Investigation of the in vivo estrogenicity of the UV-filters 4-methylbenzylidene camphor and octyl methoxy cinnamate in rainbow trout (Oncorhynchus mykiss).

The estrogenic activity of the chemical UV-filters, 4-methylbenzylidene camphor (4-MBC) and octyl methoxy cinnamate (OMC) was investigated in an in vivo rainbow trout (Oncorhynchus mykiss) assay. Plasma vitellogenin concentrations were quantified by means of an Enzyme Linked Immuno Sorbent Assay (ELISA) in juvenile rainbow trout before and after intraperitoneal injection of the test compounds. Injection of 4-MBC on day 0, 3, 6 and 10 in the exposure period caused dose and time dependent increases in the concentration of plasma vitellogenin. Significant elevation of vitellogenin concentrations in plasma was demonstrated from 151 mg 4-MBC kg-1 injection-1. Logistic regression analysis relating the percentage of responding fish to the injected dose of 4-MBC resulted in ED10, ED50 and ED90 values of 37, 115 and 194 mg kg-1 injection-1, respectively, after 14 days of exposure (4 injections). Injections with OMC (up to 202 mg kg-1 injection-1) did not result in a statistically significant response in groups of exposed fish, although some individual fish showed elevated concentrations of vitellogenin in plasma. The results confirm that 4-MBC is estrogenic in fish in vivo.

Suspended particles only marginally reduce pyrethroid toxicity to the freshwater invertebrate Gammarus pulex (L.) during pulse exposure.

Current ecotoxicological research on particle-associated pyrethroids in freshwater systems focuses almost exclusively on sediment-exposure scenarios and sediment-dwelling macroinvertebrates. We studied how suspended particles influence acute effects of lambda-cyhalothrin and bifenthrin on the epibenthic freshwater amphipod Gammarus pulex (L.) using brief pulse exposures followed by a 144 h post exposure recovery phase. Humic acid (HA) and the clay mineral montmorillonite (MM) were used as model sorbents in environmentally realistic concentrations (5, 25 and 125 mg L(-1)). Mortality of G. pulex was recorded during the post exposure recovery phase and locomotor behavior was measured during exposure to lambda-cyhalothrin. We found that HA in concentrations ≥25 mg L(-1) adsorbed the majority of pyrethroids but only reduced mortality of G. pulex up to a factor of four compared to pyrethroid-only treatments. MM suspensions adsorbed a variable fraction of pyrethroids (10% for bifenthrin and 70% for lambda-cyhalothrin) but did not significantly change the concentration-response relationship compared to pure pyrethroid treatments. Behavioral responses and immobilisation rate of G. pulex were reduced in the presence of HA, whereas behavioral responses and immobilisation rate were increased in the presence of MM. This indicates that G. pulex was capable of sensing the bioavailable fraction of lambda-cyhalothrin. Our results imply that suspended particles reduce to only a limited extent the toxicity of pyrethroids to G. pulex and that passive uptake of pyrethroids can be significant even when pyrethroids are adsorbed to suspended particles.

New model for gain control of signal intensity to object distance in echolocating bats.

Echolocating bats emit ultrasonic calls and listen for the returning echoes to orient and localize prey in darkness. The emitted source level, SL (estimated signal intensity 10 cm from the mouth), is adjusted dynamically from call to call in response to sensory feedback as bats approach objects. A logarithmic relationship of SL=20 log(10)(x), i.e. 6 dB output reduction per halving of distance, x, has been proposed as a model for the relationship between emitted intensity and object distance, not only for bats but also for echolocating toothed whales. This logarithmic model suggests that the approaching echolocator maintains a constant intensity impinging upon the object, but it also implies ever-increasing source levels with distance, a physical and biological impossibility. We developed a new model for intensity compensation with an exponential rise to the maximum source level: SL=SL(max)-ae(-)(bx). In addition to providing a method for estimating maximum output, the new model also offers a tool for estimating a minimum detection distance where intensity compensation starts. We tested the new exponential model against the 'conventional' logarithmic model on data from five bat species. The new model performed better in 77% of the trials and as good as the conventional model in the rest (23%). We found much steeper rates of compensation when fitting the model to individual rather than pooled data, with slopes often steeper than -20 dB per halving of distance. This emphasizes the importance of analyzing individual events. The results are discussed in light of habitat constraints and the interaction between bats and their eared prey.

