Biofilm formation on polyethylene microplastics and their role as transfer vector of emerging organic pollutants.

Microplastic (MP)-colonizing microorganisms are important links for the potential impacts on environmental, health, and biochemical circulation in various ecosystems but are not yet well understood. In addition, biofilms serve as bioindicators for the evaluation of pollutant effects on ecosystems. This study describes the ability of three polyethylene-type microplastics, white (W-), blue (B-), and fluorescent blue (FB-) MPs, to support microbial colonization of Pseudomonas aeruginosa, the effect of mixed organic contaminants (OCs: amoxicillin, ibuprofen, sertraline, and simazine) on plastic-associated biofilms, and the role of biofilms as transfer vectors of such emerging pollutants. Our results showed that P. aeruginosa had a strong ability to produce biofilms on MPs, although the protein amount of biomass formed on FB-MP was 1.6- and 2.4-fold higher than that on B- and W-MP, respectively. When OCs were present in the culture medium, a decrease in cell viability was observed in the W-MP biofilm (65.0%), although a general impairing effect of OCs on biofilm formation was ruled out. Microbial colonization influenced the ability of MPs to accumulate OCs, which was higher for FB-MP. In particular, the sorption of amoxicillin was lower for all bacterial-colonized MPs than for the bare MPs. Moreover, we analysed oxidative stress production to assess the impact of MPs or MPs/OCs on biofilm development. The exposure of biofilms to OCs induced an adaptive stress response reflected in the upregulation of the katB gene and ROS production, particularly on B- and FB-MP. This study improves our understanding of MP biofilm formation, which modifies the ability of MPs to interact with some organic pollutants. However, such pollutants could hinder microbial colonization through oxidative stress production, and thus, considering the key role of biofilms in biogeochemical cycles or plastic degradation, the co-occurrence of MPs/OCs should be considered to assess the potential risks of MPs in the environment.

Transcriptomic and physiological effects of polyethylene microplastics on Zea mays seedlings and their role as a vector for organic pollutants.

The widespread employment of plastics in recent decades has resulted in the accumulation of plastic residues in all ecosystems. Their presence and degradation into small particles such as microplastics (MPs) may have a negative effect on plant development and therefore on crop production. In this study, the effects of two types of polyethylene MPs on Zea mays seedlings cultured in vitro were analysed. In addition, four organic pollutants (ibuprofen, simazine, sertraline, and amoxicillin) were adsorbed by the MPs to evaluate their capacity as other contaminant vectors. The development of the plants was negatively affected by MPs alone or with the organic compounds. The strongest effect was observed in the W-MPs treatments, with a reduction in leaf and root length near 70%. Chlorophyll content was also differentially affected depending on the treatment. Transcriptome analysis showed that MPs affected gene expression in the roots of maize seedlings. As observed in the physiological parameters analysed, some gene expression changes were associated with specific treatments, such as changes in sugar transport genes in the B-MIX treatment. These results contribute to a better understanding of the molecular mechanisms of plants in regard to plastic stress responses.

Copper and Chromium toxicity is mediated by oxidative stress in Caenorhabditis elegans: The use of nanoparticles as an immobilization strategy.

Environmental contamination by heavy metals (HMs) has impelled searching for stabilization strategies, where the use of zero-valent iron nanoparticles (nZVI) is considered a promising option. We have evaluated the combined effect of Cu(II)-Cr(VI) on two Caenorhabditis elegans strains (N2 and RB1072 sod-2 mutant) in aqueous solutions and in a standard soil, prior and after treatment with nZVI (5% w/w). The results showed that HMs aqueous solutions had an intense toxic effect on both strains. Production of reactive oxygen species and enhanced expression of the heat shock protein Hsp-16.2 was observed, indicating increased HM-mediated oxidative stress. Toxic effects of HM-polluted soil on worms were higher for sod-2 mutant than for N2 strain. However, nZVI treatment significantly diminished all these effects. Our findings highlighted C. elegans as a sensitive indicator for HMs pollution and its usefulness to assess the efficiency of the nanoremediation strategy to decrease the toxicity of Cu(II)-Cr(VI) polluted environments.

