In vivo effect confirmation of anti-androgenic compounds in sediment contact tests with Potamopyrgus antipodarum.

In order to investigate the sensitivity of Potamopyrgus antipodarum to anti-androgenic compounds, three spiked sediment tests were performed. The substances benzanthrone (7H-benz[de]anthracen-7-one), traseolide (ATII) and androstenone (5α-Androst-16-en-3-one) were previously identified in an effect-directed analysis study of the river Schijn in the north of Belgium. Although, in previous studies, all of the three compounds exhibited anti-androgenic activities in vitro, only the oxy-PAH benzanthrone had significant stimulating effects on the snails' reproduction. The reproduction of P. antipodarum was significantly stimulated, following a sigmoidal dose response curve, whereby an EC(50) of 10 ng/g dry sediment was calculated. Mortality was significantly increased at the highest concentration (69 ng/g dry sediment). The results indicate different relative potencies for the in vivo test with P. antipodarum and the in vitro anti-AR-CALUX assay, performed in a previous study. This highlights the importance of combined in vitro and in vivo assays for the effect assessment of field sediments.

In situ cage experiments with Potamopyrgus antipodarum--a novel tool for real life exposure assessment in freshwater ecosystems.

In situ experiments are an important tool within ecotoxicological research but there is a lack of suitable methodologies especially for freshwater invertebrate species. Within this study, a novel in situ methodology with Potamopyrgus antipodarum was developed. Snails were inserted into cages, made of Plexiglas measuring 7 × 9 × 7 cm(3) and fixed with stainless steel pins into the sediment at the relevant sampling sites. During the experiment physico-chemical properties of the water and concentrations of metals, PAHs and PCBs were measured in the sediment. The growth and survival of the snails was not affected, but the reproduction increased significantly at one of the most polluted sites. The increase in reproduction was neither correlated with physico-chemical parameters, nor with the concentrations of the different compounds, but maybe related to certain groups of estrogenic compounds. The study demonstrates the excellent applicability of this novel in situ test.

Endocrine modulation and toxic effects of two commonly used UV screens on the aquatic invertebrates Potamopyrgus antipodarum and Lumbriculus variegatus.

The two UV screens 3-benzylidene-camphor (3-BC) and 3-(4'-methylbenzylidene)-camphor (4-MBC) were tested regarding their toxicity and estrogenic activity. The Yeast Estrogen Screen (YES) and two sediment assays with the freshwater invertebrates Lumbriculus variegatus and Potamopyrgus antipodarum were performed. In the YES, both substances activated the human estrogen receptor alpha with EC50 values of 44.2 microM for 3-BC and 44.3 microM for 4-MBC, whereby 4-MBC attained only 8% of the maximal response of 17beta-estradiol. For P. antipodarum embryo production increased after exposure to both substances (EC50 of 4.60 microM 4-MBC=1.17 mg kg(-1)dw) while mortality increased at high concentrations. The reproduction of L. variegatus was decreased by 3-BC with an EC50 of 5.95 microM (=1.43 mg kg(-1)dw) and also by 4-MBC, where no EC50 could be calculated. While reproduction decreased, the worms' weight increased after exposure to 3-BC with an EC50 of 26.9 microM (=6.46 mg kg(-1) dw), hence the total biomass remained unaffected.

Development and validation of an OECD reproductive toxicity test guideline with the mudsnail Potamopyrgus antipodarum (Mollusca, Gastropoda).

Mollusks are known to be uniquely sensitive to a number of reproductive toxicants including some vertebrate endocrine disrupting chemicals. However, they have widely been ignored in environmental risk assessment procedures for chemicals. This study describes the validation of the Potamopyrgus antipodarum reproduction test within the OECD Conceptual Framework for Endocrine Disrupters Testing and Assessment. The number of embryos in the brood pouch and adult mortality serve as main endpoints. The experiments are conducted as static systems in beakers filled with artificial medium, which is aerated trough glass pipettes. The test chemical is dispersed into the medium, and adult snails are subsequently introduced into the beakers. After 28 days the reproductive success is determined by opening the brood pouch and embryo counting. This study presents the results of two validation studies of the reproduction test with eleven laboratories and the chemicals tributyltin (TBT) with nominal concentrations ranging from 10 to 1000 ng TBT-Sn/L and cadmium with concentrations from 1.56 to 25 µg/L. The test design could be implemented by all laboratories resulting in comparable effect concentrations for the endpoint number of embryos in the brood pouch. After TBT exposure mean EC10, EC50, NOEC and LOEC were 35.6, 127, 39.2 and 75.7 ng Sn/L, respectively. Mean effect concentrations in cadmium exposed snails were, respectively, 6.53, 14.2, 6.45 and 12.6 µg/L. The effect concentrations are in good accordance with already published data. Both validation studies show that the reproduction test with P. antipodarum is a well-suited tool to assess reproductive effects of chemicals.

Aquatic acute species sensitivity distributions of ZnO and CuO nanoparticles.

