4-n-Nonylphenol and 17-beta estradiol may induce common DNA effects in developing barnacle larvae

There is a growing concern over the potential effects of environmental endocrine disrupters on both human and wildlife populations. However, to date, minimal research has been conducted to determine the effect of estrogens and xenoestrogens at the DNA level. In this study, we used the random amplified polymorphic DNA (RAPD) assay to evaluate the effects on the genomic DNA of barnacle larvae that had been exposed to 17beta-estradiol (E2) and low concentrations of 4-n-nonylphenol (NP). DNA effects include DNA damage as well as mutations and possibly other effects at the DNA level that can be induced by chemical or physical agents that directly and/or indirectly interact with genomic DNA. Not only did exposure to NP and E2 induce changes in RAPD profiles in the exposed barnacle larvae when compared to control patterns, but also, and more importantly, there were similarities in the RAPD modifications in the exposed populations that had been treated to either chemical. We propose that NP and E2 induced some common DNA effects in barnacle larvae and that these specific modifications in RAPD patterns may arise as a consequence of hot spot DNA damage (e.g. DNA adducts) and/or mutations (point mutations or genomic rearrangements). This could help to explain how xenoestrogens mimic the effects produced by natural estrogens. In conclusion, in the field of endocrine disruption, the study of DNA effects induced by estrogens and/or xenoestrogens warrants further investigation. Indeed, changes at the DNA levcl may be the precursors of some of the numerous effects reported at higher levels of biological organisation such as the feminization of males, developmental abnormalities, and infertility.     

Evaluation of chlorophyll fluorescence, in vivo spectrophotometric pigment absorption and ion leakage as biomarkers of UV-B exposure in marine macroalgae

The photosynthetic fluorescence ratio F_v:F_m, in vivo absorption spectra and ion leakage were evaluated as biomarkers of ambient and elevated UV-B (280 to 320 nm) exposure of the intertidal alga Enteromorpha intestinalis (Chlorophyta) and the sublittoral alga Palmaria palmata (Rhodophyta). Measurements of thallus growth were also used to assess adverse biological effects. Ambient and elevated UV-B significantly inhibited photosynthesis in both species. It was shown that the F_v:F_m ratio is a sensitive, non-specific general biomarker of UV-B exposure in both species. Moreover, the in vivo absorption of what was tentatively identified as chlorophylls a and b as well as phycoerythrin and/or carotenoids, phycoerythrobilin and phycocyanin decreased in a dose-response dependent manner and was associated with a decrease in growth rate in P. palmata. The intertidal alga E. intestinalis showed a greater degree of tolerance to UV-B exposure. These results indicate that changes in the F_v:F_m ratio together with reductions in in vivo pigment absorption could provide an early quantitative warning of the detrimental effects of UV-B in marine macroalgae. Received: 16 May 1997 / Accepted: 16 July 1997     

Biomarker responses at different levels of biological organisation in crabs (Carcinus aestuarii) experimentally exposed to benzo(alpha)pyrene

The aim of this study was to validate a multi-trial biomarker approach for the evaluation of toxicological risk due to benzo(alpha)pyrene. Carcinus aestuarii, exposed to increasing concentrations of B(alpha)P in the water, was used as the bioindicator organism. A set of biomarkers were tested in order to: identify biological materials for biomarker and residue analysis; determine a group of sensitive techniques for the assessment of PAH contamination; investigate correlation between responses at different levels of biological organisation. The results underlined that BPMO activities in hepatopancreas and gills were a good biomarker of exposure to PAH-type compounds. B esterases activities in hemolymph and porphyrin patterns in excreta could be proposed as a non-destructive approach for evaluating chemical exposure in this species.     

