Effects of malathion and carbendazim on Amazonian freshwater organisms: comparison of tropical and temperate species sensitivity distributions

The risk assessment of pesticides for freshwater ecosystems in the Amazon has relied on the use of toxicity data and water quality criteria derived for temperate regions due to a lack of ecotoxicological studies performed with indigenous species. This leaves an unknown margin of uncertainty for the protection of Amazonian ecosystems, as differences in environmental conditions and species sensitivity are not taken into account. To address this issue, the acute toxic effects of malathion (an organophosphorus insecticide) and carbendazim (a benzimidazole fungicide) were assessed on five fish and five freshwater invertebrates endemic to the Amazonian region. Subsequently, the intrinsic sensitivity of Amazonian and temperate freshwater species was compared using the species sensitivity distribution (SSD) concept. Amazonian species sensitivity to malathion was found to be similar to that of their temperate counterparts, with LC50 values ranging between 111 and 1507 μg/l for fish species and 2.1–426 μg/l for arthropod species. However, Amazonian fish appeared to be slightly less sensitive for carbendazim than temperate fish with LC50 values ranging between 1648 and 4238 μg/l, and Amazonian invertebrates were found to be significantly more resistant than their temperate counterparts, with LC50 values higher than 16000 μg/l. The results of this study suggest that for these compounds, the use of water quality criteria derived with laboratory toxicity data for temperate species will result in a sufficient protection level for Amazonian freshwater organisms. Recommendations for further research include the validation of threshold concentrations derived with temperate standard test species and with the SSD model with semi-field experiments considering larger assemblages of indigenous species under local environmental conditions.     

An assessment of the potential of the microbial assay for risk assessment (MARA) for ecotoxicological testing

Rapid microscale toxicity tests make it possible to screen large numbers of compounds and greatly simplify toxicity identification evaluation and other effect directed chemical analyses of effluents or environmental samples. Tests using Vibrio fischeri (such as Microtox?) detect toxicants that cause non-specific narcosis, but are insensitive to other important classes of contaminants. The microbial assay for risk assessment (MARA) is a 24 h multi-species test that seeks to address this problem by using a battery of ten bacteria and a fungus. But there has been little independent evaluation of this test, and there is no published information on its sensitivity to pesticides. Here, we assess the performance of MARA using a range of toxicants including reference chemicals, fungicides and environmental samples. Mean MARA microbial toxic concentrations and IC₂₀s (20% Inhibitory concentrations) indicate the toxicant concentrations affecting the more sensitive micro-organisms, while the mean IC₅₀ (50% Inhibitory concentration) was found to be the concentration that was toxic to most MARA species. For the two fungicides tested, the yeast (Pichia anomalia) was the most sensitive of the ten MARA species, and was more sensitive than the nine other yeasts tested. The test may be particularly valuable for work with fungicides. Mean MARA IC₅₀s were comparable to values for nine other yeast species and the lowest individual IC₅₀s for each toxicant were comparable to reported IC₅₀s for Daphnia magna, Selenastrum capricornutum and Microtox? bioassays. MARA organisms exhibited more variable sensitivities, with the most sensitive organism being different for different samples, enhancing the likelihood of toxicity detection and giving a toxicity “fingerprint” that may help identify toxicants. The test, therefore, has great potential and would be valuable for ecotoxicological testing of pollutants.     

Effects of Cd,Co, Cu,Ni and Zn on asexual reproduction and early development of the tropical sea anemone Aiptasia pulchella

Currently few studies present sub-lethal toxicity data for tropical marine species, and there are no routine toxicity tests using marine cnidarians. The symbiotic sea anemone Aiptasia pulchella has been identified as a useful species for ecotoxicological risk assessment, and would provide a tropical marine cnidarian representative. Chronic sub-lethal toxicity tests assessing the effects of 28-day trace metal exposure on asexual reproduction in A. pulchella were investigated, and concentration-dependant reductions in the number of offspring that were produced were evident for all metal exposures. Metal concentration estimates causing 50 % reductions in the numbers of asexually-reproduced juveniles after 28-day exposures (28-day effect concentrations 50 %: EC50s) were 14 µg/L for copper, 63 µg/L for zinc, 107 µg/L for cobalt, 145 µg/L for cadmium, and 369 µg/L for nickel. Slightly higher 28-day EC50s of 16 µg/L for copper, 192 µg/L for zinc, 172 µg/L for cobalt, 185 µg/L for cadmium, and 404 µg/L for nickel exposures and were estimated based on reductions in the total number of live developed and undeveloped offspring. These sensitive and chronic sub-lethal toxicity estimates help fill the knowledge gap related to metal effects on cnidarians over longer exposure periods, and this newly-developed bioassay may provide a much needed tool for ecotoxicological risk assessment relevant to tropical marine environments.     

