Malformed Larvae Research Articles

A Cause of Anormogenesis and Malformed Larvae in the Seed Production of Cockle, Fulvia mutica

A Cause of Anormogenesis and Malformed Larvae in the Seed Production of Cockle, Fulvia mutica

Malformations of the endangered Chinese sturgeon, Acipenser sinensis , and its causal agent

The anadromous Chinese sturgeon (Acipenser sinensis) is endangered and listed among the first class of protected animals in China. The possible causes for the decline of this species are the effects of synthetic chemicals, and loss of critical habitat. Chinese sturgeon in the Yangtze River have accumulated triphenyltin (TPT) to 31-128 ng/g wet weigh (ww) in liver, which is greater than the concentrations of tributyltin (<1.0 ng/g ww). Maternal transfer of TPT has resulted in concentrations of 25.5 +/- 13.0 ng/g ww in eggs of wild Chinese sturgeon, which poses a significant risk to the larvae naturally fertilized or hatched in the Yangtze River. The incidence of deformities in fry was 7.5%, with 1.2% of individuals exhibiting ocular abnormal development, and 6.3% exhibited skeletal/morphological deformations. The incidences of both ocular and skeletal/morphological deformations were directly proportional to the TPT concentration in the eggs of both the Chinese sturgeon and the Siberian sturgeon (Acipenser baerii) in controlled laboratory studies. The rates of deformities in the controlled studies were consistent with the rates caused at the similar concentrations in eggs collected from the field. Thus, TPT is the causal agent to induce the malformation of larvae of Chinese sturgeon. The incidence of deformed larvae of Chinese sturgeon is an indicator of overall population-level effects of TPT on Chinese sturgeon, because TPT at environmentally relevant concentrations can result in significantly decrease both quality and quantity of eggs and spawning frequency of fish.

Effects of bisphenol A on the embryonic development of sea urchin (Paracentrotus lividus)

Bisphenol A (BPA), is one of the most important industrial chemicals synthesized for diverse applications. In this study, tests for embryotoxic and spermiotoxic effects of BPA were utilized in the sperms and embryos of the sea urchin Paracentrotus lividus. The sperm and eggs of sea urchins were exposed to increasing concentrations of BPA (300-3500 microg/L) under static conditions. The endpoints were successful sperm fertilization, larval malformations, developmental arrest, and embryonic/larval mortality. BPA concentration (300 microg/L) had spermiotoxic and embryotoxic effects on this species. A dose-response related reduction was observed in fertilization success and significant increases in the number of larvae with skeleton malformations at the pluteus stage when the sperms were exposed BPA. The embryotoxicity of BPA is concentration-dependent and significant growth reduction at the early life stages and an increase in larval malformations as skeleton deformities at the pluteus stage were observed. It can be concluded that BPA adversely affects the reproduction and embryonic developmental stages of the P. lividus and this is of great ecological importance due to the hazard at the population level.

The Relationships among CYP1A Induction, Toxicity, and Eye Pathology in Early Life Stages of Fish Exposed to Oil Sands

Exposure of the early life stages of fish to oil sands constituents is associated with mortality and larval malformations such as edemas, hemorrhages, and skeletal, craniofacial, and eye defects. In fathead minnow (Pimephales promelas) and white sucker (Catostomus commersoni) larvae, indices of total eye pathology increased significantly following oil sands exposure. Structural, cytoplasmic, inflammatory, and degenerative eye alterations included poor retinal differentiation, microphthalmia, optic fissures, dysphasic retinas and lenses, inflammatory infiltrates, retinal epithelial lifting, and necrotic foci. Cytochrome P-4501A (CYP1A) was expressed in ocular (retina, lens) and kidney endothelial tissues, as indicated by immunohistochemistry. Although the kinetics of exposure-response curves for mortality and CYP1A expression were similar in both species, species differences in the magnitude and sensitivity of the responses were observed. Oil sands were twofold more toxic to fathead minnows (TPAH LC50 = 47–330 μg/g) than to white sucker (TPAH LC50 = 95–860 μg/g) larvae. For both species, larval mortality was significantly related to CYP1A protein concentrations in kidneys, and severity of these effects rose with oil sands exposure. The relationships among eye damage, mortality, and CYP1A indices warrants further investigation, and may lead to the use of CYP1A induction as an indicator of adverse effects rather than just contaminant exposure.