Repeated insecticide pulses increase harmful effects on stream macroinvertebrate biodiversity and function.

We exposed twelve mesocosm stream channels and four instream channels to one, two, and four pulses of the insecticide lambda-cyhalothrin (0.1 µg L-1) applied at two day intervals, each pulse lasting 90 min. Unexposed controls were included. We monitored macroinvertebrate taxonomic composition in the channels and in deployed leaf packs one day before and 29 days after the first exposure. Further, we measured drift in and out of the channels and leaf litter decomposition. Lambda-cyhalothrin exposures induced significantly increased drift in both experiments especially for Gammarus pulex, Amphinemura standfussi, and Leuctra spp. Macroinvertebrate taxonomic composition increasingly changed with increasing number of lambda-cyhalothrin exposures being most pronounced in the mesocosm channels. Further, leaf decomposition significantly decreased with increasing number of exposures in the mesocosm channels. Our study showed that species with predicted highest sensitivity to lambda-cyhalothrin were primary drivers of significant changes in taxonomic composition lasting for at least one month despite continuous recolonization of exposed channels from upstream parts of the natural stream and from the water inlet in the mesocosm channels. The overall results highlight the importance of sequential exposures to insecticides for understanding the full impact of insecticides on macroinvertebrates at the community level in streams.

Cadmium in the shore crab Carcinus maenas: seasonal variation in cadmium content and uptake and elimination of cadmium after administration via food.

The uptake and assimilation efficiency of cadmium administered via the food in the shore crab Carcinus maenas were investigated together with elimination kinetics and seasonal variations in cadmium content. The majority of shore crabs assimilated between 41 and 86% of the cadmium administered in their food. More than 90% of the cadmium taken up from food was retained in midgut gland. Elimination of cadmium after uptake from one meal of radioactively labelled soft parts of blue mussels could be described by a three-compartment model (percent 109Cd-retained = 64 x e(-0.001107 x t) + 25 x e(-0.0385 x t)+11 x e(-0.888 x t)). The biological half-life for cadmium in the most slowly exchanging compartment (containing 64% of the body burden) was 626 days. Groups of male and female shore crabs were collected from an uncontaminated site in the period May till October and the concentrations of cadmium in midgut gland and gills were determined. Male crabs had higher cadmium concentrations in the midgut gland in June and August (mean 2.7 microg Cd g(-1) dry weight) than they had in May, September and October (mean 1.7 microg Cd g(-1) dry weight). Females generally had slightly lower cadmium concentrations in the midgut gland than the males, except for a relatively high concentration in May. The cadmium concentrations in gills generally ranged between 0.3 and 0.5 microg Cd g(-1) dry weight) except for male values in October (mean 1 microg Cd g(-1) dry weight). Some of the seasonal changes in cadmium content of the crabs might plausibly be explained by changes in cadmium uptake from water, i.e. changes during the moult cycle and changes in cadmium uptake rates from water brought about by changes in ambient factors such as salinity and temperature. However, uptake of cadmium from water and transfer to the midgut gland take place at a rate that is two orders of magnitude too low to account for the increase in the cadmium concentrations in midgut gland in male crabs between May and June. The distribution of cadmium among tissues in crabs collected at uncontaminated sites also corresponds better with results obtained after administration of cadmium via the food than via water, and the exposure of the crabs to cadmium via the food is large enough to explain the increase in concentration between May and June.

In vivo and in vitro cadmium accumulation during the moult cycle of the male shore crab Carcinus maenas--interaction with calcium metabolism.