Toxicity assessment of sediments containing tributyltin around Hong Kong harbour

Rather high levels of tributyltin (TBT) in sediments were found in certain areas around the Hong Kong coastline. Sediments from two contaminated sites (Tsing Yi and Yam O) and two clean sites were used to study the release of TBT contaminants from resuspended sediment to the water column by elutriate tests. The chemical analysis data of elutriates were compared with the effective or lethal concentrations from the toxicity tests of spiked solution using marine microalga (Dunaliella tertiolecta) and benthic invertebrate (Melita koreana) to assess the potential ecotoxicological impacts arising from the dredging work. Concentrations of TBT in filtered sediment elutriates were mostly below 0.002 microg/l which suggested that very little of TBT was released in soluble form. The lethal toxicity of TBT for the invertebrate expressing as 10 day-LC(50) was 2.31 microg/l for TBT and the alga expressing as 48 h-EC(50) was 0.46 microg/l. The results of this evaluation demonstrated that the release of TBT from two contaminated sites during dredging was unlikely to attain the effective or lethal concentrations on the two test species.     

Olive oil mill wastewaters before and after treatment: a critical review from the ecotoxicological point of view

The olive oil mill wastewater (OMW) is a problematic and polluting effluent which may degrade the soil and water quality, with critical negative impacts on ecosystems functions and services provided. The main purpose of this review paper is presenting the state of the art of OMW treatments focusing on their efficiency to reduce OMW toxicity, and emphasizing the role of ecotoxicological tests on the evaluation of such efficiency before the up-scale of treatment methodologies being considered. In the majority of research works, the reduction of OMW toxicity is related to the degradation of phenolic compounds (considered as the main responsible for the toxic effects of OMW on seed germination, on bacteria, and on different species of soil and aquatic invertebrates) or the decrease of chemical oxygen demand content, which is not scientifically sound. Batteries of ecotoxicological tests are not applied before and after OMW treatments as they should be, thus leading to knowledge gaps in terms of accurate and real assessment of OMW toxicity. Although the toxicity of OMW is usually high, the evaluation of effects on sub-lethal endpoints, on individual and multispecies test systems, are currently lacking, and the real impacts yielded by its dilution, in freshwater trophic chains of receiving systems can not be assessed. As far as the terrestrial compartment is considered, ecotoxicological data available include tests only with plants and the evaluation of soil microbial parameters, reflecting concerns with the impacts on crops when using OMW for irrigation purposes. The evaluation of its ecotoxicity to other edaphic species were not performed giving rise to a completely lack of knowledge about the consequences of such practice on other soil functions. OMW production is a great environmental problem in Mediterranean countries; hence, engineers, chemists and ecotoxicologists should face this problem together to find an ecologically friend solution.     

Novel probiotic dissolvable carboxymethyl cellulose films as oral health biotherapeutics: in vitro preparation and characterization

Objectives: Oral health is influenced by the mouth's resident microorganisms. Dental caries and periodontitis are oral disorders caused by imbalances in the oral microbiota. Probiotics have potential for the prevention and treatment of oral disorders. Current formulations, including supplements and foods, have limitations for oral delivery including short storage time, low residence time in the mouth, effects on food consistency, and low patient compliance. Oral thin films (OTFs) may be efficient in delivering probiotics to the mouth. This research aims to develop a novel carboxymethyl cellulose (CMC)-probiotic-OTF to deliver probiotics for the treatment/prevention of oral disorders.

Methods: CMC-OTFs were developed with varying CMC concentration (1.25 – 10 mg/mL), weight (5 – 40 g), thickness (16 – 262 μm), hygroscopicity (30.8 – 78.9 mg/cm² film), and dissolving time (135 – 600 s). The 10 g 5 mg/mL CMC-OTF was selected and used to incorporate Lactobacillus fermentum NCIMB 5221 (6.75 × 10⁸ cells/film), a probiotic with anti-inflammatory potential for periodontitis treatment and capable of inhibiting microorganisms responsible for dental caries and oral candidiasis.

