Interaction of toxic trace metals and mechanisms of detoxification in the planktonic diatoms Ditylum brightwellii and Thalassiosira pseudonana

Abstract: Effects of cadmium (10 nM), copper (80 nM) and zinc (150 nM) additions were studied in the marine diatom Ditylum brightwellii and the riverine diatom Thalassiosira pseudonana . Defense against oxidative stress via cellular thiol (SH) pools and superoxide dismutase (SOD) activation, detoxification via phytochelatins and cell damage were monitored in metal-exposed exponential-phase cells and controls, grown in estuarine medium. Total SH and reduced + oxidized glutathione (GSH + GSSG) in T. pseudonana were much higher than in D. brightwellii . In T. pseudonana , total SH and GSH decreased at 322 nM Zn, and GSH increased at 80 nM Cu but decreased at 119 nM Cu. GSH:GSSG ratios were low, while phytochelatins were not detectable in metal-exposed D. brightwellii . Cd-exposed T. pseudonana made more phytochelatins than Cu-exposed cells, and in different proportions. At 322 nM Zn, SOD activity decreased in T. pseudonana . Zn caused a major, and Cu a minor increase of SOD activity in D. brightwellii ; inhibition of photosynthesis was observed in Cu-exposed D. brightwellii , probably due to oxidative damage. The C:N ratios were higher and protein contents lower in Cu-exposed cells of both species, which might indicate excretion due to a loss of cell membrane integrity. From these results, it is hypothesized that T. pseudonana has evolved an effective detoxification mechanism as a result of a more severe exposure to toxic metals in rivers and estuaries. In contrast, D. brightwellii , a marine-estuarine species, cannot adjust well to metal exposure. Its poor defense against metal toxicity was marked by low SH-contents.     

Trace metal interactions in the macroalga Enteromorpha prolifera (O. F. Müller) J.Ag., grown in water of the Scheldt estuary (Belgium & SW Netherlands), in response to cadmium exposure

The littoral macroalga Enteromorpha prolifera was sampled along the Scheldt estuary at Lillo (Belgium), Bath and Ellewoutsdijk (SW Netherlands). The algae were incubated in water from the same locations with salinities of 7, 14 and 23%, at about 50 molquanta m^–2 s^–1 irradiance and 15 °C. One series of cultures was exposed to 400 nm Cd, a second series served as a control. Temporal changes in cadmium, copper, iron, lead and zinc contents of E. prolifera were monitored with atomic absorption spectroscopy in a 21 day experiment. Both synergistic and antagonistic metal interactions became evident in response to cadmium uptake. During the first 8 days of incubation iron contents increased in both cadmium-exposed and control algae. However, iron contents were relatively higher in cadmium-exposed algae. Similar, but weaker synergisms were also found between lead/cadmium and coper/cadmium. Zinc contents decreased in controls and cadmium-exposed algae, but were significantly lower in cadmium-exposed algae. A distinct antagonism between cadmium and zinc was observed in cadmium-exposed algae.     

Phytoplankton composition and spatial distribution of copper and zinc in the Fal Estuary (Cornwall,UK)

In the estuaries near Falmouth (Cornwall, UK) levels of dissolved copper and zinc are high, due to drainage of copper and tin mines. Phytoplankton species composition in the autumn of 1989 deviated in the metal-contaminated Restronguet Creek from that in other estuarine branches,viz. Fal, Tresillian and Percuil. In the riverine part of Restronguet Creek (Carnon River)Euglena mutabilis, known as an acidophilic (pH 3) metal-resistant flagellate, occurred at micromolar Cu and Zn, whereas in the clean riversChlamydomonas sp. andOocystis sp. occurred at nanomolar Cu and Zn. An ordination analysis revealed the following patterns in Cu, Zn and phytoplankton species composition in the poly- and euhaline waters. Seston-bound Zn and dissolved Zn were in equilibrium,Katodinium rotundatum is Zn-tolerant, andSkeletonema costatum occurred in water with high contents of Cu in seston. However, none of the variables in these patterns correlated at a significant level (p>0.05). The results show that algae-metal interactions are complicated, and that statistical correlations foundin situ need experimental verification.Communication no. 542 of the Delta Institute for Hydrobiological Research.     

