Losing a winner: thermal stress and local pressures outweigh the positive effects of ocean acidification for tropical seagrasses.

Seagrasses are globally important coastal habitat-forming species, yet it is unknown how seagrasses respond to the combined pressures of ocean acidification and warming of sea surface temperature. We exposed three tropical species of seagrass (Cymodocea serrulata, Halodule uninervis, and Zostera muelleri) to increasing temperature (21, 25, 30, and 35°C) and pCO2 (401, 1014, and 1949 µatm) for 7 wk in mesocosms using a controlled factorial design. Shoot density and leaf extension rates were recorded, and plant productivity and respiration were measured at increasing light levels (photosynthesis-irradiance curves) using oxygen optodes. Shoot density, growth, photosynthetic rates, and plant-scale net productivity occurred at 25°C or 30°C under saturating light levels. High pCO2 enhanced maximum net productivity for Z. muelleri, but not in other species. Z. muelleri was the most thermally tolerant as it maintained positive net production to 35°C, yet for the other species there was a sharp decline in productivity, growth, and shoot density at 35°C, which was exacerbated by pCO2 . These results suggest that thermal stress will not be offset by ocean acidification during future extreme heat events and challenges the current hypothesis that tropical seagrass will be a 'winner' under future climate change conditions.

Simultaneous measurements of H+ and O2 fluxes in Zostera marina and its physiological implications.

Zostera marina (eelgrass) is an important ecological component of many shallow, temperate lagoons. Evidence suggests that Z. marina has a high bicarbonate utilization capability, which could be promoted by possible proton extrusion and the consequent formation of an 'acid zone' in the apoplastic space (unstirred layer) of its leaves. It has been found that 50 mM of the buffer Tris significantly inhibited the photosynthetic O(2) evolution of Z. marina and it was proposed that this was because of Tris's ability to bond with protons outside the cell wall. To investigate if H(+) played an important role in the photosynthetic carbon utilization of Z. marina, it is very important to simultaneously monitor the photosynthesis status and possible H(+) fluxes. However, probably because of the lack of suitable techniques, this has never been attempted. In this study, experiments were undertaken on Z. marina by monitoring H(+) and O(2) fluxes and the relative electron transport rates during light-dark transition. During stable photosynthesis, in addition to an obvious O(2) outflow, there was a significant net H(+) influx connected to Z. marina photosynthesis. The inhibitory effects of both Tris and respiration inhibitors on apparent O(2) evolution of Z. marina were confirmed. However, evidence did not support the proposed Tris inhibition mechanism.

Cadmium and/or copper excess induce interdependent metal accumulation, DNA methylation, induction of metal chelators and antioxidant defences in the seagrass Zostera marina.

In this investigation, we assessed the effects of Cu and/or Cd excess on physiological and metabolic processes of the widespread seagrass Zostera marina. Adult were exposed to low Cd and Cu (0.89 and 0.8 µM, respectively) and high Cd and Cu (8.9 and 2.4 µM, respectively) for 6 d at: Control conditions; low Cu; high Cu; low Cd; high Cd; low Cd and low Cu; and high Cd and high Cu. Photosynthetic performance decreased under single and combined treatments, although effects were more negative under Cu than Cd. Total Cu accumulation was higher than Cd, under single and combined treatments; however, their accumulation was generally lower when applied together, suggesting competition among them. Levels of glutathione (GSH) and phytochelatins (PCs) followed patterns similar to metal accumulation, with up to PC5, displaying adaptations in tolerance. A metallothionein (MET) gene showed upregulation only at high Cd, low Cu, and high Cu. The expression of the enzymes glutathione reductase (GR), ascorbate peroxidase (APX), and catalase (CAT) was greatest at high Cu, and at high Cd and Cu together; the highest expression was under Cu, alone and combined. Both metals induced upregulation of the DNA methyltransferases CMT3 and DRM2, with the highest expression at single Cu. The DNA demethylation ROS1 was overexpressed in treatments containing high Cu, suggesting epigenetic modifications. The results show that under copper and/or cadmium, Z. marina was still biologically viable; certainly based, at least in part, on the induction of metal chelators, antioxidant defences and methylation/demethylation pathways of gene regulation.