The Effects of Nonylphenol and Octylphenol on Embryonic Development of Sea Urchin (Paracentrotus lividus)

In this study, embryotoxic and genotoxic effects of nonylphenol (NP) and octylphenol (OP), which are the derivates of alkylphenol (APs), were evaluated using the gametes and embryos of the sea urchin Paracentrotus lividus. The sperm and eggs of sea urchins were exposed to increasing concentrations of NP (0.937-18.74 microg/L) and OP (5-160 microg/L) under static conditions. The endpoints were sperm fertilization success, quantitative and morphologic changes in mitotic activity, larval malformations, developmental arrest, and embryonic/larval mortality. A dose-response-related reduction (approximately 20%) was observed in fertilization success and significant increases in the number of larvae with skeleton malformations at the pluteus stage of the contaminated sperms. The spermiotoxic and embryotoxic concentrations were determined as 0.937 microg/L for NP and 4.685 microg/L for OP for this species. The embryotoxicity of NP and OP is concentration dependent, and significant growth reduction at the early life stages and an increase in larval malformations as skeleton deformities at the pluteus stage were observed. Cytogenetic analysis of embryos showed a decreasing curve in mitotic indexes (number of mitosis per embryo) with increasing concentrations of NP and OP. It can be concluded that NP and OP adversely affect the reproduction and embryonic developmental stages of the P. lividus and this is of great ecological importance because of the hazard at the population level.

Embryotoxic effects of nonylphenol and octylphenol in sea urchin Arbacia lixula

Nonylphenol (NP) and octylphenol (OP), both of which are biodegradation products of alkylphenols, are widely used in industrial applications and in some domestic products. These chemicals are found widely in surface water and aquatic sediments. We have carried out a comparative embryotoxicity analysis of the effects of increasing concentrations of NP (seven concentrations ranging from 0.937 to 18.74 microg/l) and OP (six concentrations ranging from 5 to 160 microg/l) on embryos of the sea urchin Arbacia lixula. The indicators evaluated were larval malformations, developmental arrest and embryonic/larval mortality. The results revealed that low concentrations of these chemicals (NP, OP) generally caused malformations in the skeletal system. High concentrations (18.74 microg NP/l, 160 microg OP/l) were found to inhibit the growth of embryos in the early life stages by preventing mitosis. We conclude that NP and OP present a major risk to the normal development of A. lixula at the low concentrations that have been recorded in the environment. These chemicals are therefore most likely to represent an ecological hazard at the population level given the cumulative effects of other environmental pollutants.

Evaluation of aflatoxin B1 embryotoxicity using the frog embryo teratogenesis assay‐Xenopus and bio‐activation with microsome activation systems

Aflatoxins are a group of mycotoxins produced by Aspergillus, A. flavus, and A. parasiticus. Aflatoxin B1 (AFB1) should be a strong teratogen in hamsters, but its effect in rats is equivocal and extremely limited in mice. Therefore, the AFB1 embryotoxic potential in mammals remains unclear. Little is known about the AFB1 effects on amphibians, therefore its embryotoxic potential was evaluated using the frog embryo teratogenesis assay-Xenopus (FETAX). X. laevis blastulae were exposed to: 1) positive controls for bio-activation (4 g/L cyclophosphamide monohydrate, Cy, and 4 g/L Cy+30 mg/L MAS-rat; 4 g/L Cy+30 mg/L MAS-human); 2) positive controls for MAS (30 mg/L MAS-rat and 30 mg/L MAS-human); 3) exposed groups to AFB1 (1 mg/L AFB1); and 4) AFB1 bio-activation (1 mg/L AFB1+30 mg/L MAS-rat and 1 mg/L AFB1 +30 mg/L MAS-human). In MAS-rat and human, Cy did not induce a statistically significant increase of mortality and malformed larvae percentage, but when bio-activated Cy increased the percentage of mortality. Instead, MAS-rat and human alone did not show any increase of mortality and malformed larvae percentages. When bio-activated by MAS-rat and human, AFB1 increased significantly both the mortality and malformed larvae percentages. The malformed larvae were mainly plurimalformed, i.e., affected by generalized edema, abnormal gut coiling, and microphthalmia. This research shows that AFB1 alone is not embryotoxic but, when bio-activated with MAS-rat or MAS-human the percentage of mortality and malformed larvae increased significantly. These results also show that AFB1 must be bio-activated to exert its embryotoxic effects.