Assessing the role of polyethylene microplastics as a vector for organic pollutants in soil: Ecotoxicological and molecular approaches.

Microplastics (MPs), pharmaceuticals and pesticides are emerging pollutants with proposed negative impacts on the environment. Rising interest in investigations of MPs is likely related to their potential to accumulate in agricultural systems as the base of the food chain. We applied an integrated approach using classic bioassays and molecular methods to evaluate the impact associated with a mixture of three types of polyethylene (PE) microbeads, namely, white (W), blue (B), and fluorescent blue (FB), and their interactions with pollutants (OCs), including ibuprofen (IB), sertraline (STR), amoxicillin (AMX) and simazine (SZ), on different soil organisms. PE-MPs exhibited different abilities for the adsorption of each OC; W selectively adsorbed higher amounts of SZ, whereas B and FB preferably retained AMX. Standard soil was artificially contaminated with OCs and MPs (alone or combined with OCs) and incubated for 30 days. The presence of MPs or MPs and OCs (MIX) in soil did not produce any effect on Caenorhabditis elegans endpoint growth, reproduction, or survival. Inhibition of leaf growth in Zea mays was detected, but this negative effect declined over time, while the inhibition of root growth increased, especially when OCs (32%) or MIX (47%) were added. Moreover, the expression of the antioxidant genes CAT 1, SOD-1A and GST 1 on plants was affected by the treatments studied. The addition of MPs or MIX significantly affected the soil bacterial phylogenetic profile, which selectively enriched members of the bacterial community (particularly Proteobacteria). The predicted functional profiles of MP/MIX samples indicated a potential impact on the carbon and nitrogen cycle within the soil environment. Our results indicate that MPs and their capability to act as pollutant carriers affect soil biota; further studies should be carried out on the bioavailability of OCs adsorbed by microplastics and how long it takes to leach these OCs into different organisms and/or ecosystems.

Prediction of the impact induced by Cd in binary interactions with other divalent metals on wild-type and Cd-resistant strains of Dictyosphaerium chlorelloides.

The metals present in freshwater have a toxic profile with bioaccumulation and are biomagnified along the aquatic food chain. The metals induce high sensitivity in most aquatic organisms, while others, such as some microalgae species, evolve towards resistance. Therefore, this research predicted through the Combination Index method the binary interaction exposed to divalent metals by inhibiting population growth in a Cd-resistant strain (DcRCd100) compared to the wild-type strain (Dc1Mwt) of Dictyosphaerium chlorelloides and evaluate the specific resistance level obtained by DcRCd100 to Cd relative to other divalent metals.The results showed that DcRCd100 presents resistance compared to Dc1Mwt in individual exposure in the order of Fe2+ > Ni2+ > Cd2+ > Co2+ > Zn2+ > Cu2+ > Hg2+ with 50% inhibitory concentration at 72 h of exposure (IC50(72)) values 1253, 644.4, 423, 162.7, 141.3, 35.1, and 9.9 µM, respectively. It induces cross-resistance with high antagonistic rates (Combination Index (CI); CI > > 1) in the Cd/Zn and Cd/Cu. Cd/Ni, its initial response, is antagonistic, and it ends in an additive (CI = 1). DcRCd100 showed a lower resistance in Co, and Cd/Fe resistance was reduced individually. The interaction with Hg increased its resistance ten times more than individually.This research highlights the use of the CI as a highly efficient prediction method of the binary metal interactions in wild-type and Cd-resistant strains of D. chlorelloides. It may have the potential for metal accumulation, allowing the development of new methods of bioremediation of metals in effluents, and to monitor the concentration of metals in wastewater, its relative availability, transport, and mechanisms on resistant strains of microalgae.