Transcriptome profiling analysis of the seagrass, Zostera muelleri under copper stress.

Copper (Cu) in an essential trace metal but it can also contaminate coastal waters at high concentrations mainly from agricultural run-off and mining activities which are detrimental to marine organisms including seagrasses. The molecular mechanisms driving Cu toxicity in seagrasses are not clearly understood yet. Here, we investigated the molecular responses of the Australian seagrass, Z. muelleri at the whole transcriptomic level after 7 days of exposure to 250 µg Cu L-1 and 500 µg Cu L-1. The leaf-specific whole transcriptome results showed a concentration-dependent disturbance in chloroplast function, regulatory stress responses and defense mechanisms. This study provided new insights into the responses of seagrasses to trace metal stress and reports possible candidate genes which can be considered as biomarkers to improve conservation and management of seagrass meadows.

Seed selection and storage with nano-silver and copper as potential antibacterial agents for the seagrass Zostera marina: implications for habitat restoration.

Globally, seagrass meadows are extremely important marine ecosystems that are disappearing at an alarming rate. Therefore, research into seagrass restoration has become increasingly important. Various strategies have been used in Zostera marina L. (eelgrass) restoration, including planting seeds. To improve the efficiency of restoration by planting seeds, it is necessary to select high-quality seeds. In addition, a suitable antibacterial agent is necessary for wet storage of desiccation sensitive seeds to reduce or inhibit microorganism infection and seed decay. In the present study, an efficient method for selecting for high-quality eelgrass seeds using different specific gravities of salt water was developed, and potential antibacterial agents (nano-silver and copper sulfate) for seed storage were assessed. The results showed that the highest proportion of intact seeds (72.91 ± 0.50%) was recorded at specific gravities greater than 1.20. Therefore, specific gravities greater than 1.20 can be used for selecting high-quality eelgrass seeds. During seed storage at 0 °C, the proportion of intact seeds after storage with nano-silver agent was over 90%, and also higher than 80% with copper sulfate agent, which was significantly higher than control treatments. The findings revealed a potential selection method for high-quality seeds and long-term seed storage conditions for Z. marina, which could facilitate conservation and habitat restoration.

Interaction of short-term copper pollution and ocean acidification in seagrass ecosystems: Toxicity, bioconcentration and dietary transfer.

We aimed to show how the predicted pH decrease in the ocean would alter the toxicity, bioconcentration and dietary transfer of trace metal copper on seagrass ecosystems, on a short-term basis. Seagrass Zostera noltei was exposed to two pH levels (8.36 and 8.03) and three copper levels (nominal concentrations, <3, 30 and 300 µg Cu L-1) in a factorial design during 21 days, while Gammarus locusta amphipods were continuously fed with the treated seagrass leaves. We found that the toxicity and bioconcentration of copper in seagrasses were not affected by pH, yet complex copper-pH interactions were observed in the seagrass photosynthesis. We demostrated that seagrasses can act as a copper source in the food web via direct consumption by herbivores. Future research need to investigate the interactive effects on a long-term basis, and to include biochemical and molecular endpoints to provide additional insights to the complex phisiological interactions observed.

Physiological responses of eelgrass (Zostera marina) to ambient stresses such as herbicide, insufficient light, and high water temperature.

This study aimed to elucidate the biological responses of eelgrass (Zostera marina) to artificially induced stresses such as herbicide (Irgarol 1051, Irg) exposure, insufficient light, and high water temperature (27 ± 1.0 °C) by evaluating growth inhibition, photosynthetic activity, and metabolomic profiles. After 14 days, all treatments inhibited growth, but photosynthetic activity was only reduced in the Irg-exposed group. In the Irg-exposed and insufficient light groups, the metabolomic profiles were characterized by decreased levels of sugar (sucrose) and increased levels of amino acids such as glutamine, glycine, and leucine. Biochemical and ultrastructural analyses revealed that the loss of sugar-derived metabolic energy was compensated for by energy generated during autophagic protein degradation. Furthermore, the level of myo-inositol, which has various biological roles and participates in several cellular processes such as cell wall synthesis, stress response, and mineral nutrient storage, was markedly increased in the Irg-exposed and insufficient light groups. A combination of metabolomic analysis with other analyses such as measurement of photosynthetic activity might further elucidate the response of eelgrass to ambient stresses in the natural environment.