Accumulation of organotin in Littorina littorea and Mya arenaria from Danish coastal waters

Organotin concentrations were determined in seawater, sediment and selected molluscs collected from the vicinity of the island of Fyn, Denmark. The extent of organotin contamination varied with the proximity of sampling sites to marinas and commercial shipping activities. Seasonal reductions in environmental TBT concentrations coincided with removal of pleasure craft from marinas during the winter season. At marina sites, concentration factors for TBT were 150-1500 for sediment and 500-10,000 for Littorina littorea (compared with seawater concentrations). Away from marina sites, Mya arenaria concentrated TBT by a factor of 57,000 to 220,000. The concentrations of TBT in M. arenaria from relatively clean sites were more than 10 times higher than found in L. littorea from heavily polluted marinas. M. arenaria were absent from marina sites and their immediate surroundings. Of the total organotin found in M. arenaria throughout the year, 80-90% was in the form of TBT. In contrast, in L. littorea approximately 60-70% of the total organotin was present as TBT in spring, but this proportion fell to 20-40% during autumn and winter. M. arenaria shows potential as a bioindicator organism of TBT pollution as it appears to have a very limited ability to metabolise and eliminate TBT, yet can tolerate quite high levels of TBT exposure. L. littorea might be used as a bioindicator of short-term fluctuations in TBT concentrations at heavily polluted sites. It is concluded that continued presence of TBT in the Danish coastal ecosystem may still pose an ecotoxicological threat.     

Evaluation of cadmium, copper, zinc, and iron concentrations and tissue distributions in the benthic crab, Dorippe granulata (De Haan, 1841) from Tolo Harbour, Hong Kong

The distributions of copper, zinc, iron, and cadmium among the tissues of Dorippe granulata were determined. The highest copper concentrations were found in the haemolymph (c. 53 microg ml(-1)) while the highest iron concentrations occurred in the gills (c. 720 microg g(-1) dry weight) and the highest zinc concentrations in the exoskeleton (c. 200 microg g(-1) dry weight). By comparison, concentrations of the non-essential metal, cadmium, were low in all tissues (mean = 10 microg g(-1) dry weight). The highest value was recorded from the midgut gland of a female crab (18.5 microg Cd g(-1) dry weight). Concentrations of copper, zinc, and iron were positively correlated with tissue-hydration levels. Such a relationship was not found for cadmium. The findings are discussed with regard to trace-metal levels found in temperate and tropical brachyurans from clean and polluted localities.     

Simultaneous, long-term monitoring of valve and cardiac activity in the blue mussel Mytilus edulis exposed to copper

Valve and cardiac activity were simultaneously measured in the blue mussel (Mytilus edulis) in response to 10?d copper exposure. Valve movements, heart rates and heart-rate variability were obtained non-invasively using a Musselmonitor^® (valve activity) and a modified version of the Computer-Aided Physiological Monitoring system (CAPMON; cardiac activity). After 2?d exposure of mussels (4 individuals per treatment group) to a range of dissolved copper concentrations (0 to 12.5?M as CuCl₂) median valve positions (% open) and median heart rates (beats per minute) declined as a function of copper concentration. Heart-rate variability (coefficient of variation for interpulse durations) rose in a concentration-dependent manner. The 48?h EC₅₀ values (concentrations of copper causing 50% change) for valve positions, heart rates and heart-rate variability were 2.1, 0.8, and 0.06?M, respectively. Valve activity was weakly correlated with both heart rate (r?=?0.48?±?0.02) and heart-rate variability (r?=?0.32?±?0.06) for control individuals (0?M Cu²⁺). This resulted from a number of short enclosure events that did not coincide with a change in cardiac activity. Exposure of mussels to increasing copper concentrations (≥0.8?M) progressively reduced the correlation between valve activity and heart rates (r?=?0 for individuals dosed with ≥6.3?M Cu²⁺), while correlations between valve activity and heart-rate variability were unaffected. The poor correlations resulted from periods of valve flapping that were not mimicked by similar fluctuations in heart rate or heart-rate variability. The data suggest that the copper-induced bradycardia observed in mussels is not a consequence of prolonged valve closure.     