Cryopreservation of common carp ( Cyprinus carpio) sperm in 1.2 and 5 ml straws and occurrence of haploids among larvae produced with cryopreserved sperm

Experiments were carried out on the cryopreservation of common carp ( Cyprinus carpio) sperm in order to test the suitability of using 1.2 and 5 ml straws and to investigate the ploidy of malformed larvae found among the hatched progeny. In the first set of experiments, the effect of freezing time was investigated on the hatch rate of embryos. The highest hatch rate for 1.2 ml straws was 69 ± 16% at the freezing time of 4 min, and 39 ± 27% for 5 ml straws at 5 min. In the second set, the effect different egg volumes fertilized with one straw of sperm on the hatch rate and the rate of malformed larvae was investigated. The highest hatch rate with 1.2 ml straws (86 ± 12%) was observed when 10 g of eggs were fertilized with one straw, whereas with 5 ml straws the hatch rate was highest (65 ± 18%) when 40 g of eggs were fertilized. The highest rate of malformed larvae (15 ± 9%) was found in the control, whereas the highest rate of malformed larvae among the groups fertilized with cryopreserved sperm (13 ± 7%) was found in the 1× dose group fertilized with 5 ml straw. The chromosome numbers of malformed larvae were investigated and haploids were found among those hatched from eggs fertilized with cryopreserved sperm whereas only diploids were found in the controls.

Tire debris organic extract affects Xenopus development

Tire debris (TD) and its organic components were identified as a main source of PM 10 atmospheric and water pollution. Because few data are available on the embryotoxic effects of TD organic components, the lethal and teratogenic potential of tire debris organic extract (TDOE) was evaluated using the frog embryo teratogenesis assay- Xenopus (FETAX), coupled with a histopathological screening of the survived larvae. From stage 8 to stage 47, Xenopus laevis embryos were exposed to TDOE at concentrations of 50, 80, 100, 120 and 140 mg/L. The results showed 50 mg/L TDOE to be the non-observable effect concentration (NOEC). TDOE mortality at 80 mg/L was significantly higher than the control, but did not increase further with higher concentrations. A good concentration-response was observed for percentages of malformed larva and from 80 mg/L on these percentages were significantly higher than the control. Therefore, probit analysis gave a 144.6 mg/L TC50. At 120 and 140 mg/L, many larvae were plurimalformed. The most frequent alterations observed were abnormal gut coiling, microphthalmia, monolateral anophthalmia, and narrowing eyes. The histological screening mainly revealed ocular malformations such as double retina, retina nervous cell layer coiling, and altered lens. Moreover severe vacuolisation and necrosis were scored in liver and axial musculature. These results strongly support the assumption that TDOE is a powerful teratogen for X. laevis.

Toxic effects of two pesticides, Imazalil and Triadimefon, on the early development of the ascidian Phallusia mammillata (Chordata, Ascidiacea)

Azole compounds are fungicides used in agriculture and in clinical area and are suspected to produce craniofacial malformations in vertebrates. Toxicity tests on sperm viability, fertilization and embryogenesis of the solitary ascidian Phallusia mammillata were performed to evaluate the effects of two azole derivatives, Imazalil and Triadimefon. Ascidian (Chordata, Ascidiacea) embryos and larvae could provide biological criteria for seawater quality standards because the larvae are lecitotrophic and have a short pelagic period, allowing to run the larval toxicity tests over a short period of time. Imazalil and Triadimefon proved to have strong consequences on P. mammillata. They could influence the reproductive rate of the animal exerting their effects at different levels: acting as spermiotoxic agents, inhibiting fertilization and impairing embryological development. Fertilization rate significantly decreased after 30 min exposure of sperm to 25 μM Imazalil ( P < 0.0001) and after exposure of both gametes to 50 μM Imazalil ( P < 0.05) and 1 mM Triadimefon ( P < 0.0001) as compared to controls. Malformations caused by exposure of embryos to both substances were dose dependent. Imazalil median teratogenic concentration (TC 50 concentration, the concentration that resulted in 50% malformed larvae) value was 0.67 μM and median lethal concentration (LC 50, the concentration that resulted in 50% embryos dead before completing the development) value was 10.23 μM while for Triadimefon TC 50 value was 29.56 and LC 50 value was 173.7 μM. Larvae developed from embryos treated with Imazalil and Triadimefon showed alterations of the anterior structures of the trunk: papillary nerves and the anterior central nervous system failed to correctly differentiate, as showed by immunostaining with anti-β-tubulin antibody. Comparing the anomalies caused by retinoic acid, reported in a previous study, it was possible to hypothesize that malformations induced by Imazalil and Triadimefon could be due to a perturbation of the endogenous retinoid content, as it has been proposed for mammals. Ascidians proved to be good models to study the toxic effects of pesticides since they offered both the convenience of working with an invertebrate species and the tissue sensitivity to chemical compound comparable to vertebrates.