The effect of moult stage on cadmium accumulation and distribution was investigated in vivo in male shore crabs Carcinus maenas exposed to 1 mg Cd l(-1) for 7 days. The accumulation of cadmium in all tissues examined was markedly higher in postmoult (A(1-2) and B(1-2)) compared to intermoult (C1, C3 and C4) and premoult (D(0-3)). In addition, elevated levels of cadmium were found in gills of late premoult (D(2-3)) animals. The total amount of cadmium accumulated in the tissues (haemolymph, gills, midgut gland and muscle) increased from 43 microg Cd in early premoult (D(0-1)) to 391 microg Cd in late postmoult (B(1-2)). Gills and midgut gland were the primary cadmium accumulating tissues in C4-intermoult and premoult (D(0-3)); in early postmoult (A(1-2)) haemolymph and midgut gland were the main cadmium containing tissues, while midgut gland dominated in late postmoult (B(1-2)) and early intermoult (C1 and C3). A detailed account of calcium distribution in haemolymph, gills, midgut gland, muscle and exoskeleton during the moult cycle is presented. Mechanistic links between cadmium and calcium uptake in posterior gills of C4-intermoult and early postmoult (A(1-2)) crabs were explored using an in vitro gill perfusion technique. Calcium and cadmium influxes were markedly higher in postmoult compared to intermoult. No differences between intermoult and postmoult effluxes were found for either calcium or cadmium. From intermoult to postmoult net influx increased from 2.4 to 29 micromol Ca2+ g(-1) ww(gill) h(-1) and from 0.24 to 25 nmol Cd2+ g(-1) ww(gill) h(-1). The results indicate that the postmoult increase in cadmium influx is due to increased active transport of cadmium, at least partly, by accidental uptake via calcium transporting proteins. The in vitro net influx rates corresponded accurately to the observed in vivo accumulation of both cadmium and calcium. Although cadmium accumulation and distribution are clearly linked to changes in calcium requirements, cadmium did not interfere with calcium accumulation or distribution at any stage during the moult cycle.

Trace element distribution during the reproductive cycle of female and male spiny and Pacific scallops, with implications for biomonitoring.

Trace element concentrations and contents in gills, gonad, kidneys, mantle, muscle and remainder during the reproductive cycle of female and male spiny and Pacific scallops, from the Strait of Georgia, BC, Canada, were quantified by using ICPMS. The elements investigated were chromium, manganese, iron, cobalt, nickel, selenium, molybdenum, cadmium, tin and mercury. For all ten elements, the tissue distribution was to some extent influenced by species, sex and reproductive status. The implications of the present study in relation to the design of biomonitoring programmes are: (1) care should be taken to ensure an equal/constant sex composition when making interannual comparisons of pooled samples. Preferably the sexes should be monitored separately. (2) the practice of obtaining pooled samples in the interspawn phase is applicable only to monitoring long-term trends in contaminant levels, while the reproductive status should be heeded when studying short-term changes. (3) the present study confirms that direct temporal or spatial comparisons of absolute accumulated element concentrations are only valid intraspecifically.

Pesticide impacts on predator-prey interactions across two levels of organisation.