Mechanisms of copper toxicity in the shore crab,Carcinus maenas

The influence of copper(II)chloride on selected key enzymes and metabolites in intermediary metabolism and the energy charge potential of the hyperregulating shore crab, Carcinus maenas (L.) were investigated. Crabs, collected in Odense Fjord, Denmark between September 1989 and May 1990, were exposed to 10 ppm copper(II)chloride, in 10 ppt salinity seawater at 15°C for up to 1 wk. Hexokinase activity was 77% lower in the posterior gills and 60% lower in the midgut gland than in control crabs. Phosphofructokinase activity was reduced in the midgut gland by 82% but was unaffected in the gills. Pyruvate kinase activity was undetectable in the gills of exposed crabs and reduced by 47% in the midgut gland. Citrate synthase activity was depressed by 30% in the posterior gills and unaffected in other tissues. Copper exposure had no apparent effect on tissue cytochrome c oxidase activity. Chela muscle was the only tissue in which copper exposure did not bring about reductions in the enzyme activities tested. As a result of 1 wk of copper exposure the average lactate level increased by 3.5-fold in haemolymph, 9.3-fold in posterior gills and 6.5-fold in midgut gland. The average glucose level was raised by a factor of 6 in the haemolymph, 3.4 in the posterior gills and 1.2 in the midgut gland. Energy charge potential was at a very high level (ca. 0.9) and was unaffected by copper exposure.     

Automated interpulse duration assessment (AIDA) in the shore crab Carcinus maenas in response to copper exposure

The effects of acute (24 h) and chronic (4␣weeks) copper exposure and subsequent recovery in “clean” conditions on cardiac activity were examined in shore crabs ( Carcinus maenas). Cardiac activity was monitored by automated interpulse duration assessment (AIDA) which permits detection of irregularities in cardiac rhythms in selected invertebrates. Mean interpulse durations, maximum interpulse durations and coefficients of variation were significantly affected by exposure to copper for 24 h and 4 weeks. Mean interpulse durations decreased reflecting an increase in heart rate, whereas maximum interpulse durations and coefficients of variation increased with increasing exposure concentration. Recovery of normal cardiac activity was evident following a 6-week recovery period in “clean” conditions. However, high levels of mortality were seen among crabs exposed to the two highest copper concentrations (0.6 and 0.8 mg l⁻¹ Cu). The extent of recovery was assessed by subjecting the surviving crabs to a physiological challenge. There were no apparent differences in circulatory response to physiological challenge between crabs previously exposed to copper and control crabs. Gill copper concentrations were significantly elevated in crabs that had previously been exposed to copper, despite their return to “clean” sea water for 6 weeks. Midgut gland wet weight/dry weight ratios, haemolymph total protein and midgut gland metallothionein concentrations did not reflect the previous copper exposure. The findings of the present study indicate that AIDA may be a valuable tool for use in toxicity testing. Received: 24 March 1997 / Accepted: 21 October 1997     

4-n-Nonylphenol and 17-β estradiol may induce common DNA effects in developing barnacle larvae

There is a growing concern over the potential effects of environmental endocrine disrupters on both human and wildlife populations. However, to date, minimal research has been conducted to determine the effect of estrogens and xenoestrogens at the DNA level. In this study, we used the random amplified polymorphic DNA (RAPD) assay to evaluate the effects on the genomic DNA of barnacle larvae that had been exposed to 17β-estradiol (E₂) and low concentrations of 4-n-nonylphenol (NP). DNA effects include DNA damage as well as mutations and possibly other effects at the DNA level that can be induced by chemical or physical agents that directly and/or indirectly interact with genomic DNA. Not only did exposure to NP and E₂ induce changes in RAPD profiles in the exposed barnacle larvae when compared to control patterns, but also, and more importantly, there were similarities in the RAPD modifications in the exposed populations that had been treated to either chemical. We propose that NP and E₂ induced some common DNA effects in barnacle larvae and that these specific modifications in RAPD patterns may arise as a consequence of hot spot DNA damage (e.g. DNA adducts) and/or mutations (point mutations or genomic rearrangements). This could help to explain how xenoestrogens mimic the effects produced by natural estrogens. In conclusion, in the field of endocrine disruption, the study of DNA effects induced by estrogens and/or xenoestrogens warrants further investigation. Indeed, changes at the DNA level may be the precursors of some of the numerous effects reported at higher levels of biological organisation such as the feminization of males, developmental abnormalities, and infertility.