Metal oxide nanoparticles are increasingly being produced and will inevitably end up in the aquatic environment. Up till now, most papers have studied individual nanoparticle effects. However, the implementation of these data into a risk assessment tool, needed to characterise their risk to the aquatic environment, is still largely lacking. Therefore, aquatic species sensitivity distributions (SSDs) were constructed for ZnO and CuO nanoparticles and 5% hazard concentrations (HC5) were calculated in this study. The effect of individual nanoparticles on these SSDs was estimated by comparison with bulk SSDs. Additionally, the effect of nanoparticle dynamics (aggregation and dissolution) was considered by evaluating the effect of aggregate size on the toxicity, by estimation of the dissolved fraction and comparison with SSDs for ZnCl2 and CuCl2 inorganic salt. Bacteria, protozoa, yeast, rotifera, algae, nematoda, crustacea, hexapoda, fish and amphibia species were included in the analysis. The results show that algae (Zn) and crustacea (Zn, Cu) are the most sensitive species when exposed to the chemicals. Similar acute sensitivity distributions were obtained for ZnO nanoparticles (HC5: 0.06 with 90% confidence interval: 0.03-0.15 mg Zn/l; 43 data points), bulk ZnO (HC5: 0.06 with CI: 0.03-0.20 mg Zn/l; 23 dps) and ZnCl2 (HC5: 0.03 with CI: 0.02-0.05 mg Zn/l; 261 dps). CuO nanoparticles (HC5: 0.15 with CI: 0.05-0.47 mg Cu/l; 43 dps) are more toxic than the bulk materials (HC5: 6.19 with CI: 2.15-38.11 mg Cu/l; 12 dps) but less toxic than CuCl2 (HC5: 0.009 with CI: 0.007-0.012 mg Cu/l; 594 dps) to aquatic species. However, the combined dissolution and SSD results indicate that the toxicity of these nanoparticles is mainly caused by dissolved metal ions. Based on the available information, no current risk of these nanoparticles to the aquatic environment is expected.

The chronic toxicity of ZnO nanoparticles and ZnCl2 to Daphnia magna and the use of different methods to assess nanoparticle aggregation and dissolution.

In this study, the effect of ZnO nanoparticles and ZnCl2 on growth, reproduction and accumulation of zinc in Daphnia magna was determined in a 21-day chronic toxicity test. A variety of techniques were used to distinguish the free zinc ion, dissolved, nanoparticle and aggregated zinc fraction in the Daphnia test medium. The results showed similar chronic effects on growth, reproduction and accumulation for the ZnO nanoparticles (EC10, 20, 50 reproduction: 0.030, 0.049, 0.112 mg Zn/l) and the ZnCl2 (EC10, 20, 50 reproduction: 0.014, 0.027, 0.082 mg Zn/l). A large fraction of the nanoparticles rapidly dissolved after introduction in the exposure medium. Aggregation of nanoparticles was also observed but within 48 h of exposure most of these ZnO aggregates were dissolved. Based on the combined dissolution kinetics and toxicity results, it can be concluded that the toxicological effects of ZnO nanoparticles at the chronic level can be largely attributed to the dissolved fraction rather than the nanoparticles or initially formed aggregates.

Effects of inbreeding on mouthpart deformities of Chironomus riparius under sublethal pesticide exposure.

Mouthpart deformities in chironomids have been reported to indicate adverse effects of environmental pollutants. The authors assessed rates of mouthpart deformities in tributyltin-exposed, inbred, and outcrossed Chironomus riparius larvae over multiple generations. The authors found that the occurrence of mouthpart deformities was significantly correlated with inbreeding, whereas no correlation was found with the tributyltin exposure. The present study confirms the strong effect of high inbreeding rates on developmental deformities in chironomids.

Sediment contact test with Potamopyrgus antipodarum in effect-directed analyses-challenges and opportunities.

BACKGROUND AND SCOPE: Effect-directed analysis is increasingly used for the identification of key toxicants in environmental samples and there is a growing need for in vivo biotests as diagnostic tools. Within this study, we performed an in vivo sediment contact test, applicable on both native field samples and their extracts or fractions, in order to be able to compare the results from both field and laboratory studies. MATERIAL AND METHODS: A sediment contact test with the prosobranch snail, Potamopyrgus antipodarum, was carried out on extracts and fractions of field sediments from three European river basins. The results were compared with previous results of the native field samples. RESULTS: In contrast to the native sediments, the extracts of the samples led to an overall decrease in reproduction. Even the chosen reference sites had an adverse effect on the snails' reproduction. It appeared that a higher bioavailability in the organic extracts, together with a changing composition of compounds could have lead to this change in effects. The fractionation of the extracts partly led to a more differentiated picture, but the resolution was not high enough to see any distinct effects on the snails' reproduction. DISCUSSION AND CONCLUSION: Our results highlight the importance of the use of in vivo biotests and point out the relevance of bioavailability in native sediments. For further fractionation studies, a more realistic extraction procedure, together with a higher resolution fractionation, would be appropriate in order to separate individual bioavailable compounds more efficient.