Aflatoxin M1 effects on Xenopus laevis development

The principal Aflatoxin B(1) (AFB(1)) hydroxylated metabolite excreted in milk is Aflatoxin M(1) (AFM(1)) classified in group 2B by the International Agency for Research on Cancer (IARC). Human exposure to AFM(1) is due to the consumption of contaminated dairy products and partly to endogenous production through AFB(1) liver metabolism. Since no data are available on AFM(1) embryotoxicity, its lethal and teratogenic potential was investigated using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). Stage-8 blastulae were exposed to AFM(1) at 1, 4, 16, 64, and 256 microg/L concentrations until stage 47, free-swimming larva. A slight increase of mortality and malformed larva percents was found in AFM(1)-exposed groups but these differences were not statistically significant in comparison with the controls. Therefore, AFM(1) is a non-embryotoxic compound when evaluated with a FETAX model at concentrations under the conditions tested. However, AFM(1) merits further studies using mammals as experimental models to identify a possible risk during human pregnancy.

H2O2 induces abnormal tail flexure in Xenopus embryos: similarities with Paraquat teratogenic effects

As previously shown, Paraquat (PQ) treatments of Xenopus developing embryos mainly induce a characteristic developmental alteration we named "abnormal tail flexure." PQ oxidative activity has been indicated as the cause of this malformation. Since PQ evokes reactive oxygen species (ROS), among which hydroxyl radicals (OH(*)), and H(2)O(2) can be converted to (OH(*)) via Fenton reaction, we compared here the lethal and teratogenic potentials of both oxidants by using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX), in order to grasp eventual similarities in their teratogenic activity. Xenopus embryos were exposed, from stage 8 to stage 47, at 368, 491, 612, and 735 microM H(2)O(2) and 0.388 microM PQ. The probit analysis of H(2)O(2) mortality and malformed larva percents gave a 598.82 microM Lethal Concentration 50% (LC(50)) and 536.04 microM Teratogenic Concentration 50% (TC(50)) from which a 1.11 Teratogenic Index (T.I.) has been calculated. This T.I. value should allow the classification of H(2)O(2) as a non-teratogenic compound. A comparison of H(2)O(2) mortality and malformed larva percents with those obtained from PQ exposure showed the higher embryotoxicity of PQ, but, markedly, both compounds mainly induced the "abnormal tail flexure." Histological analysis of both H(2)O(2) and PQ malformed embryo tails showed a similar distorted morphology of both somites and myocytes. Some of muscle cells were necrotic and affected by an apical enlargement as well as a detachment from the connective tissue of intersomitic boundaries. In our opinion, both of the tested chemicals likely weaken the mechanical bridge connecting the myocyte contractile apparatus to the extracellular matrix, therefore causing the detachment of some of tail myocytes from their connectival septum as well as their apical enlargement. This could lead to the unbalance of tail tensional forces and, in turn, to the appearance of the "abnormal tail flexure."

CYP1A Induction and Blue Sac Disease in Early Life Stages of White Suckers (Catostomus commersoni) Exposed to Oil Sands