Bioassays to assess the ecotoxicological impact of polyethylene microplastics and two organic pollutants, simazine and ibuprofen.

Research on the environmental impact of plastics, especially on the effect of microplastics (MPs), has become a priority issue in recent years, mainly in terrestrial ecosystems where there is a lack of studies. This work aims to assess the impact of two types of polyethylene MPs, white microbeads (W) and fluorescent blue microbeads (FB), and their interactions with two contaminants, ibuprofen (Ib) and simazine (Sz), on different organisms. A set of bioassays for Vibrio fischeri, Caenorhabditis elegans and Lactuca sativa was carried out, which helped to establish the ecotoxicological impact of those pollutants. C. elegans showed the least sensitivity, while V. fischeri and L. sativa showed a high toxicological response to MPs alone. We found that W and FB induced an inhibition of 27% and 5.79%, respectively, in V. fischeri, and the growth inhibition rates were near 70% in L. sativa for both MPs. MPs exhibited a potential role as contaminant vectors in V. fischeri since the inhibition caused by W-Ib or W-Sz complexes was near 39%. The W-Sz complex significantly reduced leaf development in L. sativa, and a reduction of 30% in seed germination was detected when the complex FB-Sz was tested. This study reveals the importance of designing a complete set of analyses with organisms from different trophic levels, considering the great variability in the effects of MPs and the high number of relevant factors.

Evaluation of nanoremediation strategy in a Pb, Zn and Cd contaminated soil.

We addressed the efficiency of a nanoremediation strategy using zero-valent iron nanoparticles (nZVI), in a case of co-mingled heavy metals (HM) pollution (Pb, Cd and Zn). We applied a combined set of physical-chemical, toxicological and molecular analyses to assess the effectiveness and ecosafety of nZVI (5% w/w) for environmental restoration. After 120 days, nZVI showed immobilization capacity for Pb (20%), it was scarcely effective for Zn (8%) and negligibly effective for Cd. The HMs immobilization in the nZVI treated soils (compared to control soil), reaches its maximum after 15 days (T3) as reflected in the decrease of HM toxicity towards V. fischeri. The overall abundance of the microbial community was similar in both sets of samples during all experiment, although an increase in the number of metabolically active bacteria was recorded 15 days post treatment. We studied the induced impact of nanoremediation on the soil microbial community structure by Next Generation Sequencing (NGS). Even when higher HM immobilization was recorded, no significant recovery of the microbial community structure was found in nZVI-treated soil. The most marked nZVI-induced structural shifts were observed at T3 (increase in the Firmicutes population with a decrease in Gram-negative bacteria). Predictive metagenomic analysis using PICRUSt showed differences among the predicted metagenomes of nZVI-treated and control soils. At T3 we found decrease in detoxification-related proteins or over-representation of germination-related proteins; after 120 days of nZVI exposure, higher abundance of proteins involved in regulation of cellular processes or sporulation-related proteins was detected. This study highlights the partial effectiveness of nanoremediation in multiple-metal contaminated soil in the short term. The apparent lack of recovery of biodiversity after application of nZVI and the decreased effectiveness of nanoremediation over time must be carefully considered to validate this technology when assurance of medium- to long-term immobilization of HMs is required.

Coffee Silverskin Extract: Nutritional Value, Safety and Effect on Key Biological Functions.