Cellular responses to elevated light levels in Fucus spiralis embryos during the first days after fertilization

Cellular responses of 1‐, 2‐ and 4‐d‐old Fucus spiralis embryos subjected to a single dose of elevated photosynthetically active photon flux density (PPFD), with or without ultraviolet (UV) radiation, were investigated by measuring the effects on the effective quantum yield of photosystem II (ΔF / F_m′) and intracellular production of active oxygen species (AOS). Production of AOS was determined by the in vivo conversion of 5‐(and‐6)‐chloromethyl‐2′,7′‐dichlorodihydrofluorescein diacetate (CM‐DCFH₂‐DA) to the fluorescent compound dichlorofluorescein (DCF) using confocal laser scan microscopy (CLSM) and image analysis. The role of xanthophyll cycle pigments in photoprotection was also assessed. A rapid decline in ΔF / F_m′ was observed under all elevated light conditions. A correlation was found between non‐photochemical quenching and the de‐epoxidation ratio zeaxanthin/(zeaxanthin + violoxanthin). Active oxygen formation increased with PPFD and was higher in older embryos and when UVB was present. Two photoinhibition responses were recognized: (i) a rapid decline of the PSII yield due to the violoxanthin–zeaxanthin cycle (photoprotection), and (ii) a slower second‐phase decline, correlated with active oxygen production. Electron transport rate (ETR) increased with embryo age, and was correlated with AOS production. As a result of enhanced AOS production, there was a slow recovery of the PSII yield, in particular with increased effective UV dose. In general, embryos were able to recover from the imposed light conditions, but UVB had a more damaging effect. Overall, our data suggest that under natural conditions, embryos of F. spiralis are susceptible to elevated light levels, and that UVB radiation is an important stress factor.     

Assessment of oxidative stress in the planktonic diatom Thalassiosira pseudonana in response to UVA and UVB radiation

Induction of oxidative stress by UVA and UVB in the diatom Thalassiosirapseudonana was experimentally studied. Cells, pre-grown in alight-limited continuous culture, were incubated for 4 h at175 µmol m^-2s^-1photosynthetically active radiation, withoptional supplementary UVA at an unweighted dose rate of 0.70W m^-2, or 2.79 W m^-2UVA plus 0.45 W m^-2UVB (unweighted). A fluorescence-basedmeasure of photosynthetic efficiency (F_v/F_m) decreased from0.69 to 0.58 in the presence of UVB, whereas UVA caused a minordecrease of F_v/F_m. Quantitative analysis of confocal imagesshowed a minor increase of active oxygen production associatedwith supplemental UVA alone, and a 100% increase with additionalUVB. Cellular malondialdehyde, an indicator of lipid peroxidationby active oxygen, almost doubled under UVA and increased three-foldwith additional UVB. Activities of superoxide dismutase (scavengingactive oxygen) and glutathione reductase (reducing GSSG to GSH)increased in response to UVB exposure, whereas ascorbate peroxidaseactivities did not. UVB caused a minor decrease in the glutathioneratio GSH : (GSH + 0.5GSSG), which indicates a moderate oxidativestress.     

Impacts of anthropogenic stresses on the early development stages of seaweeds

Seaweeds are important primary producers, and as such contributesignificantly to nearshore ecosystems. Studies on the effects ofanthropogenic stresses on these organisms have largely been concernedwith the vegetative adult stages of the life cycle. Here we review thelimited information on the sensitivity of early stages in the life cycleof seaweeds to global change (UV increase; global warming;increased storm frequencies) and pollution (eutrophication, trace metalsand oil). Impacts on fertility, substrate attachment, development,photosynthesis, growth and mortality are highlighted. In their naturalhabitats, early stages are shade-adapted, as they live shel tered underadult canopies and in pores of the substrata. Although some acclimationunder increased moderate irradiance is seen, higher solar irradiance,and especially ultraviolet-B, inhibits early development. Global warmingmay decrease the fertility and shorten the fertile period of somespecies. With the increasing likelihood of storms associated with globalwarming, gamete release may be inhibited while scouring by suspendedsediments may detach newly settled stages. Succession and localdistribution patterns are likely to be affected. Eutrophication canresult in accelerated development of the early stages of some algalspecies but sewage discharges have a negative impact on sperm motility,fertilisation and can cause increased mortality in germlings. Impacts ofother, indirect effects of eutrophication, including increased sedimentcover of substrata, scouring caused by wind-induced resuspension ofsediments, and grazing, are also expected to be negative. Toxic tracemetals affect gamete viability, inhibit fertilisation and development,and reduce growth rates. Gametes are particularly susceptible to oilpollution and interactions between hydrocarbons and the adhesive mucussurrounding the embryonic stages seem to inhibit settlement.Recommendations for future studies are provided that are aimed atgaining greater insight into the effects of anthropogenic stress on theweakest links in the development cycle of seaweeds.     