Effect directed analysis of riverine sediments--the usefulness of Potamopyrgus antipodarum for in vivo effect confirmation of endocrine disruption.

In vivo tests are not commonly used in effect directed analysis (EDA) approaches. In the present study, a novel methodology was developed whereby Potamopyrgus antipodarum, which is known to be sensitive to endocrine disrupting compounds, was used as test organism. Field sediments from a polluted site in the north of Belgium were extracted and fractionated using three coupled and automatically switched normal-phase HPLC columns. Part of the fractions were spiked to artificial sediments and tested in a sediment contact test with P. antipodarum. The other part was used for an in vitro effect confirmation with the ER-LUC and anti-AR CALUX assays. Two of the six tested fractions stimulated the reproduction of the snails, while two others inhibited the reproduction. The fractions that caused an increase in reproduction also showed an increased estrogenic potency in the ER-LUC assay. Chemical analysis revealed that one of the most prominent compounds in those fractions was bisphenol-A, which has already been reported to have a stimulating effect on the reproduction of P. antipodarum by other authors. Due to the fact that previous studies have shown that this snail is also present in the field at this certain site, it was possible to directly link the results with effects that were observed in the field. This study indicates that effect directed analyses, supported by in vivo biotests, are very useful tools in order to identify the compounds that cause adverse effects on organisms or even population level.

Characterizing field sediments from three European river basins with special emphasis on endocrine effects - A recommendation for Potamopyrgus antipodarum as test organism.

The assessment of endocrine disrupting potentials of field sediments has until now been mostly limited to classical chemical analysis, in vitro assays and in vivo bioassays performed with vertebrates. There is an urgent need for easy, cheap and reproducible invertebrate tests which may be applied in certain monitoring activities. Since the mudsnail Potamopyrgus antipodarum is known to be tolerant to natural stressors, but also sensitive to endocrine disrupting chemicals, it is very likely that this organism could be suitable for the assessment of endocrine effects of e.g. field sediments. Within this study the endocrine potential of sediments in three European river basins was assessed. The yeast estrogen screen (YES) and a sediment contact test with P. antipodarum were performed. Furthermore, analyses of physico-chemical properties and concentrations of heavy metals, PAHs, organotins, natural steroids and alkylphenols were done. In the sediment contact test, the reproduction of the snail was promoted by a part of the sediments. This phenomenon could not be explained by their physico-chemical properties. However, at some of those sites a high estrogenic activity was detected in the YES, leading to the assumption that endocrine disrupting compounds could be responsible for those effects. This assumption could be confirmed to some extent with partially high concentrations of xeno-estrogens (e.g. nonylphenol) at the certain sites. Our study demonstrates the applicability of the test with P. antipodarum for a variety of sediments and once again points out the need of suitable in vivo biotests for the risk assessment of field sediments.

Prosobranch snails as test organisms for the assessment of endocrine active chemicals--an overview and a guideline proposal for a reproduction test with the freshwater mudsnail Potamopyrgus antipodarum.

Recently, prosobranch snails have been recommended as promising candidates for test organisms for the assessment of endocrine active chemicals. Three prosobranch snail species, the freshwater mudsnail Potamopyrgus antipodarum, the freshwater ramshorn snail Marisa cornuarietis, and the marine netted whelk Nassarius reticulatus are portrayed and their respective biotests are presented together with results of laboratory experiments and biological effect monitoring surveys in the field. All characterized species are highly sensitive toward xeno-androgens [triphenyltin (TPT), tributyltin (TBT), methyltestosterone (MT) and fenarimol (FEN)], and xeno-estrogens [bisphenol A (BPA), octylphenol (OP), ethinylestradiol], and show effects at environmentally relevant, rather low concentrations in laboratory experiments. For exposure to the xeno-androgen TPT, EC(10) values range between 15.9 and 29.0 ng as Sn/L (sediment 0.03 mug as Sn/kg), for TBT, EC(10) values are found between 3.42 and 37.8 ng as Sn/L (sediment 2.98 microg as Sn/kg) and effect concentrations for FEN are calculated as 18.6 ng/L (EC(10)) and 0.19 microg/kg (EC(50) sediment; EC(10) not calculable). Exposure to xeno-estrogens yielded EC(10 )values of 13.9 ng/L (0.19 microg/kg) for BPA, a NOEC of <1 microg/L (EC(10) of 0.004 microg/kg) for OP and a NOEC of 1 ng/l (EC(10) sediment of 2.2 microg/kg) for ethinylestradiol. Responses to androgens comprised the development of imposex and the reduction of fertility or embryo production, effects of estrogens included the stimulation of egg production and embryo production, and the increased weight of glands. Also, biological effect monitoring studies with P. antipodarum and N. reticulatus in several rivers or estuarine areas revealed the capacity of the biotests to detect an androgenic or estrogenic potential of sediment samples. A comparison of the three test species with regard to sensitivity and practical aspects in routine application favors the freshwater mudsnail P. antipodarum for a standardized procedure, and this reproduction test will be introduced into the OECD guideline program for standardization in the near future.