This study aimed to complete the scientific basis for the validation of a coffee silverskin extract (CSE) as a novel food ingredient according to European legislation. Nutritional value, safety, effects on biochemical biomarkers and excretion of short chain fatty acids (SCFAs) in vivo of CSE were assessed. Proteins, amino acids, fat, fatty acids, fiber, simple sugars and micronutrients were analyzed. For the first time, toxicological and physiological effects were evaluated in vivo by a repeated-dose study in healthy Wistar rats. Hormone secretion, antioxidant (enzymatic and no-enzymatic) and anti-inflammatory biomarkers, and dietary fiber fermentability of CSE (analysis of SCFAs in feces) were studied in biological samples. This unique research confirms the feasibility of CSE as a human dietary supplement with several nutrition claims: "source of proteins (16%), potassium, magnesium, calcium and vitamin C, low in fat (0.44%) and high in fiber (22%)". This is the first report demonstrating that its oral administration (1 g/kg) for 28 days is innocuous. Hormone secretion, antioxidant or anti-inflammatory biomarkers were not affected in heathy animals. Total SCFAs derived from CSE fiber fermentation were significantly higher (p < 0.05) in male treated rats compared to male control rats. All the new information pinpoints CSE as a natural, sustainable and safe food ingredient containing fermentable fiber able to produce SCFAs with beneficial effects on gut microbiota.

Bioaccesibility, Metabolism, and Excretion of Lipids Composing Spent Coffee Grounds.

The bioaccessibility, metabolism, and excretion of lipids composing spent coffee grounds (SCGs) were investigated. An analysis of mycotoxins and an acute toxicity study in rats were performed for safety evaluation. Total fat, fatty acids, and diterpenes (cafestol and kahweol) were determined in SCGs and their digests obtained in vitro. A pilot repeated intake study was carried out in Wistar rats using a dose of 1 g SCGs/kg b.w. for 28 days. Fat metabolism was evaluated by analysis of total fat, cholesterol, and histology in liver. The dietary fiber effect of SCGs was measured radiographically. The absence of mycotoxins and toxicity was reported in SCGs. A total of 77% of unsaturated fatty acids and low amounts of kahweol (7.09 µg/g) and cafestol (414.39 µg/g) were bioaccessible after in vitro digestion. A significantly lower (p < 0.1) accumulation of lipids in the liver and a higher excretion of these in feces was found in rats treated with SCGs for 28 days. No lipid droplets or liver damage were observed by histology. SCGs acutely accelerated intestinal motility in rats. SCGs might be considered a sustainable, safe, and healthy food ingredient with potential for preventing hepatic steatosis due to their effect as dietary fiber with a high fat-holding capacity.

Morphological and physiological changes exhibited by a Cd-resistant Dictyosphaerium chlorelloides strain and its cadmium removal capacity.

Changes induced on freshwater microalga Dictyosphaerium chlorelloides (Dc(wt)) acclimated in the laboratory until their survival in culture media enriched with cadmium 100 µM have been studied. Cadmium removal by living cells of this Cd-resistant (Dc(CdR100)) strain was tested in cultures exposed to 100 µM Cd during 30 days. Cell dimensions were measured under light microscopy, and cell growth was studied. Photosynthetic yield (ΦPSII) was analyzed and the photosynthetic oxygen development and respiration response was obtained. Results show that Dc(CdR100) strain exhibited significant cell morphology changes in comparison to Dc(wt) cells, which affected both surface area and cell biovolume. Malthusian fitness analysis showed that Dc(CdR100) strain living in Cd-enriched culture had developed a lower capacity of nearly 50% growth, and its photosynthetic oxygen development and respiration response were significantly reduced in both light and dark photosynthetic phases. Dc(CdR100) strain showed a very high capacity to remove cadmium from the aquatic environment (over 90%), although most of the removed heavy metal (≈70%) is adhered to the cell wall. These specific characteristics of Dc(CdR100) cells suggest the possibility of using this strain in conjunction with Dc(wt) strain as bioelements into a dual-head biosensor, and in bioremediation processes on freshwater polluted with Cd.

Influence of pH on the survival of Dictyosphaerium chlorelloides populations living in aquatic environments highly contaminated with chromium.

