Effect of size on concentrations and cadmium inducibility of metallothionein in the shore crab Carcinus maenas.

Metallothionein (MT) plays an important role in protecting organisms from the adverse effects of Cd, Hg, Zn and Cu. Investigations on mammals show variations in metallothionein concentrations and inducibility with age. This has never been investigated in invertebrates, and we determined the concentrations and inducibility of metallothionein in gills and midgut gland of different size classes of shore crabs from uncontaminated areas. Metallothionein concentrations in gills and midgut gland ranged between 30 and 40 µg g-1 dry weight with no differences among the different size classes. Concentrations of cadmium, copper and zinc in the midgut gland increased with increasing size of the crabs when the concentrations were expressed on a dry weight basis; water content in the midgut gland increased with the size and only the cadmium concentration increased with size when concentrations were expressed on wet weight basis. There was an inverse relationship between metallothionein and both copper and cadmium concentrations. Smaller crabs exposed to 1 mg Cd L-1 accumulated higher concentrations of cadmium in midgut gland and gills than larger ones and metallothionein concentrations in the midgut gland were higher in the smaller crabs. However, the increase in metallothionein concentration per accumulated unit of cadmium showed a linear increase with the size of the crabs. The ratio [Cd]midgut/[Cd]gills decreased with the size of the crabs. The overall conclusion is that baseline metallothionein concentrations do not change with age in shore crabs, but that the inducibility of metallothionein upon cadmium challenge does.

Use of standard test organisms for sound validation of UV-based ballast water treatment systems.

To challenge ballast water treatment system (BWTS) efficacy for organisms in the size-class 10-50 µm, intake concentration during tests must reach certain minimum requirements. Often, natural concentrations are too low to meet intake requirements and standard test organisms (STOs) are added. We tested the robustness of Tetraselmis suecica and Odontella sp. to a range of UV-treatments to explore fluences needed to meet the IMO discharge standard (<10 org. ml-1) evaluated using two viability assessment methods. To meet discharge standards, fluences of >1000 mJ cm-2 were required using vital stain whereas 135-500 mJ cm-2 were needed using regrowth assays. Besides, results suggest that T. suecica and Odontella sp. were at least as robust as natural algae towards UV-treatments. We suggest the advantageous use of these species as STOs in test water to support intake water requirements and to obtain more conservative validation of UV-based BWTS to ensure more environmental protective procedures.

UV fluences required for compliance with ballast water discharge standards using two approved methods for algal viability assessment.

This study investigates the extra UV fluence needed to meet the International Maritime Organisation's ballast water discharge standards for the 10-50 µm size-class using the approved vital stain (VS) method compared to the Most Probable Number (MPN) method for organism viability assessment. Low- and medium pressure UV collimated beam treatments were applied to natural algae collected in temperate and tropical water environments and analysed using both methods. About 10 times higher UV fluence was required to meet discharge standards when using VS compared to MPN. Implementing a dark-hold period after UV treatments decreased algal viability. Length of dark-hold period to meet discharge standards decreased with increasing UV fluence. No significant differences between temperate and tropical samples were observed. The results showed that UV treated algae assessed using the VS method could meet discharge standards by increasing fluence and/or introducing a dark-hold period.

Retention and distribution of methylmercury administered in the food in marine invertebrates: Effect of dietary selenium.

Methylmercury is transported along aquatic food chains from the lower trophic levels and selenium modulates the biokinetics of mercury in organisms in complex ways. We investigated the retention of orally administered methylmercury in various marine invertebrates and the effect of selenium hereon. Shrimps (Palaemon adpersus and P. elegans), blue mussels (Mytilus edulis), shore crabs (Carcinus maenas) and sea stars (Asterias rubens) eliminated methylmercury slowly (t½â€¯= ½ to >1 year) and the copepod (Acartia tonsa) faster (t½â€¯âˆ¼â€¯12-24 h). Orally administered selenite augmented elimination of methylmercury in the copepod (in one of two experiments) and blue mussels, but not in shrimps, crabs and sea stars. Selenium generally alters the distribution of the body burden of mercury, leaving more mercury in muscle and less mercury in digestive glands or rest of the body - also in the species where total body retention is not affected.

Amount and metal composition of midgut gland metallothionein in shore crabs (Carcinus maenas) after exposure to cadmium in the food.

Accumulation of cadmium in aquatic invertebrates may compromise human food safety and anthropogenic additions of cadmium to coastal areas cause concern. Induction of crustacean metallothionein has been suggested as a useful biomarker for contamination of the aquatic environment with cadmium. We investigated how exposure to low concentrations of cadmium in the food affects the subcellular binding of cadmium with the shore crab Carcinus maenas as model organism. Approximately 80% of the assimilated cadmium was bound in the soluble fraction of the midgut gland and of this, 82% was found in the metallothionein fraction. Metallothionein synthesis was only induced at the highest exposure level. However, the number of cadmium atoms bound per molecule of metallothionein increased linearly with exposure, from approximately 0.18 in the control group to 1.4 in a group administered food containing 5.1 µg Cd g(-1). We noted a marked interaction between the presence of copper and zinc in the midgut gland and the binding of cadmium. The usefulness of crustacean midgut gland metallothionein as a biomarker for cadmium exposure at modest levels was questioned since exposures at levels producing significant increases in the tissue contents of the metal did not result in elevated concentrations of metallothionein in the midgut gland.