Excess copper promotes photoinhibition and modulates the expression of antioxidant-related genes in Zostera muelleri.

Copper (Cu) is an essential micronutrient for plants and as such is vital to many metabolic processes. Nevertheless, when present at elevated concentrations, Cu can exert toxic effects on plants by disrupting protein functions and promoting oxidative stress. Due to their proximity to the urbanised estuaries, seagrasses are vulnerable to chemical contamination via industrial runoff, waste discharges and leachates. Zostera muelleri is a common seagrass species that forms habitats in the intertidal areas along the temperate coast of Australia. Previous studies have shown the detrimental effects of Cu exposure on photosynthetic efficiency of Z. muelleri. The present study focuses on the impacts of sublethal Cu exposure on the physiological and molecular responses. By means of a single addition, plants were exposed to 250 and 500 µg Cu L-1 (corresponding to 3.9 and 7.8 µM, respectively) as well as uncontaminated artificial seawater (control) for 7 days. Chlorophyll fluorescence parameters, measured as the effective quantum yield (ϕPSII), the maximum quantum yield (Fv/Fm) and non-photochemical quenching (NPQ) were assessed daily, while Cu accumulation in leaf tissue, total reactive oxygen species (ROS) and the expression of genes involved in antioxidant activities and trace metal binding were determined after 1, 3 and 7 days of exposure. Z. muelleri accumulated Cu in the leaf tissue in a concentration-dependent manner and the bioaccumulation was saturated by day 3. Cu exposure resulted in an acute suppression of ϕPSII and Fv/Fm. These two parameters also showed a concentration- and time-dependent decline. NPQ increased sharply during the first few days before subsequently decreasing towards the end of the experiment. Cu accumulation induced oxidative stress in Z. muelleri as an elevated level of ROS was detected on day 7. Lower Cu concentration promoted an up-regulation of genes encoding Cu/Zn-superoxide dismutase (Cu/Zn-sod), ascorbate peroxidase (apx), catalase (cat) and glutathione peroxidase (gpx), whereas no significant change was detected with higher Cu concentration. Exposure to Cu at any concentration failed to induce regulation in the expression level of genes encoding metallothionein type 2 (mt2), metallothionein type 3 (mt3) and cytochrome c oxidase copper chaperone (cox17). It is concluded that chlorophyll fluorescence parameters provide timely probe of the status of photosynthetic machinery under Cu stress. In addition, when exposed to a moderate level of Cu, Z. muelleri mitigates any induced oxidative stress by up-regulating transcripts coding for antioxidant enzymes.

Seagrass wasting disease: Nitrate enrichment and exposure to a herbicide (Diuron) increases susceptibility of Zostera marina to infection.

Seagrass meadows suffered large-scale declines in the past century. The 'wasting disease', pathognomonically associated with Labyrinthula zosterae, reduced populations of Zostera marina on both sides of the North Atlantic in, and since, the 1930s, coinciding with intensive agricultural use of artificial fertilizers and herbicides. This study tests the long-standing hypothesis that nutrient enrichment and a herbicide increases vulnerability to pathogens. Z. marina shoots from the Thames Estuary grown in elevated nitrate concentrations had significantly higher rates of infection by L. zosterae than controls, but not by Aplanochytrium sp., another slime-mould like protist. Z. marina shoots grown in 2µg·l-1 Diuron solutions and infected separately by L. zosterae and Aplanochytrium sp. had significantly higher wasting indices than controls. The results identified Aplanochytrium sp. as another opportunistic pathogen causing a seagrass wasting-type disease and support the hypothesis that pollution by herbicides and nitrate increases the susceptibility of Z. marina to infections.

Transcriptomic analysis reveals the molecular adaptation to NaCl stress in Zostera marina L.