The objectives of this study were to evaluate the influence of natural oil sands on the early developmental stages of white sucker (Catostomus commersoni) and to determine whether biochemical responses in this species were similar to native fish caught in the Athabasca Oil Sands area. Early life stage (ELS) sediment toxicity tests were conducted using controls, reference sediments, natural oil sands, and industrially contaminated (wastewater pond) sediments collected from sites along the Athabasca River, Alberta (Canada). Eggs and larvae were observed for mortality, hatching, deformities, growth, and cytochrome P-4501A (CYP1A) activity using immunohistochemistry. E-Nat-, S-Nat-, and wastewater pond sediment-exposed groups showed significant premature hatching, reduced growth, and exposure-dependent increases in ELS mortality and larval malformations relative to controls. The most common larval deformities included edemas (pericardial, yolk sac, and subepidermal), hemorrhages, and spinal defects. Juveniles exposed to oil sands and wastewater pond sediments (96 h) demonstrated significantly increased 7-ethoxyresorufin–O-deethylase (EROD) activity (30- to 50-fold) as compared to controls. Reference sediment-exposed groups and water controls demonstrated reliable embryo and larval survival, minimal malformations, and negligible CYP1A staining. These observed signs of blue sac disease (ELS mortality, malformations, growth reductions, CYP1A activity induction) may produce deleterious reproductive effects in natural fish populations exposed to oil sands mixtures.This research was funded by grants from the Toxic Substances Research Initiative (Project 187) through Health Canada, Panel on Energy Research and Development, and the Natural Sciences and Engineering Council of Canada (PVH). M. Colavecchia was supported by scholarships awarded by OGS, Canadian Network of Toxicology Centres, and Petro-Canada. The authors thank the following individuals for their technical assistance with the field sampling program: R. Neurtherander (NWRI), B. Crosley, M.Conly (CWS), Golder Associates (K. Allen and M. Ezekiel), Regional Aquatics Monitoring Program, Suncor Energy (A. Cummins), Syncrude Canada (T. VanMeer, N. Rutley), M. Bowerman, and A. Winchester (Queen's University). We thank G. Fodor (DFO), Dr. P. Akhtar (Queen's University), B. Blunt, M. Baker (NWRI), S. Cagampan, and S. Backus (NLET) for their enthusiastic laboratory assistance. Portions of this research were presented at the 2003 Annual Aquatic Toxicity Workshop (Thirtieth Annual Meeting s, p. 90). Useful comments by Dr. S. Kacew and two anonymous reviewers helped improve an earlier draft of this article.

Toxic Effects of Irgarol and Diuron on Sea Urchin Paracentrotus lividus Early Development, Fertilization, and Offspring Quality

Irgarol and Diuron are the most representative "organic booster biocides" that replaced organotin compounds in antifouling paints. It cannot be assumed beforehand that their use will have no environmental impact: more ecotoxicological data and a significant environmental monitoring are required. Spermio and embryotoxicities of the biocides Irgarol and Diuron were investigated on Paracentrotus lividus, the dominant echinoid species of the Mediterranean Sea. Spermiotoxicity was studied by assessing the effects of sperm exposure on fertilization rate as well as on the induction of transmissible damages to the offspring. Embryotoxicity was studied by assessing the developmental defects in the exposed larvae. The experimental results show a Diuron EC50 of 2.39 (+/- 0.21) mg/L with a NOEL of 0.25 mg/L for embryos, and of 5.09 (+/- 0.45) mg/L with a NOEL of 0.5 mg/L for sperms, respectively. Data obtained from the embryotoxicity test on Irgarol [EC50 0.99 (+/- 0.69) mg/L] are of the same order of magnitude as the literature data about Japanese urchins. Spermiotoxicity tests show an Irgarol EC50 of 9.04 (+/- 0.45) mg/L with a NOEL of 0.1 mg/L. These data show the different sensitivities of the two tests: embryos are more sensitive than sperms for both the tested chemicals and Diuron seems to be the less toxic one. Moreover, as a major output of the experimental work, tested herbicides exert transmissible damage to spermatozoa evidenced by larval malformations in the offspring, mainly P1 type (skeletal alterations). The comparison of the endpoints results offers an interesting indication of a probable different mode of action (Irgarol seems to interact with calcium homeostasis) of the two biocides.

Developmental disorders in larvae of eelpout ( Zoarces viviparus) from German and Swedish Baltic coastal waters

Pregnant eelpout were collected for several years at German and Swedish Baltic coastal stations with different impact of pollutants and the prevalence of fry abnormalities compared between sites. Mortality of apparently normal developed larvae was observed with high variability in both German and Swedish sites. Malformation of larvae was a prominent disorder at all German stations with a prevalence ranging from about 50% up to a maximum of 90% in almost all analysed samples. At the Swedish stations malformation rates were significantly lower (range: 0–6%). Growth retardation of fry was clearly more prominent in eelpout from German coastal waters as well. Taken together the field data suggests that impaired larval development was associated with the state of environmental pollution at the sampling locations.