In this study, we aimed to evaluate the effects of a short pulse exposure of the pyrethroid lambda-cyhalothrin (LC) on the predator and anti-predator behaviour of the same species; Gammarus pulex. Predator behaviour, at the level of the individual, was studied in indoor microcosms using video tracking equipment during simultaneous exposure of the predator (G. pulex) and its prey (Leuctra nigra) during 90 min exposure of 1, 6.6 or 62.1 ngL(-1) LC. During an initial 30 min of exposure, the predator and prey organisms were maintained physically separated, and the actual interaction was studied through the subsequent 60 min of exposure. The anti-predator behaviour of G. pulex (drift suppression in response to the presence of brown trout) was studied in outdoor stream channels during a 90 min pulse exposure to LC (7.4 or 79.5 ngL(-1)) with, or without, brown trout. Based on survival curves for L. nigra we found that the mortality rate for L. nigra significantly decreased during exposure to 6.6 and 62.1 ngL(-1) LC (P<0.05 and P<0.001, respectively). We found no significant effects suggesting that G. pulex was repelled by contaminated prey items (P>0.05). We found that the exposure of G. pulex to 7.4 and 79.5 ngL(-1) LC significantly increased drift (from ∼0% to ∼100% in both treatments; P<0.001) independent of the presence of brown trout (P<0.05). In other words, the natural anti-predator behaviour of G. pulex was overruled by the stress response to LC exposure increasing G. pulex predation risk from drift feeding brown trouts. Our results show that the anti-predator and predator behaviour of G. pulex were significantly changed during exposure to very low and environmentally realistic LC concentrations and exposure duration. The potential implications for the field scenario are discussed.

Behavioural changes in three species of freshwater macroinvertebrates exposed to the pyrethroid lambda-cyhalothrin: laboratory and stream microcosm studies.

Pesticides are transported from crop fields to adjacent streams via surface run-off, drains, groundwater, wind drift and atmospheric deposition and give rise to transient pulse contamination. Although the concentrations observed, typically <10 microg L(-1), cannot be expected to be acutely lethal, effects in streams at the population and ecosystem level have been reported. One of the most conspicuous phenomena associated with these transient pesticide pulses is drift, where large numbers of freshwater invertebrates are carried along by the current and disappear from the contaminated stretch of the stream. The aim of the present study was to evaluate the feasibility of linking laboratory studies of the sublethal effects of pulse exposure to the pyrethroid lambda-cyhalothrin on the locomotory behaviour of stream invertebrates with effects on drift behaviour under more environmentally realistic conditions in stream microcosms. In the laboratory as well as in the microcosms, the order of sensitivities of the three species tested was (with Leuctra nigra being the most sensitive): L. nigra>Gammarus pulex>Heptagenia sulphurea. The LOECs determined for L. nigra (1 ng L(-1)), G. pulex (10 ng L(-1)) and H. sulphurea (100 ng L(-1)) are all within expected environmental concentrations. For the species of invertebrates investigated, it was possible to extrapolate directly from pyrethroid-induced behavioural changes observed in the laboratory to drift under more realistic conditions in stream microcosms. Consequently, the fast and cost-effective video tracking methodology may be applied for screening for potential effects of a wider range of pesticides and other stressors on the locomotory behaviour of freshwater invertebrates. The results indicate that such behavioural changes may be predictive of effects at the ecosystem level.

Arsenic compounds in the haemolymph of the Dungeness crab, Cancer magister, as determined by using HPLC on-line with inductively coupled plasma mass spectrometry.

Arsenobetaine, two arsenosugars, dimethylarsinate and several unidentified arsenic species were detected in extracts of the haemolymph of the Dungeness crab, Cancer magister, by using HPLC-ICP-MS. This is the first report of the presence of arsenosugars in the haemolymph/blood of marine animals. Total, extractable and residual arsenic concentrations were determined by ICP-MS. The concentration of total arsenic was in the range of 1.4-3.8 [micro sign]g ml(-1). Nearly all (98%) the arsenic was found to be extractable, and accounted for primarily by arsenobetaine, two arsenosugars and dimethylarsinate. The results demonstrate that arsenic compounds present in the diet of crabs are not fully metabolized in the gut. They are, at least partly, taken up into the haemolymph. The concurrence of arsenobetaine and arsenosugars suggests that the use of repeated haemolymph sampling in crustaceans could facilitate investigations into the kinetics of the biotransformation pathways of arsenic compounds. Finally, the present study clearly demonstrates the unique capabilities of HPLC-ICP-MS for the detection and identification of minor arsenic components amongst the predominant arsenobetaine.