The seagrass Zostera marina L. shows optimal growth in marine water and reduced growth under low salinity conditions. However, little is known about the molecular mechanisms underlying its adaptation to high salinity in Z. marina. In this study, transcriptomic analyses were performed using RNA-seq of the following two groups with different NaCl content: the CK group (seagrasses grown in the absence of NaCl) and the NaCl group (seagrasses grown in the presence of 400 mM NaCl for 6 h). Approximately 316 million high-quality reads were generated, and 87.9% of the data were mapped to the reference genome. Moreover, differentially expressed genes between the CK and NaCl groups were identified. According to a functional analysis, the up-regulated genes after the NaCl treatment were significantly enriched in nitrogen metabolism, calcium signalling and DNA replication while the down-regulated genes were significantly enriched in photosynthesis. A comparative transcriptomic analysis detected many differentially expressed genes and pathways required for adaptation to NaCl stress, providing a foundation for future studies investigating the molecular mechanisms of salt adaptation in Z. marina. We discuss how molecular changes in these processes may have contributed to the NaCl adaptation.

Seasonal dynamics of trace elements in sediment and seagrass tissues in the largest Zostera japonica habitat, the Yellow River Estuary, northern China.

Trace element accumulation is an anthropogenic threat to seagrass ecosystems, which in turn may affect the health of humans who depend on these ecosystems. Trace element accumulation in seagrass meadows may vary temporally due to, e.g., seasonal patterns in sediment discharge from upstream areas. In addition, when several trace elements are present in sufficiently high concentrations, the risk of seagrass loss due to the cumulative impact of these trace elements is increased. To assess the seasonal variation and cumulative risk of trace element contamination to seagrass meadows, trace element (As, Cd, Cr, Cu, Pb, Hg, Mn and Zn) levels in surface sediment and seagrass tissues were measured in the largest Chinese Zostera japonica habitat, located in the Yellow River Estuary, at three sites and three seasons (fall, spring and summer) in 2014-2015. In all three seasons, trace element accumulation in the sediment exceeded background levels for Cd and Hg. Cumulative risk to Z. japonica habitat in the Yellow River Estuary, from all trace elements together, was assessed as "moderate" in all three seasons examined. Bioaccumulation of trace elements by seagrass tissues was highly variable between seasons and between above-ground and below-ground biomass. The variation in trace element concentration of seagrass tissues was much higher than the variation in trace element concentration of the sediment. In addition, for trace elements which tended to accumulate more in above-ground biomass than below-ground biomass (Cd and Mn), the ratio of above-ground to below-ground trace element concentration peaked at times corresponding to high water discharge and high sediment loads in the Yellow River Estuary. Overall, our results suggest that trace element accumulation in the sediment may not vary between seasons, but bioaccumulation in seagrass tissues is highly variable and may respond directly to trace elements in the water column.

Impacts on the seagrass, Zostera nigricaulis, from the herbicide Fusilade Forte® used in the management of Spartina anglica infestations.

The herbicide Fusilade Forte® (FF) is widely applied in agricultural weed management and in the management of the invasive saltmarsh grass, Spartina anglica (ricegrass or cordgrass). FF (active ingredient fluazifop-P acid, FPA) is selective for poaceous grasses. Its primary mode of action is inhibition of the acetyl coenzyme-A carboxylase (ACCase) specific to this taxonomic group, and its secondary mode is by promotion of oxidative stress. FF is applied to S. anglica infestations in the intertidal zone, in proximity to seagrass meadows. Despite the potential for vital seagrass ecosystems to be exposed to FF, there is limited knowledge of any potential impacts. We investigated impacts of FPA on the endemic Australian seagrass, Zostera nigricaulis, measuring ACCase activity and parameters that reflect oxidative stress: photosynthetic performance, lipid peroxidation and photosynthetic pigment content. Seagrass was exposed to FF (0.01-10mgL-1 FPA and a control) for 7d, followed by a 7-d recovery in uncontaminated seawater. An enzyme assay demonstrated that FPA ≤10mgL-1 did not inhibit the activity of ACCase isolated from Z. nigricaulis, demonstrating that this seagrass is resistant to FF's primary mode of action. However, physiological impacts occurred following 7 days exposure to ≥0.1mgL-1 FPA, including up to a 72% reduction in photosynthetic pigment concentration. After 7-d recovery, photosynthetic pigment content improved in treatment plants; however, treated plants exhibited higher levels of lipid peroxidation. This study demonstrates that while Z. nigricaulis is resistant to FF's primary mode of action, significant physiological impacts occur following 7 days exposure to ≥0.1mgL-1 FPA. This study provides valuable information on the effects of FF on a non-target species that can better inform approaches to Spartina management in coastal seagrass ecosystems.