POPK-1/Sad-1 kinase is required for the proper translocation of maternal mRNAs and putative germ plasm at the posterior pole of the ascidian embryo

Maternal mRNAs localized to specific regions in eggs play important roles in the establishment of embryonic axes and germ layers in various species. Type I postplasmic/PEM mRNAs, which are localized to the posterior-vegetal cortex (PVC) of fertilized ascidian eggs, such as the muscle determinant macho-1 mRNA, play key roles in embryonic development. In the present study, we analyzed the function of the postplasmic/PEM RNA Hr-POPK-1, which encodes a kinase of Halocynthia roretzi. When the function of POPK-1 was suppressed by morpholino antisense oligonucleotides, the resulting malformed larvae did not form muscle or mesenchyme, as in macho-1-deficient embryos. Epistatic analysis indicated that POPK-1 acts upstream of macho-1. When POPK-1 was knocked down, localization of every Type I postplasmic/PEM mRNA examined, including macho-1, was perturbed, showing diffuse early distribution and eventual concentration into a smaller area. This is the probable reason for the macho-1 dysfunction. The postplasmic/PEM mRNAs such as macho-1 and Hr-PEM1 are co-localized with the cortical endoplasmic reticulum (cER) and move with it after fertilization. Eventually they become highly concentrated into a subcellular structure, the centrosome-attracting body (CAB), at the posterior pole of the cleaving embryos. The suppression of POPK-1 function reduced the size of the domain of concentrated cER at the posterior pole, indicating that POPK-1 is involved in the movement of postplasmic/PEM RNAs via relocalization of cER. The CAB also shrank. These results suggest that Hr-POPK-1 plays roles in concentration and positioning of the cER, as well as in the concentration of CAB materials, such as putative germ plasm, in the posterior blastomeres.

Egg quality determinants in cod (Gadus morhua L.): egg performance and lipids in eggs from farmed and wild broodstock

Lipids and essential fatty acids, particularly the highly unsaturated fatty acids, 20:5n-3 (eicosapentaenoic acid; EPA), 22:6n-3 (docosahexaenoic acid; DHA) and 20:4n-6 (arachidonic acid, AA) have been shown to be crucial determinants of marine fish reproduction directly affecting fecundity, egg quality, hatching success, larval malformation and pigmentation. In Atlantic cod (Gadus morhua L.) culture, eggs from farmed broodstock can have much lower fertilization and hatching rates than eggs from wild broodstock. The present study aimed to test the hypothesis that potential quality and performance differences between eggs from different cod broodstock would be reflected in differences in lipid and fatty acid composition. Thus eggs were obtained from three broodstock, farmed, wild/fed and wild/unfed, and lipid content, lipid class composition, fatty acid composition and pigment content were determined and related to performance parameters including fertilization rate, symmetry of cell division and survival to hatching. Eggs from farmed broodstock showed significantly lower fertilization rates, cell symmetry and survival to hatching rates than eggs from wild broodstock. There were no differences in total lipid content or the proportions of the major lipid classes between eggs from the different broodstock. However, eggs from farmed broodstock were characterized by having significantly lower levels of some quantitatively minor phospholipid classes, particularly phosphatidylinositol. There were no differences between eggs from farmed and wild broodstock in the proportions of saturated, monounsaturated and total polyunsaturated fatty acids. The DHA content was also similar. However, eggs from farmed broodstock had significantly lower levels of AA, and consequently significantly higher EPA/AA ratios than eggs from wild broodstock. Total pigment and astaxanthin levels were significantly higher in eggs from wild broodstock. Therefore, the levels of AA and phosphatidylinositol, the predominant AA-containing lipid class, and egg pigment content were positively related to egg quality or performance parameters such as fertilization and hatching success rates, and cell symmetry.

Examination of larval malformations in African catfish Clarias gariepinus following fertilization with cryopreserved sperm

