24(R, S)-Saringosterol - From artefact to a biological medical agent.

Enhancing the cholesterol turnover in the brain via activation of liver x receptors can restore memory in a mouse model for Alzheimer's disease. The edible Asian brown alga Sargassum fusiforme (Hijiki) contains high amounts of oxysterols such as (3ß, 24ξ)-stigmasta-5, 28-dien-3, 24-diol (24[R, S]-saringosterol) that are a potent liver x receptor agonists. We aimed to find native European seaweed species with contents of 24(R, S)-saringosterol that are comparable to those found in Sargassum fusiforme. Additionally, we hypothesize that seasonal variations modify the amount of 24(R, S)-saringosterol in seaweeds. Sterols and oxysterols were extracted with chloroform/methanol from various seaweed species harvested in the Eastern Scheldt in different seasons between October 2016 and September 2017. Identification and quantification of the lipids was performed by gas chromatography- mass spectrometry and gas chromatography- flame ionization detection. We confirmed that brown algae Undaria pinnatifida harvested in February and Sargassum muticum harvested in October contained the highest amounts of 24(R, S)-saringosterol (32.4 ± 15.25 µg/g, mean ± S.D. and 32.95 ± 2.91 µg/g, respectively) and its precursor fucosterol (1.48 ± 0.11 mg/g), higher than Sargassum fusiforme (20.94 ± 3.00 µg/g, mean ± S.D.), while Ascophyllum nodosum and Fucus vesiculosus and Fucus serratus contained amounts of 24(R, S)-saringosterol (22.09 ± 3.45 µg/g, 18.04 ± 0.52 µg/g and 19.47 ± 9.01 µg/g, mean ± S.D., respectively) comparable to Sargassum fusiforme. In other algae only minor amounts of these sterols were observed. The green algae Ulva lactuca contained only 0.29 mg/g fucosterol and 10.3 µg/g 24 (R, S)-saringosterol, while all investigated red algae did not contain any 24(R, S)-saringosterol or fucosterol. In the Eastern Scheldt algae harvested in September/October delivered the highest yield for 24(R, S)-saringosterol, with the exception of Undaria pinnatifida that showed the highest levels in February. We showed that exposure of lipid extracts of Ulva lactuca to sunlight at room temperature or in the presence of oxygen to UV-C light lead to the quantitative conversion of fucosterol into 24(R, S)-saringosterol. Exposing pure fucosterol to UV-light did not convert any fucosterol into 24(R, S)-saringosterol underscoring the requirement of seaweed constituents in the conversion of fucosterol into 24(R, S)-saringosterol. In conclusion, we showed that brown seaweeds harvested from the Eastern Scheldt contain amounts of 24(R, S)-saringosterol comparable to Sargassum fusiforme, varying per season and showing the highest amounts in spring. In accordance with these observations the amount of 24(R, S)-saringosterol in the brown seaweeds can be modulated by light.

Early life developmental effects of marine persistent organic pollutants on the sea urchin Psammechinus miliaris.

A new 16-day echinoid early life stage (ELS) bioassay was developed to allow for prolonged observation of possible adverse effects during embryogenesis and larval development of the sea urchin Psammechinus miliaris. Subsequently, the newly developed bioassay was applied to study the effects of key marine persistent organic pollutants (POPs). Mortality, morphological abnormalities and larval development stages were quantified at specific time points during the 16-day experimental period. In contrast to amphibians and fish, P. miliaris early life development was not sensitive to dioxin-like toxicity in the prolonged early life stage test. Triclosan (TCS) levels higher than 500 nM were acutely toxic during embryo development. Morphological abnormalities were induced at concentrations higher than 50 nM hexabromocyclododecane (HBCD) and 1000 nM tetrabromobisphenol A (TBBPA). Larval development was delayed above 25 nM HBCD and 500 nM TBBPA. Heptadecafluorooctane sulfonic acid (PFOS) exposure slightly accelerated larval development at 9 days post-fertilization (dpf). However, the accelerated development was no longer observed at the end of the test period (16 dpf). The newly developed 16-day echinoid ELS bioassay proved to be sensitive to toxic effects of POPs that can be monitored for individual echinoid larvae. The most sensitive and dose related endpoint was the number of developmental penalty points. By manipulation of the housing conditions, the reproductive season could be extended from 3 to 9 months per year and the ELS experiments could be performed in artificial sea water as well.

Toxicity of analytically cleaned pentabromodiphenylether after prolonged exposure in estuarine European flounder (Platichthys flesus), and partial life-cycle exposure in fresh water zebrafish (Danio rerio).