Can pesticides, copper and seasonal water temperature explain the seagrass Zostera noltei decline in the Arcachon bay?

Dwarf eelgrasses (Zostera noltei) populations have decreased since 2005 in Arcachon Bay (southwest France). Various stressors have been pointed out, however the role of xenobiotics like pesticides or copper (Cu) and of parameters like water temperature warming have not yet been explored. To determine their impact, Z. noltei individuals were collected in a pollution-free site and transferred to the laboratory in seawater microcosms. This dwarf eelgrass was exposed to a pesticide cocktail and copper, alone or simultaneously, at temperatures (10°C, 20°C, 28°C) representative of different seasons. After a two-week contamination, leaf growth, leaf bioaccumulation of Cu, and differential expression of target genes were studied. Eelgrasses bioaccumulated Cu regardless of the temperature, with reduced efficiency in the presence of the Cu and pesticide cocktail at the two higher temperatures. High temperature also exacerbated the effect of contaminants, leading to growth inhibition and differential gene expression. Mitochondrial activity was strongly impacted and higher mortality rates occurred. Experimental results have been confirmed during field survey. This is the first report on the impacts on Z. noltei of pesticides and Cu associate to temperature.

Metal concentrations in seagrass (Halophila ovalis) tissue and ambient sediment in a highly modified estuarine environment (Sydney estuary, Australia).

Research into sediment-seagrass tissue metal relationships has been undertaken in Sydney estuary due to the recognized role contamination plays in threats to seagrass health. Seagrass (Halophila ovalis) leaf tissue concentrations are elevated in Cu, Pb and Zn and contain the highest reported root Cr concentrations. Seagrass metal concentrations were significantly different between species H. ovalis and Zostera capricorni; between root and leaf tissue; and between sampling locations. Greatest tissue enrichment was for Pb, however metals were not enriched in seagrass relative to surficial sediment. Fine and total sediment metal concentrations were temporally consistent between collection years 2013/15, whereas root tissue metals changed between years and sites and leaf metal contents were temporally inconsistent. Extractable metal concentrations in fine sediment (<62.5 µm) showed moderate significant correlation with root tissue and a weak significant relationship with leaf tissue, whereas total sediment metal showed no such relationships. Management implications are provided.

Toxicity effects of an environmental realistic herbicide mixture on the seagrass Zostera noltei.

Worldwide seagrass declines have been observed due to multiple stressors. One of them is the mixture of pesticides used in intensive agriculture and boat antifouling paints in coastal areas. Effects of mixture toxicity are complex and poorly understood. However, consideration of mixture toxicity is more realistic and ecologically relevant for environmental risk assessment (ERA). The first aim of this study was to determine short-term effects of realistic herbicide mixture exposure on physiological endpoints of Zostera noltei. The second aim was to assess the environmental risks of this mixture, by comparing the results to previously published data. Z. noltei was exposed to a mixture of four herbicides: atrazine, diuron, irgarol and S-metolachlor, simulating the composition of typical cocktail of contaminants in the Arcachon bay (Atlantic coast, France). Three stress biomarkers were measured: enzymatic activity of glutathione reductase, effective quantum yield (EQY) and photosynthetic pigment composition after 6, 24 and 96 h. Short term exposure to realistic herbicide mixtures affected EQY, with almost 100% inhibition for the two highest concentrations, and photosynthetic pigments. Effect on pigment composition was detected after 6 h with a no observed effect concentration (NOEC) of 1 µg/L total mixture concentration. The lowest EQY effect concentration at 10% (EC10) (2 µg/L) and pigment composition NOEC with an assessment factor of 10 were above the maximal field concentrations along the French Atlantic coast, suggesting that there are no potential short term adverse effects of this particular mixture on Z. noltei. However, chronic effects on photosynthesis may lead to reduced energy reserves, which could thus lead to effects at whole plant and population level. Understanding the consequences of chemical mixtures could help to improve ERA and enhance management strategies to prevent further declines of seagrass meadows worldwide.