Vitellogenin as biomarker for estrogenicity in flounder Platichthys flesus in the field and exposed to 17α-ethinylestradiol via food and water in the laboratory.

The ability of 17α-ethinylestradiol (EE2) to elevate vitellogenin levels were investigated in male flounder Platichthys flesus and vitellogenin concentrations in flounders from the Danish coastal environment were determined. Male flounders were exposed to 17α-ethinylestradiol (EE2) via food or water. Average vitellogenin concentrations in the control fish ranged between 25 and 100 ng mL(-)(1). Exposure to 5.1, 8.1 and 16.8 ng EE2 L(-)(1) in water and 500 and 5000 ng EE2 kg(-)(1) body weight (bw) every second day in the food increased the plasma vitellogenin concentration in a concentration and time dependent manner, whereas exposure to 2.7 ng EE2 L(-)(1) in water for 21 d and 5 and 50 ng EE2 kg(-)(1) bw for 12 days in the food did not. EE2 could be detected in liver and testes (but not in muscle) after exposure to 8.1 and 16.8 ng EE2 L(-)(1) in the water and 5000 ng EE2 kg(-)(1) bw in the food; the highest concentration was 6 ng g(-)(1) wet weight in liver. The majority of the male flounders collected from nine coastal Danish sites from 1999 to 2004 had vitellogenin concentrations below 100 ng mL(-)(1), and only at two sites moderate estrogenic inputs were indicated.

Genetic adaptation of earthworms to copper pollution: is adaptation associated with fitness costs in Dendrobaena octaedra?

Exposure to copper pollution affects reproduction, growth and survival of earthworms. It is known that earthworms can cope with high copper burdens, but the distinction between physiological acclimation and evolutionary heritable changes and associated fitness consequences of the adaption to long-term copper exposure has rarely been studied. To investigate adaptation of earthworm populations of Dendrobaena octaedra to copper contamination, three populations from polluted soil were studied and compared to three unpolluted reference sites. Adult worms were collected at all six sites and cultured in uncontaminated control soil in the laboratory, where life-history traits were studied and F1-generations were produced. The newly hatched F1-generation worms were placed in uncontaminated control or copper-spiked soil to study if the adaptation was due to acclimation or genetic inheritance. This experiment showed that populations from polluted areas generally had a higher individual growth rate, reduced time to maturity, increased reproduction, and also increased mortality compared to the reference populations in both control and copper-spiked soil. The differences in life-history traits indicate that natural selection has resulted in genetic adaptation to copper pollution in the exposed populations. The population growth rates suggest a weak detrimental effect on population growth rate of being exposed to copper for both type of populations, but no sign of cost. On the contrary, estimates of population growth rates integrating all life-history traits showed that copper adapted populations perform on average relatively better than reference populations in both uncontaminated and copper-spiked soil.

Distribution of the UV filter 3-benzylidene camphor in rat following topical application.

A straightforward analytical method for determination of 3-benzylidene camphor (3-BC) in rat adipose tissue, brain, liver, muscle, plasma and testis following topical application was developed and validated. Three exposure levels (60, 180 and 540 mg kg(-1) day(-1)) were tested for 65 days in male Sprague-Dawley rats (24 days postnatal). Sample preparation involving homogenization and n-heptane or methanol extraction of the tissue was applied before injection into the LC-ESI-MS-MS system. The response was linear from 2 to 100 microg l(-1) for the qualifier and the quantifier MRM transitions (R(2) (quantifier) > 0.994). Detection limit of the method corresponded to 0.005 microg g(-1) tissue and 12.5 microg l(-1) plasma, respectively. Recovery was determined for all tissues (adipose tissue: 40%; all other tissues: 80-100%) at three individual levels. 3-(4-Methyl benzylidene camphor) (4-MBC) was used throughout the study as internal standard. 3-Benzylidene camphor was detected in all tissues at all exposure levels at concentrations between 0.05 microg g(-1) (liver) and 36 microg g(-1) (adipose tissue) and in plasma at 16-89 microg l(-1). The method allowed for the quantification of 3-benzylidene camphor in all tested tissues following topical application. Furthermore, it was shown that 3-benzylidene camphor can be found in various tissues in the rat following topical application. These findings may suggest that following use of 3-benzylidene camphor containing sunscreen, similar disposition and distribution may occur in humans.