Effects of anti-androgens cyproterone acetate, linuron, vinclozolin, and p,p'-DDE on the reproductive organs of the copepod Acartia tonsa.

The study was performed to detect the effects of anti-androgenic compounds on the reproduction. In this paper alterations observed in the marine calanoid copepod Acartia tonsa exposed to environmental concentrations of cyproterone acetate (CPA), linuron (LIN), vinclozolin (VIN), and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE) for 21 days covering a full life cycle are described. Histological alterations were studied with a focus on reproductive organs, gonad and accessory sexual glands. Exposure to ≥1.2 µg L(-1) CPA caused degeneration of spermatocytes and deformation of the spermatophore in males. In a single male exposed to 33 µg L(-1) CPA, an ovotestis was observed. In CPA exposed females, enhancement of oogenesis, increase in apoptosis and a decrease in proliferation occurred. Exposure of males to ≥12 µg L(-1) LIN caused degenerative effects in spermatogonia, spermatocytes and spermatids, and at 4.7 µg L(-1) LIN, the spermatophore wall displayed an irregular formation. In LIN exposed females, no such structural alterations were found; however, the proliferation index was reduced at 29 µg L(-1) LIN. At an exposure concentration of ≥100 µg L(-1) VIN, distinct areas in male gonad were stimulated, whereas others displayed a disturbed spermatogenesis and a deformed spermatophore wall. In VIN exposed female A. tonsa, no effects were observed. Male A. tonsa exposed to p,p'-DDE displayed an impairment of spermatogenesis in all stages with increased degrees of apoptosis. In p,p'-DDE-exposed females, a statistical significant increase of the proliferation index and an intensification of oogenesis were observed at 0.0088 µg L(-1).

Effects of methyltestosterone, letrozole, triphenyltin and fenarimol on histology of reproductive organs of the copepod Acartia tonsa.

The marine calanoid copepod Acartia tonsa was exposed to methyltestosterone (MET, 1.6-126 µg L(-1)), letrozole (LET, 10-1000 µg L(-1)), triphenyltin chloride (TPT, 0.0014-0.0088 µg L(-1) TPT-Sn) and fenarimol (FEN, 2.8-105 µg L(-1)) for 21 d covering a full life-cycle. All four compounds investigated are known to act as androgens in vertebrates. The digestive tract, musculature, nervous system, reproductive organs, gonad and accessory sexual glands were examined by light microscopy after routine staining and immune-labelling for detection of apoptosis and determination of proliferation activities. MET induced an inhibition of oogenesis, oocyte maturation and yolk formation, respectively, which was most pronounced at the lowest concentrations tested. In LET exposed males, spermatogenesis was enhanced with very prominent gamete stages; in some stages apoptosis occurred. The spermatophore was hypertrophied and displayed deformations. In females, LET induced a disorder of oogenesis and disturbances in yolk synthesis. TPT stimulated the male reproductive system at 0.0014 and 0.0035 µg TPT-SnL(-1), whereas inhibiting effects were observed in the female gonad at 0.0088 µg TPT-SnL(-1). In FEN exposed females proliferation of gametes was reduced and yolk formation showed irregular features at 2.8-105 µgL(-1). In FEN exposed males an elevated proliferation activity was observed. No pathological alterations in other organ systems, e.g. the digestive tract including the hindgut acting as respiratory organ, the nervous system, or the musculature were seen. This indicates that the effects on gonads might be caused rather by disturbance of endocrine signalling or interference with hormone metabolism than by general toxicity.

Effect of 2,4-dihydroxybenzophenone (BP1) on early life-stage development of the marine copepod Acartia tonsa at different temperatures and salinities.

Benzophenone (BP)-type ultraviolet (UV) filters are widely used in cosmetic and sunscreen products and can enter the aquatic environment. Therefore, we investigated the subchronic toxicity of 2,4-dihydroxybenzophenone (BP1) on the marine calanoid copepod Acartia tonsa in an early life-stage development study. Since developmental endpoints depend on environmental conditions, a preceding study of A. tonsa development was performed at three temperatures, four salinities, four light:dark regimes, six food densities, and four culture densities. Times elapsed until 50% of the population had reached a copepodite stage (DT(½) ) at the different conditions were calculated. The DT(½) values decreased from 296 h at 15°C to 89 h at 25°C and were also affected by salinity (126 h at 15‰ and 167 h at 30‰), whereas the light:dark regime and culture density influenced development only to a minor extent. BP1 was found acutely toxic at 2.6 mg/L (48-h median lethal concentration [LC50]). The toxicity of BP1 on early life-stage development was studied in combinations of three temperatures (15, 20, 25°C) and three salinities (15, 20, 25‰) using five toxicant concentrations between 0.051 and 2 mg/L in each scenario. Concentrations causing 10 and 50% inhibition of development (EC10 and EC50) were determined. Acartia tonsa was most resistant towards BP1 at 20°C where an EC50 of 1.1 mg/L was found, whereas EC50 values were significantly lower at 15°C (0.49 mg/L) and 25°C (0.77 mg/L), respectively. The EC50 also decreased with increasing salinity. Our results demonstrate that environmental conditions do influence toxicity test results; thus, they need to be considered carefully when developing test protocols as well as for environmental risk assessments of chemicals.