In our earlier experiments on the cryopreservation of African catfish ( Clarias gariepinus) sperm a high number of malformed individuals were found among the larvae hatched from eggs fertilized with frozenthawed sperm. In our present study we explored one of the possible reasons of this malformation by examining the ploidy of the larvae. Sperm was frozen in a 6% fructose extender containing 10% methanol and 10% DMSO as cryoprotectant. Sperm was drawn to straw of different volumes (0.25, 0.5 and 1.2 ml). Freezing was carried out in liquid nitrogen vapor. Fifteen grams or thirty grams of eggs were used for fertilization. Chromosome preparations were made of non-feeding larvae. Fifty to one hundred larvae were incubated in 0.05% colchicine for 3 h then in a hypotonic 0.075M KCl solution for 25 min. Larvae were fixed in a 3:1 solution of methanol and acetic acid, then cell suspensions were made in 50% acetic acid. The suspensions were spread on slides and stained in 4% Giemsa for 8 min. Chromosome counting and evaluation was done at 1200× magnification on well spread metaphases. A majority of the hatched larvae were diploid, with 56 chromosomes. Surprisingly some of the malformed larvae hatched from eggs fertilized with cryopreserved sperm were haploids. Haploids occurred only when 0.25 or 0.5 ml straws were used for freezing. One possible explanation of haploidy is that the genome of the fertilizing spermatozoon is damaged during the process of freezing thus it can still move and fertilize the egg but its genome does not take part in the development of the embryo. This hypothesis is supported by the fact that no haploids were found among the malformed larvae of the control group which was fertilized with fresh sperm.

Impact of tire debris on in vitro and in vivo systems

BackgroundIt is estimated that over 80% of respirable particulate matter (PM10) in cities comes from road transport and that tire and brake wear are responsible for the 3–7% emission of it. Data on the indicators of environmental impact of tire debris (TD), originated from the tire abrasion on roads, are extremely scarce, even though TD contains chemicals (zinc and organic compounds) which can be released in the environment.MethodsTD particle morphology was analysed with SEM, TEM and FIB instruments. TD eluates and TD organic extracts were tested at dilution series on human cell lines and Xenopus laevis embryos. 50 and 100 g/L TD were used for the eluates obtained after 24 h at pH 3 and the quantity of zinc present was measured with a ICP-AES. Eluates diluted to 1%, 10%, 50% in culture media and undiluted were used on X. laevis embryos in the FETAX test. HepG2 cells were exposed for 24 h to 0.05 – 50 μg/ml of zinc salt while A549 cells were exposed for 24, 48 and 72 h to 10, 50, 60, or 75 μg/ml of TD extract. X. laevis embryos were exposed to 50, 80, 100, or 120 μg/ml TD extract.ResultsThe solution of undiluted 50 g/L TD produced 80.2% mortality (p < 0.01) in X. laevis embryos and this toxic effect was three times greater than that produced by 100 g/L TD. Zn accumulation in HepG2 cells was evident after 4 h exposure. A549 cells exposed to TD organic extract for 72 h presented a modified morphology, a decrease in cell proliferation and an increase in DNA damage as shown by comet assay. The dose 80 μg/ml of TD extract produced 14.6% mortality in X. laevis embryos and 15.9% mortality at 120 μg/ml. Treatment with 80, 100, or 120 μg/ml TD organic extract increased from 14.8% to 37.8% malformed larvae percentages compared to 5.6% in the control.ConclusionSince the amount of Zn leached from TD is related to pH, aggregation of particles and elution process, the quantity of TD present in the environment has to be taken into account. Moreover the atmospheric conditions, which may deeply influence the particle properties, have to be considered. The TD organic fraction was toxic for cells and organisms. Thus, because of its chemical components, TD may have a potential environmental impact and has to be further investigated.

Comparative teratogenicity of Chlorpyrifos and Malathion on Xenopus laevis development

The embryotoxic potential of chlorpyrifos (CPF) and malathion (MTN), two organophosphorus insecticides (OPs), was evaluated by modified Frog Embryo Teratogenesis Assay- Xenopus (FETAX). CPF and MTN were not embryolethal even at the highest concentration tested (6000 μg/l), but both exhibited a powerful teratogenicity. The probit analysis of malformed larva percentages showed a TC 50 of 161.54 μg/l for CPF, and a TC 50 of 2394.01 μg/l for MTN. Therefore, CPF teratogenicity was about 15 times higher than MTN. Larvae of both exposed groups were mainly affected by ventral and/or lateral tail flexure coupled with abnormal gut coiling. Histopathological diagnosis displayed abnormal myotomes and myocytes with marked hypertrophies localized at the cell extremity, probably due to a break away of myofibril extremities at the intersomitic junction level. We speculate that this muscular damage was related to inhibition of acetylcholinesterase that showed a clear concentration-response in CPF and MTN exposed larvae. The teratogenic effects of these anti-cholinesterase compounds on Xenopus laevis myogenesis suggest a possible role played by OPs on induction of congenital muscular dystrophy.