Trace elements in Mediterranean seagrasses: Accumulation, tolerance and biomonitoring. A review.

This study investigated the state of the art on trace elements in Mediterranean seagrasses, and their close environment (seawater and sediment). The analyzed species were Posidonia oceanica, Cymodocea nodosa, Halophila stipulacea, Zostera marina and Zostera noltei. All these species showed high tolerance to pollution and high capacity of accumulation of trace elements. Seagrasses also showed similar patterns of accumulation: the highest concentrations of As, Hg and Pb were found in the roots, whereas those of Cd, Cr, Cu, Mn, Ni and Zn were found in the leaves. Phytotoxic levels in seagrasses are unknown for most trace elements. The accumulation of trace elements in seagrasses is widely recognized as a risk to the whole food web, but the real magnitude of this risk is still uncertain. Seagrasses are known to act as trace element bioindicators, but this potential is still poorly valued for the creation of biomonitoring networks.

Lethal and sub-lethal chronic effects of the herbicide diuron on seagrass.

Photosystem II herbicides from agricultural sources have been detected throughout nearshore tropical habitats including seagrass meadows. While PSII herbicides have been shown to inhibit growth in microalgae at low concentrations, the potential impacts of chronic low concentration exposures to seagrass health and growth have not been investigated. Here we exposed two tropical seagrass species Halodule uninervis and Zostera muelleri to elevated diuron concentrations (from 0.3 to 7.2µgl(-1)) over a 79-day period followed by a 2-week recovery period in uncontaminated seawater. PAM fluorometry demonstrated rapid effect of diuron on photosystem II (PSII) in both seagrass species at 0.3µgl(-1). This effect included significant inhibition of photosynthetic efficiency (ΔF/Fm') and inactivation of PSII (Fv/Fm) over the 11 week exposure period. Significant mortality and reductions in growth was only observed at the highest exposure concentration of 7.2µgl(-1) diuron. However, biochemical indicators demonstrated that the health of seagrass after this prolonged exposure was significantly compromised at lower concentrations. For example, the drop in C:N ratios (0.6µgl(-1)) and reduced δ(13)C (1.7µgl(-1)) in seagrass leaves indicated reduced C-assimilation from photosynthesis. Critically, the energetic reserves of the plants (as measured by starch content in the root-rhizome complex) were approximately halved following diuron exposure at and above 1.7µgl(-1). During the 2-week recovery period, the photosynthetic capacity of the seagrass improved with only plants from the highest diuron treatment still exhibiting chronic damage to PSII. This study shows that, although seagrass may survive prolonged herbicide exposures, concentrations ≥0.6µgl(-1) diuron equivalents cause measureable impacts on energetic status that may leave the plants vulnerable to other simultaneous stressors. For example, tropical seagrasses have been heavily impacted by reduced light from coastal flood plumes and the effects on plant energetics from light limitation and diuron exposure (highest in flood plumes) are very similar, potentially leading to cumulative negative effects.

In situ impact of multiple pulses of metal and herbicide on the seagrass, Zostera capricorni.