A critical analysis of the biological impacts of plasticizers on wildlife.

This review provides a critical analysis of the biological effects of the most widely used plasticizers, including dibutyl phthalate, diethylhexyl phthalate, dimethyl phthalate, butyl benzyl phthalate and bisphenol A (BPA), on wildlife, with a focus on annelids (both aquatic and terrestrial), molluscs, crustaceans, insects, fish and amphibians. Moreover, the paper provides novel data on the biological effects of some of these plasticizers in invertebrates, fish and amphibians. Phthalates and BPA have been shown to affect reproduction in all studied animal groups, to impair development in crustaceans and amphibians and to induce genetic aberrations. Molluscs, crustaceans and amphibians appear to be especially sensitive to these compounds, and biological effects are observed at environmentally relevant exposures in the low ng l(-1) to microg l(-1) range. In contrast, most effects in fish (except for disturbance in spermatogenesis) occur at higher concentrations. Most plasticizers appear to act by interfering with the functioning of various hormone systems, but some phthalates have wider pathways of disruption. Effect concentrations of plasticizers in laboratory experiments coincide with measured environmental concentrations, and thus there is a very real potential for effects of these chemicals on some wildlife populations. The most striking gaps in our current knowledge on the impacts of plasticizers on wildlife are the lack of data for long-term exposures to environmentally relevant concentrations and their ecotoxicity when part of complex mixtures. Furthermore, the hazard of plasticizers has been investigated in annelids, molluscs and arthropods only, and given the sensitivity of some invertebrates, effects assessments are warranted in other invertebrate phyla.

Towards an internationally harmonized test method for reproductive and developmental effects of endocrine disrupters in marine copepods.

New and updated methods to detect and characterize endocrine disrupting chemicals (EDCs) are urgently needed for the purpose of environmental risk assessment since these substances are often not detected using existing chronic toxicity tests. Numerous reports on the effects of EDCs on crustacean development and reproduction have been published and the development of life-cycle tests with crustaceans has been prioritized within the OECD work program for endocrine disrupter testing and assessment. As a result, Sweden, and Denmark initiated a proposal for development of a full life-cycle test with marine copepods (Acartia tonsa, Nitocra spinipes, Tisbe battagliai, and Amphiascus tenuiremis). The present paper gives an overview on the endocrine system of crustaceans with special emphasis on development and reproduction, which are targets for endocrine disruption, and reviews available methods for detecting effects on development and reproduction in calanoid and harpacticoid copepods. A draft OECD guideline Copepod Development and Reproduction Test has been developed, and a pre-validation of this draft guideline was completed in 2005. An updated draft guideline, taking into account the results of the pre-validation, is now under validation in an international ring-test, which is running till the end of 2006.

COMPRENDO: Focus and approach.

Tens of thousands of man-made chemicals are in regular use and discharged into the environment. Many of them are known to interfere with the hormonal systems in humans and wildlife. Given the complexity of endocrine systems, there are many ways in which endocrine-disrupting chemicals (EDCs) can affect the body's signaling system, and this makes unraveling the mechanisms of action of these chemicals difficult. A major concern is that some of these EDCs appear to be biologically active at extremely low concentrations. There is growing evidence to indicate that the guiding principle of traditional toxicology that "the dose makes the poison" may not always be the case because some EDCs do not induce the classical dose-response relationships. The European Union project COMPRENDO (Comparative Research on Endocrine Disrupters--Phylogenetic Approach and Common Principles focussing on Androgenic/Antiandrogenic Compounds) therefore aims to develop an understanding of potential health problems posed by androgenic and antiandrogenic compounds (AACs) to wildlife and humans by focusing on the commonalities and differences in responses to AACs across the animal kingdom (from invertebrates to vertebrates) .

Acute and chronic effects of pulse exposure of Daphnia magna to dimethoate and pirimicarb.

Short-term (<48 h) and long-term (21 d) effects of dimethoate and pirimicarb were studied in Daphnia magna exposed to pulses of 0.5 to 8 h in duration. During a 21-d postexposure observation period, the following parameters were monitored: Mortality, mobility, day for first offspring, animal size, weight of offspring and adults, and number of offspring produced. In general, animals exposed to a single pulse of dimethoate or pirimicarb regained mobility after 24 to 48 h in clean media. Animals exposed to repeated pulses of dimethoate did not recover mobility during a 48-h postexposure observation period, and mortality was significantly increased. Animals exposed to two pulses of pirimicarb showed less recovery of mobility compared with those exposed to one pulse. Exposure of D. magna to 30 mg/L of dimethoate or 100 microg/L of pirimicarb for 2 to 6 h resulted in a significant reduction in the number of offspring and in the average weight of offspring. The average body length was reduced after pulse exposure to 30 mg/L of dimethoate for 3 h or 70 microg/L of pirimicarb for 4 h, and these exposure concentrations caused a delay in the day for first offspring at exposure durations of 2 to 6 h. The most important new findings in the present study are that short-term (<4 h) pulse exposure of neonates to acetylcholinesterase-inhibiting pesticides caused reproductive damage in D. magna and that repeated-pulse exposure significantly increased mortality in animals that apparently had recovered after a single-pulse exposure.