OXIDATIVE STRESS RESPONSES IN THE MARINE ANTARCTIC DIATOM CHAETOCEROS BREVIS (BACILLARIOPHYCEAE) DURING PHOTOACCLIMATION1

The enzyme superoxide dismutase (SOD) holds a key position in the microalgal antioxidant network. The present research focused on oxidative stress responses in the Antarctic diatom Chaetoceros brevis F. Schütt during transition to excess (including ultraviolet radiation [UVR]) and limiting irradiance conditions. Over a 4 d period, cellular responses of thiobarbituric acid reactive substances (TBARS, a general oxidative stress indicator), SOD activity, photosynthetic and xanthophyll cycle pigments, PSII efficiency, and growth were determined. In addition, oxidative responses were measured during a daily cycle. Changing irradiance conditions significantly affected growth rates of C. brevis. PSII efficiency decreased significantly during periodic excess irradiance and increased under low irradiance conditions. Transition to excess irradiance increased the ratio of xanthophyll to light‐harvesting pigments, whereas the opposite was observed for cultures transferred to low irradiance. This acclimation process was completed after 2 d in the new irradiance environment. SOD activity increased significantly after the first day regardless of the new irradiance environment but returned to preexposure values on the fourth day. We hypothesize that SOD activity may be temporarily elevated in C. brevis after irradiance shifts, thereby reducing oxidative stress when photoacclimation is in progress.     

Spatial and temporal variation in community composition and photosynthetic characteristics of phytoplankton in the upper Westerschelde estuary (Belgium,SW Netherlands)

Community composition, biomass and primary production of phytoplankton were studied in the east- ernmost section of the Westerschelde estuary in 1984. Photosynthetic characteristics were compared with distribution of some dominant phytoplankton species along a salinity gradient from 5 to 22 Spring phytoplankton, with Cyclotella meneghiniana (freshwater) and Skeletonema costatum (marine) as the dominant species grew faster than summer phytoplankton. In summer, biomass achieved its maximum, due to the riverine Scenedesmus species and the marine diatoms Thalassiosira levanderi and Ditylum brightwellii, as dominants. Growth conditions were more favourable to phytoplankton communities above 15%, than below this salinity. The data were compared with previous studies (1972) of species composition in the area.     

Saline seepage and vertical distribution of oxygen in a brackish ditch

Summary Seepage caused salinity gradients in a 120 cm deep ditch adjacent to Lake Veere in the Dutch Delta area. The water was mixed by strong horizontal currents, forced by a pumping engine. The restoration of vertical chloride and oxygen gradients was studied.During summer when the level of Lake Veere was raised by 70 cm, a rapid seepage into the ditch was observed, ranging from 6 to 10 mm.h^–1. This caused an anoxic saline layer. At the anaerobic-aerobic interface 1.4 to 2.4 mg O₂.l^–1.h^–1 of the oxygen present in the upper water column was taken up. Sulfide oxidation required one quarter of this amount of oxygen.During winter the level of Lake Veere was lowered again. Normally seepage was absent then, and chloride entered the water column by diffusion; the mud chlorinity decreased from 15 to 3 Cl^– on its way to the ditch. Only after maximum discharge of polder water a moderate seepage of less than 4.4 mm.h^–1 restored a chloride gradient; about 1.6 mg O₂.l^–1.h^–1, produced by benthic diatoms, had to be supplied to the reducing materials in the seepage water in less than 12 hours.A saline seepage was simulated in the laboratory. These simulation tests showed that seepage below 1 mm.h^–1, as well as benthic respiration only, had a small impact on the vertical oxygen profile in the water column. Bottom oxygen demand increased with seepage velocity. At a strong seepage the water column could become entirely anaerobic.Communication no. 226.     

Short-term antioxidative responses of 15 microalgae exposed to excessive irradiance including ultraviolet radiation

Short-term photosensitivity and oxidative stress responses were compared for three groups of marine microalgae: Antarctic microalgae, temperate diatoms and temperate flagellates. In total, 15 low-light-acclimated species were exposed to simulated surface irradiance including ultraviolet radiation (SSI). Photosensitivity was assessed as the rate of recovery of Fv/Fm in the hours following SSI treatment. Before, during and after the SSI treatment, oxidative stress responses were assessed by following xanthophyll content and cycling, and activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase, and glutathione redox status. When acclimated to low irradiance, antioxidant levels were not group specific. Superoxide dismutase activity was positively correlated with cell size, whereas in general, ascorbate peroxidase activity appeared to be lower and glutathione redox status appeared to be higher in the Antarctic than in the temperate species. After SSI exposure, the strong inhibition of PSII was followed by variable rates of recovery, although four species remained photosynthetically inactive. SSI tolerance appeared unrelated to geographic or taxonomic background, or to cell size. PSII recovery was enhanced in species with decreasing superoxide dismutase activity, glutathione redox status and increased xanthophyll cycle activity. We conclude that antioxidant responses are highly species specific and not related to the geographic or taxonomic background. Furthermore, xanthophyll cycling seems more important than antioxidants. Finally, it can be hypothesized that glutathione could function as a stress sensor and response regulator.