitive enough to detect and quantify acute EtHg exposure after shots of thimerosal-containing vaccines. Based on work with MeHg and Hg^II, estimated detection limits for this method are 0.050, 0.10, and 0.10 ng g⁻¹ for MeHg, Hg^II, and EtHg, respectively, for a 20-mg sample. Specific limits depend on the amount of sample extracted and the amount of extract injected.     

Mercury in blood,hair, and feces from subsistence fish-eating riverines of the Madeira River Basin (Western Amazon)

BackgroundThe Madeira River (Amazon Basin) has been impacted by activities related to artisanal and small-scale gold mining (ASGM), deforestation and burning (for timber, agriculture, and hydroelectric dam projects). All these activities contribute to environmental mercury (Hg) release and cycling into the Amazon ecosystem and thus to changing lifestyles.MethodWe assessed exposure to total and MeHg in two small riverine communities of the Madeira River (Amazon): Lago Puruzinho (LP, n = 26 families) and São Sebastião do Tapurú (SST, n = 31 families). Samples of human hair (n = 137), blood (n = 39), and feces (n = 41) were collected from adults and children (0–15 years of age).ResultsIn women of childbearing age from LP village, the mean blood total-Hg (THg) (45.54 ± 24.76 μg.L⁻¹) and MeHg (10.79 ± 4.36 μg.L⁻¹) concentrations were significantly (p = 0.0024; p < 0.0001, respectively) higher than in women from SST village (THg: 25.32 ± 16.75 μg.L⁻¹; MeHg: 2.32 ± 1.56 μg.L⁻¹) village; the trend in hair-Hg persisted but was statistically significant (p < 0.0145) only for THg (LP, 11.34 ± 5.03 μg. g⁻¹; SST, 7.97 ± 3.51 μg. g⁻¹). In women, the median hair:blood ratio of total Hg was 269. In children, the mean hair THg concentrations were 6.07 ± 3.60 μg. g⁻¹ and 6.47 ± 4.16 μg. g⁻¹ in LP and SST; thus, not significantly different (p = 0.8006). There was a significant association (p < 0.001) between hair-Hg concentrations of mothers and their respective children. The excretion of Hg in feces of women (0.52 μg. g⁻¹ dw) was not significantly different from children (0.49 μg. g⁻¹ dw). The only statistically significant correlation between Hg in feces and in hair was found in children, (n = 16, r_s = 0.38, p = 0.005). Significant relationship was seen between the levels of THg in blood and hair of women from LP and SST. Based on hair-Hg concentrations, fish consumption rate ranged from 94.5 to 212.3 g.day⁻¹.ConclusionWomen and children excrete THg in feces in comparable concentrations. However, the mean fish consumption rate and blood MeHg are higher in the most remote villagers. Mother`s hair-Hg concentration is a good predictor of children’s hair-Hg.     

Photosynthetic parameters as early indicators of ozone injury in apple leaves

Ozone may affect leaf photosynthesis even before visible symptoms become apparent. This study had the objective to test several parameters of chlorophyll fluorescence and leaf gas exchange for their usefulness as indicators of latent ozone injury in the field. Container-grown apple trees (Malus domestica Borkh. cv. Golden Delicious) were exposed to four different ozone levels in open-top chambers. Identical leaves were analyzed in fixed-time intervals for the characteristics of fast fluorescence induction kinetics in vivo. By using high-time resolution, characteristic parameters describing the early photochemical events could be calculated according to the JIP-test. Parameters responsive to the different ozone treatments showed clear dependence on the accumulated ozone dose. Ozone exposure immediately preceding the measurements was more important for the extent of the physiological effects than the total accumulated ozone dose. The most sensitive parameters were the turnover number N (indicating how many times Q_A has to be reduced for full reduction of all acceptors; positively correlated to ozone dose) and D₀, the density of reaction centres per leaf area (negatively correlated to ozone dose). Most parameters analyzed showed clearer responses to ozone on the adaxial than on the abaxial surface of the leaf. Changes in the parameter N were better correlated to ozone doses with low cut-offs (AOT00 and AOT20), whereas changes in D₀ and in the specific electron fluxes per reaction centre were mainly influenced by ozone doses with high cut-offs (AOT80 and AOT100). Leaf gas exchange analyses revealed a higher ozone sensitivity in carboxylation efficiency than in light utilization efficiency and in the rate of light-saturated net photosynthesis. All ozone-induced photosynthetic effects were observed in leaves showing no sign of visible leaf injury. This study identified fluorescence parameters that could be useful for rapid monitoring and early detection of latent leaf injury by ozone.