Brominated flame retardants: activities in a crustacean development test and in an ecdysteroid screening assay.

Brominated flame retardants (BFRs) were investigated for toxic effects both in vivo and in vitro in two invertebrate bioassays. Subchronic effects of tetrabromobisphenol A (TBBPA), tribromophenol (TBP), and four polybrominated diphenyl ethers ([PBDEs]; BDE-28, BDE-47, BDE-99, and BDE-100) on larval development of the marine copepod Acartia tonsa were studied. For TBBPA and TBP 5-d effective median concentration (EC50) values for inhibition of the larval development rate were 125 and 810 microg/L, respectively, whereas the PBDEs were much more potent with 5-d EC50 in the low microg/L range (1.2 microg/L for BDE-100; 4.2 microg/L for BDE-99; 13 microg/L for BDE-28; and 13 microg/L for BDE-47). These concentrations were up to two orders of magnitude below the 48-h LC50 for acute adult toxicity (108 microg/L for BDE-28; 400 microg/L for TBBPA; 520 microg/L for BDE-100; 705 microg/L for BDE-99; 1,500 microg/L for TBP; and 2,370 microg/L for BDE-47). To distinguish between general toxicological and endocrine-mediated toxic effects, the BFRs were assessed in vitro for ecdysteroid agonistic/antagonistic activity with the ecdysteroid-responsive Drosophila melanogaster B(II)-cell line. The pentabrominated diphenyl ethers BDE-99 and BDE-100 showed weak ecdysteroid antagonistic activity. Thus, these PBDEs may be regarded as potential endocrine disrupters in invertebrates. The combination of in vitro assays and subchronic biotests with ecologically important crustacean species is a rapid and cost-effective tool when screening for sublethal effects of BFRs and other chemicals.

Effects of three PBDEs on development, reproduction and population growth rate of the harpacticoid copepod Nitocra spinipes.

The current knowledge concerning effects of polybrominated diphenyl ethers (PBDEs) on aquatic organisms is very limited. A full life-cycle (< or =26 days exposure) ecotoxicity test with the particle-feeding copepod Nitocra spinipes was therefore used to study effects of BDE-47, -99 and -100 on larval development rate (LDR) and population growth rate (r(m)). LDR significantly decreased in copepods exposed for 6 days to nominal concentrations > or =0.013 mg/l BDE-47 and > or =0.03 mg/l BDE-99. Large concentration ratios (< or =338) between adult acute and juvenile subchronic endpoints were observed. Exposure over the full life cycle (< or =26 days) showed that r(m) in general was a less sensitive endpoint than LDR. Still, the r(m) in copepods exposed to 0.04 mg/l BDE-47 was significantly reduced compared to the controls (***P<0.001). Partitioning experiments with 14C-BDE-47 and 14C-BDE-99 in the test system showed that the major fractions (approximately 50-80%) were associated to particulate material. Our findings indicate that development and reproduction in N. spinipes are sensitive to the tested PBDEs and that ingestion of particle-adsorbed PBDEs most likely is the predominant route of exposure in N. spinipes. However, to further improve the usefulness of laboratory effect levels of PBDEs and other lipophilic substances for environmental risk assessment, it is important to develop ecotoxicological tools, which can evaluate and rate the toxic contribution from different matrices, such as suspended particles, sediment, food, water etc.

Inhibition of larval development of the marine copepod Acartia tonsa by four synthetic musk substances.

A nitro musk (musk ketone) and three polycyclic musks (Tonalide, Galaxolide and Celestolide) were tested for acute and subchronic effects on a marine crustacean, the calanoid copepod Acartia tonsa. Sublethal effects on A. tonsa larvae were investigated with a rapid and cost effective bioassay, which is based on the easily detectable morphological change from the last nauplius to the first copepodite stage during copepod larval development. The inhibition of larval development after 5 days exposure was a very sensitive endpoint, with 5-d-EC(50)-values as low as 0.026 mg/l (Tonalide), 0.059 mg/l (Galaxolide), 0.066 mg/l (musk ketone) and 0.160 mg/l (Celestolide), respectively. These values were generally more than one order of magnitude below the 48-h-LC(50)-values found for adults, which were 0.47 mg/l (Galaxolide), 0.71 mg/l (Celestolide), 1.32 mg/l (musk ketone) and 2.5 mg/l (Tonalide). Since the synthetic musks strongly inhibited larval development in A. tonsa at low nominal concentrations, they should be considered as very toxic. The larval development test with A. tonsa is able to provide important aquatic toxicity data for the evaluation of synthetic musks, for which there is little published ecotoxicological information available regarding Crustacea. It is suggested that subchronic and chronic copepod toxicity tests should be used more frequently for risk assessment of environmental pollutants.

Effects of four synthetic musks on the life cycle of the harpacticoid copepod Nitocra spinipes.

A full life-cycle (