Tides and freshwater inflow which influence water movement in estuarine areas govern the exposure-regime of pollutants. In this experiment, we examined the in situ impact of double pulses of copper and the herbicide Irgarol 1051 on the photosynthesis of the seagrass, Zostera capricorni. Despite a 4-day recovery period between the two 10h pulses of toxicant, the effective quantum yield of photosystem II (DeltaF/Fm') and total chlorophyll concentrations indicated that multiple-pulses had a greater impact than a single pulse. During the first exposure period, samples exposed to Irgarol 1051 had DeltaF/Fm' values as low as zero while controls remained around 0.6 relative units. After the second exposure period, treated samples recovered to only 0.4 relative units. Samples exposed to copper had DeltaF/Fm' values around 0.3 relative units during the first exposure period and while these samples recovered before the second dose, they remained below 0.2 relative units after the second exposure period. Alternate samples were also exposed to one toxicant, allowed to recover and then exposed to the other toxicant. DeltaF/Fm' values indicated that copper exposure followed by Irgarol 1051 exposure was more toxic than Irgarol 1051 exposure followed by copper exposure.

The interactive effects of the antifouling herbicides Irgarol 1051 and Diuron on the seagrass Zostera marina (L.).

The herbicides Irgarol 1051 (2-(tert-butylamino)-4-cyclopropylamino)-6-(methylthio)-1,3,5-triazine) and Diuron (3-(3',4'-dichlorophenyl)-1,1-dimethylurea) are commonly incorporated into antifouling paints to boost the efficacy of the compound towards algae. Previous investigations have identified environmental concentrations of these herbicides as being a threat to non-target organisms, such as seagrasses. Their individual toxicity has been assessed, but they can co-occur and interact, potentially increasing their toxicity and the threat posed to seagrass meadows. Chlorophyll fluorescence (Fv:Fm) and leaf specific biomass ratio (representing plant growth) were examined in Zostera marina L. after a 10-day exposure to the individual herbicides. The EC20 for each herbicide was determined and these then used in herbicide mixtures to assess their interactive effects. Irgarol 1051 was found to be more toxic than Diuron with lowest observable effect concentrations for Fv:Fm reduction of 0.5 and 1.0 +/- microg/l and 10-day EC50 values of 1.1 and 3.2 microg/l, respectively. Plants exposed to Irgarol 1051 and Diuron showed a significant reduction in growth at concentrations of 1.0 and 5.0 microg/l, respectively. When Z. marina was exposed to mixtures, the herbicides commonly interacted additively or antagonistically, and no significant further reduction in photosynthetic efficiency was found at any concentration when compared to plants exposed to the individual herbicides. However, on addition of the Diuron EC20 to varying Irgarol 1051 concentrations and the Irgarol 1051 EC20 to varying Diuron concentrations, significant reductions in Fv:Fm were noted at an earlier stage. The growth of plants exposed to Diuron plus the Irgarol 1051 EC20 were significantly reduced when compared to plants exposed to Diuron alone, but only at the lower concentrations. Growth of plants exposed to Irgarol 1051 and the Diuron EC20 showed no significant reduction when compared to the growth of plants exposed to Irgarol 1051 alone. Despite the addition of the EC20 not eliciting a further significant reduction when compared to the herbicides acting alone for most of the mixtures, the lowest observable significant effect concentration for growth and photosynthetic efficiency decreased to 0.5 microg/l for both herbicides. Irgarol 1051 and Diuron have been shown to occur together in concentrations above 0.5 microg/l, suggesting that seagrasses may be experiencing reduced photosynthetic efficiency and growth as a result.

Towards a more ecologically relevant assessment of the impact of heavy metals on the photosynthesis of the seagrass, Zostera capricorni.

This in situ study used photosynthetic activity (measured as chlorophyll a fluorescence) and photosynthetic pigment concentrations to assess the effect of copper, cadmium, lead and zinc on the seagrass Zostera capricorni. Custom-made portable in situ exposure (PIE) chambers were developed so seagrasses could be dosed within the meadow. Z capricorni was exposed to 0.1 and I mg l(-1) of metal solutions for 10 h. During this time and for the subsequent four-day recovery period, the effective quantum yield of photosystem II (PS II) (deltaF/Fm') was measured. While the results were variable, copper and zinc exposed samples had a depressed deltaF/Fm' during the exposure period. Samples exposed to zinc recovered to pre-exposure levels but those exposed to copper did not. Cadmium and lead did not impact on the chlorophyll a fluorescence and the chlorophyll pigment data supported these findings. This study presents an innovative new application of chlorophyll a fluorescence stress assessment.