The accommodation of photosynthetic organisms to adverse conditions, such as pH changes in the aquatic environment, and their response to aquatic pollutants is essential to develop future biosensors. The present study reports the ability of both Cr(VI)-sensitive and tolerant Dyctiosphaerium chlorelloides strains to live in aqueous solutions highly contaminated with hexavalent chromium under varying ranges of pH, by the determination of chromium toxic effects on these strains. Studies of cell growth, photosynthetic quantum yield and gross photosynthesis rate show that both D. chlorelloides strains are able to survive in alkaline and moderately acidified (pH 4.25) aquatic environments. Below this pH value cell populations from both strains exposed for short periods of time to Cr(VI) showed alterations in the three parameters studied. There were no significant differences comparing the response of both strains at pH change in the culture medium. However, Cr(VI)-tolerant strain exhibits a better fit to maintain cell growth than Cr(VI)-sensitive strain when both were subjected to pH 4.25 in the culture medium. The absence of significant differences in photosynthetic activity results for both strains suggests that the lower sensitivity exhibited by Cr(VI)-tolerant strain would be due to cellular morphological changes rather than changes in cellular activity.

Assessment of genotoxic effects induced by selected pesticides on RTG-2 fish cells by means of a modified fast micromethod assay.

The cytotoxic and genotoxic effects induced by alachlor and dichlorvos, two pesticides broadly used, were studied on RTG-2 fish cell line. As measure of cytotoxicity, neutral red assay was used to determine the cellular viability. Toxicity ranking based on IC(50) values found that alachlor was more cytotoxic than dichlorvos. DNA damage has been evaluated on RTG-2 cultures by means of an in vitro assay based on the ability of PicoGreen fluorochrome to interact preferentially with double-stranded DNA (dsDNA), and the results indicated that alachlor induced DNA strand breaks at concentrations above 1.52 µg/mL, equivalent to 1/50-EC(50(48)) , whereas exposures to dichlorvos induced DNA damage only at the maximal concentrations tested 25 µg/mL (1/10-EC(50(48)) ). These results confirm the suitability of this method for the screening of genotoxic effects of this type of aquatic pollutants, and we suggest their use in hazard assessment for environmental risk procedures.

Toxicity of betulin derivatives and in vitro effect on promastigotes and amastigotes of Leishmania infantum and L. donovani.

The toxicity and antileishmanial activity of 20 betulin derivatives were studied. The toxicity of betulin and synthesized compounds was determined using a bacterial test (Microtox) and two mammalian cell lines (CHO-K1 and J774). The antileishmanial activity of compounds (50 µM) was examined in both the promastigote and intracellular amastigote stages of Leishmania infantum and L. donovani. No correlation was found among the toxicity tests. All the compounds showed significant antipromastigote activity. The antiproliferative capacity of derivatives was dependent on the parasite stage studied, and no substantial differences were found between Leishmania species. Betulin, 3,28-di-O-acetylbetulin and L-aspartyl amide of betulonic acid showed moderate activity against amastigotes. The highest inhibition of intracellular amastigote multiplication was achieved with a low micromolar concentration (IC(50) ca 9 µM) of heterocyclic betulin derivative 3,28-di-O-acetyllup-13(18)-ene with N-ethyltriazolo moiety 16, without significant toxicity for mammalian cells. These results point to the interest of this lead compound for further in vitro and in vivo tests.

GENETIC ADAPTATION AND ACCLIMATION OF PHYTOPLANKTON ALONG A STRESS GRADIENT IN THE EXTREME WATERS OF THE AGRIO RIVER-CAVIAHUE LAKE (ARGENTINA)(1).