Residues of polybrominated diphenylethers (PBDEs), extensively applied as flame retardants, are widely spread in the aquatic environment and biota. The present study investigates effects of the environmentally relevant lower brominated diphenylethers in two fish species in vivo under controlled laboratory conditions. Euryhaline flounder (Platichthys flesus) and freshwater zebrafish (Danio rerio) were exposed to a range of concentrations of a commercial pentabromodiphenylether mixture, DE-71. Chemical analysis of exposed fish showed a pattern of PBDE congeners that was very similar to that in wild fish. The resulting range included environmentally relevant, as well as higher levels. Animals were investigated histopathologically with emphasis on endocrine and reproductive organs. In zebrafish, hatching of embryos and larval development were assessed. Biochemical parameters were investigated in flounder as markers for suggested dioxin-like activity (ethoxyresorufin-O-deethylase=EROD), and activation of endogenous estrogen synthesis (gonad aromatase activity). Thyroid hormones were analyzed in plasma in both species. Benchmark analysis using internal PBDE concentrations showed a mild dose-dependent decrease of hepatic EROD and ovarian aromatase activities, and plasma thyroxin levels in flounder, and an increase of plasma thyroid hormone levels in zebrafish. These trends did not result in statistically significant differences from control fish, and major histopathological changes were not observed. Reproduction in zebrafish appeared to be the most sensitive parameter with statistically significantly reduced larval survival and non-significant indications for decreased egg production at internal levels that were more than 55 times the highest environmental recordings. The present results indicate limited risk for endocrine or reproductive effects of current environmental PBDE contamination in fish.

A chronic bioassay with the estuarine amphipod Corophium volutator: test method description and confounding factors.

Methods of conducting a chronic sediment toxicity test with the estuarine amphipod Corophium volutator are described. They consist of a 49-day exposure, after which mortality, growth and reproduction are determined. Pilot experiments were used to optimize test design parameters such as temperature, duration, feeding and refreshing regimes, and effects of indigenous organisms. By way of further validation, the present study focused on the effects of four different parameters: oxygen saturation, salinity, ammonium and nitrite. These confounding factors might play an important role especially if the test is used for risk assessment of field-contaminated sediments. It is concluded that the present experimental design is well suited for chronic sediment exposures with C. volutator. The test can be performed at a broad range of salinity values, provided that controls are performed at the same salinity. Results further demonstrate that with the endpoints growth and reproduction this chronic test procedure is a factor 7-18 more sensitive to ammonium and nitrate than the standardized acute bioassay (endpoint mortality).

Ammonium toxicity at high pH in a marine bioassay using Corophium volutator.

Two forms of ammonium exist in water: un-ionized ammonia NH3 and ionized ammonium NH4+. The toxicity to many aquatic organisms is primarily attributed to the NH3 (un-ionized) species, with the NH4+ ion (ionized) species being relatively less toxic. The pH level influences the degree of ionization. It is therefore very important that quality criteria be derived for total ammonium levels at several pH values in order to allow correct interpretation of the sediment bioassay with Corophium volutator. The responses of Corophium to total ammonium were studied in a series of pH-controlled experiments. The LC50 of total ammonium showed a significant decrease with increasing pH, in both water-only and sediment experiments. The results indicated a combined NH4+ and NH3 toxicity at pH levels less than 8.3. The results can be used to set pH-dependent water quality criteria for total ammonium in overlying water in a 10-day sediment bioassay with Corophium volutator.

Using marine bioassays to classify the toxicity of Dutch harbor sediments.

A procedure was developed to assess contaminated marine sediments from Dutch harbors for possible adverse biological effects using three laboratory bioassays: A 10-d survival test with the amphipod Corophium volutator, a 14-d survival test with the heart urchin Echinocardium cordatum (adults), and the bioluminescence inhibition test with the bacterium Vibrio fischeri (Microtox solid phase test LSP]). Microtox results were mathematically corrected for the modifying influence of fine sediment particles. After a validation procedure on test performance and modifying factors, respectively, 81%, 99%, and 90% of the amphipod, heart urchin, and Microtox results were approved. Lower and upper threshold limits for biological effects were set at respectively 24 and 30% mortality for C. volutator, 27 and 35% mortality for E. cordatum, and 24 and 48 toxic units for the Microtox SP based on significant differences with control sediment and the performance of reference sediments. The bioassays clearly distinguished harbor sediments that give rise to acute effects and those that do not. Threshold limits for the amphipods, heart urchins, and bacteria were exceeded in, respectively, 9 to 17%, 33 to 40%, and 23 to 50% of the sediment samples. Highest effects were observed in sediments from the northerly harbors; there was significantly less response in sediments from the Delta Region and the port of Rotterdam (The Netherlands). The procedure outlined in this paper can be used for routine screening of contaminated dredged material that is proposed for open water disposal.

A toxicity identification evaluation of silty marine harbor sediments to characterize persistent and non-persistent constituents.

Sediment toxicity in silty marine harbor sediments is frequently dominated by ammonia or sulfide, leaving the adverse effects of persistent toxic substances unnoticed. To investigate the latter, we subjected interstitial water from three contaminated silty sediments to toxicity identification evaluation (TIE) phase I manipulations and tested for toxicity with four bioassays: the amphipod Corophium volutator (survival as an endpoint), the sea urchin Psammechinus miliaris (fertilization, embryo development) and the bacterium Vibrio fischeri (bioluminescence inhibition). The graduated pH manipulations identified the prominent toxicity of ammonia in the amphipod and sea urchin embryo tests, and also sulfide toxicity in the bacterium test. In two of the three samples tested with the amphipods, sea urchin embryos and bacteria, a small but significant reduction in interstitial water toxicity was achieved by removing persistent compounds through C(18) solid phase extraction. EDTA chelation resulted in a slight detoxification of the interstitial water for the amphipods and sea urchin embryos, but this was not related to any measured trace metals. Despite the presence of toxic levels of ammonia and sulfide in the harbor sediments, we established the adverse biological effects of persistent constituents by means of the TIE manipulations and in vivo interstitial water bioassays.