Future migratory behaviour predicted from premigratory levels of gill Na+/K+-ATPase activity in individual wild brown trout (Salmo trutta).

The relationship between premigratory gill Na+/K+-ATPase activity, determined at two dates during spring, and future migratory behaviour was investigated using non-lethal gill biopsies and PIT-tagging in wild brown trout (Salmo trutta) from two tributaries. No significant relationship between future migratory strategy (individuals eventually becoming migrants or residents) and gill Na+/K+-ATPase activity was found in late February-early March. By contrast, in mid-April, a highly significant logistic regression equation identifying the migratory strategy in 93% of the 75 individuals was obtained. The ability of this regression model from the tributaries to predict future migratory behaviour in an independent group of trout caught in early April in the mainstream was evaluated. A threshold probability of migration was used to predict the behaviour of the mainstream individuals as either future migrants or residents. The maximum percentage of correct predictions of future migratory behaviour in mainstream fish was observed at threshold probabilities between approximately 0.15 and 0.45 (corresponding to threshold gill Na+/K+-ATPase activities between 2.7 and 3.7 micromol ADP mg(-1) protein h(-1)), with an average of 91% of the predictions being correct. The present study shows that a non-lethal premigratory biochemical measurement can successfully select individual brown trout with high probability of migration.

The chemical UV-filter 3-benzylidene camphor causes an oestrogenic effect in an in vivo fish assay.

Chemical UV-filters are used in sun protection products and various kinds of cosmetics. The lipophilic chemical UV-filter 3-benzylidene camphor was investigated for its capability to cause vitellogenin induction, possibly via oestrogen receptor binding, in a well-established in vivo fish assay (juvenile rainbow trout, Oncorhynchus mykiss, vitellogenin ELISA). A clear relationship was demonstrated between the dose of injected 3-benzylidene camphor and the concentration of plasma vitellogenin with a 105-times induction from 68 mg 3-benzylidene camphor /kg/injection and above compared to the control vitellogenin level. The relationship between the injected dose of 3-benzylidene camphor and the percent of responding fish (vitellogenin) was evaluated by logistic regression analysis and effective dose-values (ED-values) were determined. ED10, ED50 and ED90 of 3-benzylidene camphor after 6 days (2 injections) were 6.4, 16 and 26 mg/kg/ injection, respectively. These ED-values place 3-benzylidene camphor among the more potent xenooestrogens discovered to date and necessitates investigations on the distribution, concentration, persistence and bioaccumulation of 3-benzylidene camphor and other UV-filters in nature.

Vitellogenin induction by 17beta-estradiol and 17alpha-ethinylestradiol in male zebrafish (Danio rerio).

Adult male zebrafish (Danio rerio) were exposed to 17beta-estradiol (E2) or 17alpha-ethinylestradiol (EE2) in flow-through systems for 8 days. This was done to compare the sensitivity of the estrogen inducible vitellogenin (Vtg) biomarker system of this proposed OECD test guideline species to other relevant test species. Vtg was quantified in whole body homogenate by a species-specific ELISA. Actual water concentrations of E2 and EE2 were quantified by LC-MS, with detection limits of 1.0 and 0.6 ng/l, respectively. Vtg induction (LOEC) occurred in whole body homogenate at actual water concentrations of 21 ng E2/l and 3.0 ng EE2/l, respectively. As an alternative to the ANOVA approach, the relationship between the percentage of responding fish (Vtg) and the external E2 or EE2 concentration was determined by logistic regression analysis. Based on the regression analysis, EC-values could be determined: EC10, EC50 and EC90 were 15.4, 41.2 and 67.1 ng E2/l, respectively and 0.92, 2.51 and 4.09 ng EE2/l, respectively. Comparisons of these response limits to corresponding values for rainbow trout (Oncorhynchus mykiss), fathead minnow (Pimephales promelas) and Japanese medaka (Oryzias latipes) revealed the zebrafish as a sensitive test species.