We tested if different adaptation strategies were linked to a stress gradient in phytoplankton cells. For this purpose, we studied the adaptation and acclimation of Dictyosphaerium chlorelloides (Naumann) Komárek et Perman (Chlorophyta) and Microcystis aeruginosa (Kütz.) Kütz. (Cyanobacteria) to different water samples (from extremely acid, metal-rich water to moderate stressful conditions) of the Agrio River-Caviahue Lake system (Neuquén, Argentina). Both experimental strains were isolated from pristine, slightly alkaline waters. To distinguish between physiological acclimation and genetic adaptation (an adaptive evolution event), a modified Luria-Delbrück fluctuation analysis was carried out with both species by using as selective agent sample waters from different points along the stress gradient. M. aeruginosa did not acclimate to any of the waters tested from different points along the stress gradient nor did D. chlorelloides to the two most acidic and metal-rich waters. However, D. chlorelloides proliferated by rapid genetic adaptation, as the consequence of a single mutation (5.4 × 10(-7) resistant mutants per cell per division) at one locus, in less extreme water and also by acclimation in the least extreme water. It is hypothesized that the stress gradient resulted in different strategies of adaptation in phytoplankton cells from nonextreme waters. Thus, very extreme conditions were lethal for both organisms, but as stressful conditions decreased, adaptation of D. chlorelloides cells was possible by the selection of resistant mutants, and in less extreme conditions, by acclimation.

Toxicity and adaptation of Dictyosphaerium chlorelloides to extreme chromium contamination.

Metals are often spilled by industries into inland water environments, with adverse consequences. Numerous papers have reported that heavy metals produce massive destruction of algae. Nevertheless, algal populations seem to become tolerant when they have had previous exposures to heavy metals. Because the mechanisms allowing heavy metal tolerance of algae are not yet known, the present study analyzed the effect of hexavalent chromium on growth and photosynthetic performance of Dictyosphaerium chlorelloides, stressing on the adaptation mechanisms to chromium contamination. Growth and photosynthetic performance of algal cells were inhibited by Cr(VI) at 10 mg/L, and the 72-h median inhibition concentration was established as 1.64 and 1.54 mg/L, respectively. However, after further incubation for a three month period in an environment with 25 mg/L of chromium, some rare, chromium-resistant cells occasionally were found. A Luria-Delbrück fluctuation analysis was performed to distinguish between resistant algae arising from rare, spontaneous mutations and resistant algae arising from physiological adaptation and other adaptive mechanisms. Resistant cells arose only by spontaneous mutations before the addition of chromium, with a rate of 1.77 x 10(-6) mutants per cell division. From a practical point of view, the use of both chromium-sensitive and chromium-resistant genotypes could make possible a specific algal biosensor for chromium.

Use of a microbial toxicity test (Microtox) to determine the toxigenicity of Aspergillus fumigatus strains isolated from different sources.

The toxic activity of Aspergillus fumigatus is attributable to substances secreted by its cells. Specific toxic compounds synthesized by the fungi such as gliotoxin, can be detected by sensitive chemical procedures like TLC or HPLC. Measuring the total toxigenicity of a strain extract, however, requires a bioassay. In the present study, we evaluated the possibility of using the Microtox bioassay to determine the toxigenicity of A. fumigatus, using 32 strains from different sources. The Microtox method is based on the ability of Vibrio fischeri to produce luminescence, and their sensitivity to toxins. A. fumigatus strains, grouped according to their original sources, showed differences in toxigenicity. Strains isolated from invasive aspergillosis patients proved to be more toxigenic than environmental strains, or strains from colonized patients. Since the strains that were more toxigenic were isolated from sick patients, it is not surprising they showed more virulence than the other strains, and as expected, virulence could be correlated with high toxigenicity. The Microtox bioassay could be a useful tool in the study of toxigenicity of the mycelial fungi and their possible pathogenic roles, and for rapid assessment of secreted toxic compounds.

Protective effect induced by atropine, carbamates, and 2-pyridine aldoxime methoiodide Artemia salina larvae exposed to fonofos and phosphamidon.

The acute toxicity of fonofos and phosphamidon on three age classes of Artemia salina was evaluated. An increase in toxicity of these organophosphorous (OP) insecticides was found following longer development of A. salina. The effects of pretreatment with the nonselective muscarinic antagonist atropine, the two reversible acetylcholinesterease inhibitors physostigmine and pyridostigmine, and the cholinesterase-reactivating oxime 2-pyridine aldoxime methoiodide (2-PAM), as individual and combined pretreatments, on OP-induced lethality in 24 h Artemia were also investigated. The lethal action of both OP insecticides was prevented by pretreatment of 24 h Artemia with atropine and 2-PAM, while physostigmine proved ineffective against intoxication with both OP insecticides and pyridostigmine exhibited a low synergic effect. In both cases, the inhibitory effects of combinations of atropine (10(-5)M) plus 2-PAM were greater than those elicited by either drug alone, with the maximum protection afforded being 100%. Combined pretreatment of atropine (10(-5)M) plus physostigmine practically abolished the lethal effects induced by both insecticides. Pretreatment with 2-PAM (10(-6)M) plus physostigmine afforded maximal protection of 100% and 76% on the lethality induced by fonofos and phosphamidon, respectively. The data obtained suggest that the combination of atropine plus 2-PAM or physostigmine and the combined pretreatment of 2-PAM plus physostigmine are effective in the prevention of the lethal effects induced by fonofos and phosphamidon in A. salina larvae.

Effects of selected biocides used in the disinfection of cooling towers on toxicity and bioaccumulation in Artemia larvae.

This paper reports the acute toxicity of three biocides used in the disinfection of cooling towers, tetrakis(hydroxymethyl) phosphonium chloride (THPC), trichloroisocyanuric acid (TCIC), and sodium bromide (NaBr), on Artemia larvae, their effect on the phototactic response of this organism, and the potential of bioaccumulation in this species. The 24-h median lethal concentration (LC50) values for these biocides with respect to 24-, 48-, and 72-h-old Artemia, determined by static bioassays, showed the following rank order of toxicity: THPC < TCIC < NaBr. An age-dependent increase in sensitivity was seen for each compound. All three biocides reduced the phototactic response of 24-h-old Artemia larvae in 24-h static bioassays; the median inhibitory concentration ratios obtained were 30 to 40 times lower than their respective 24-h LC50 values. The results suggest that phototaxis bioassays could provide the speed and simplicity required for screening many potential pollutants for harmful effects. The bioconcentration factors obtained for Artemia larvae exposed to 10% LC50 for 168 h in renewal assays were 93.75, 1.67, and 0.23 for THPC, TCIC, and NaBr, respectively. This shows these biocides pose no bioaccumulation risk in this organism, although the value of 93.75 obtained for THPC is close to the threshold above which such a risk is considered to exist.

Influence of water hardening of the chorion on cadmium accumulation in medaka (Oryzias latipes) eggs.

This report describes a study in which in vitro fertilization methods were used to expose medaka (Oryzias latipes) eggs to cadmium (Cd(2+)). This approach was applied to address the differential sensitivity and cumulative potential of Cd(2+) when exposure was initiated early (before fertilization and water hardening of the chorion) versus later during embryo development (i.e., well after the chorion has undergone water hardening). Following range finding exposures (2.5, 10, 20, 40 or 80 mg/l) under artificially controlled experimental procedures, results from hatching success and embryo malformations showed the earlier exposure interval more sensitive than the assay involving only the embryonated egg. Subsequent accumulation studies have shown that the exposure initiated before fertilization apparently led to more Cd(2+) deposition in the chorion compared to the exposure during embryonated stages of the eggs. Similarly, values for total Cd(2+) indicated higher concentrations in those eggs exposed prior to--and during--water hardening. Results suggest an alteration of the properties of the zona radiata in the early-stage eggs, making it more permeable to the potential exit or entrance of waterborne agents even after water hardening. Ongoing studies must now address the development of more realistic exposure conditions of the gametes by using incubation media with osmolarities similar to surface waters, and by shortening duration for gamete exposure. Also, sensitive methods to localize Cd(2+) and to delineate the transfer from the chorion to the embryo are needed.