Results: The CMC-OTF maintained probiotic viability and antioxidant activity following 150 days of storage with a production of 549.52 ± 26.08 μM Trolox equivalents.

Conclusion: This research shows the successful development and characterization of a novel probiotic-CMC-OTF with potential as an oral health biotherapeutic.     

Toxicity of the organophosphorus defoliant def to rainbow trout (Salmo gairdneri) and channel catfish (Ictalurus punctatus)

Ninety-day partial life cycle toxicity tests were conducted to determine the effects of the organophosphorus defoliant S,S,S-tributyl phosphorotrithioate (DEF), on rainbow trout (Salmo gairdneri) and channel catfish (Ictalurus punctatus). Exposure concentrations were 0, 0.76, 1.2, 1.7, 2.7, 3.6, 6.5, and 9.5 μg/l for rainbow trout and 0, 0.77, 2.0, 4.8, 10.0, and 19.6 μg/l for channel catfish. In rainbow trout growth was reduced by DEF concentrations ≥ 1.2 and survival by concentrations ≥ 3.6 μg/l; growth and survival of channel catfish were reduced by DEF concentrations ≥ 4.8 and 19.6 μg/l, respectively. DEF adversely affected vertebral biochemical composition and mechanical properties of both species. DEF was not highly accumulative in the tissues of either species and was eliminated rapidly when the fish were transferred to contaminant-free water. The determination of safe environmental concentrations of DEF depends partly on the species tested and the biological response measured. Application factors classically used to estimate safe environmental concentrations of contaminants may not be applicable to some of the new-generation organophosphate compounds in relation to cold-water species such as rainbow trout.     

Effects of Substrate Salinity on Early Seedling Survival and Growth of Scirpus robustus Pursh and Spartina alterniflora Loisel

Rooted aquatic plants are being used increasingly to test the toxicity of sediments. However, effects of naturally occurring substrate constituents on most potential test species are not well understood even though their effects could affect the test results. The objective of this study was to determine the effect of substrate salinity (NaCl) on early seedling survival and growth of the emergent macrophytes, Scirpus robustus Pursh and Spartina alterniflora Loisel. Results of four 21- and 28-day toxicity tests, conducted in an artificial sediment, indicated interspecific differences in NaCl sensitivity when based on changes in shoot, root and whole plant dry-weight biomass. Concentrations of 7.8 g NaCl/l and 19.2 g NaCl/l first reduced early seedling biomass of S. robustus and S. alterniflora (P<0.05), respectively, when compared to plants grown in sediment containing no measurable salinity. Seedling survival was not affected at average concentrations of 17.5 g NaCl/l or less for S. robustus and 22.3 g NaCl/l or less for S. alterniflora. The results indicate that substrate salinity is an important consideration in the selection of test species for laboratory phytotoxicity tests conducted with estuarine sediments, particularly if determination of chronic toxicity attributable to anthropogenic contamination is the primary objective.     

Pro-apoptotic effect of fly ash leachates in hepatocytes of freshwater fish (Channa punctata Bloch).

The pro-apoptotic effect of fly ash leachates (FAL) was studied in the hepatocytes of an Indian freshwater fish, Channa punctata Bloch. Hepatocytes were exposed to different concentrations of '7-day' FAL for 24 and 48h and various parameters of apoptosis were studied using standardized procedures. FAL-induced apoptosis in hepatocytes was indicated by cytological examination, DNA fragmentation and DNA laddering. The induction in cytochrome-c release, caspases 3, 7, 10 and 9 activities and lactate dehydrogenase level provide mechanistic platform for FAL-induced apoptosis. Cytological examination showed an unambiguous apoptotic effect of ash leachates in fish hepatocytes. Exposed hepatocytes also showed increased production of H(2)O(2), superoxide ions and an increase in lipid peroxidation (LPO). The present study suggests a possible role of reactive oxygen species (ROS) in FAL-induced apoptosis in hepatocytes. Lactate dehydrogenase, LPO and apoptosis as biomarkers of cytotoxicity have recently been used for assessment of ecotoxicological impact of environmental chemicals. Our findings show that these biomarkers may also be used for evaluation of ecotoxicological impact of complex chemical mixture such as fly ash and its leachates.     

Acute and sub-lethal toxicities of two common pyrithione antifouling biocides to the marine amphipod Elasmopus rapax

This study investigated the acute and sub-lethal toxicities of two widely used antifouling booster biocides, copper pyrithione (CuPT) and zinc pyrithione (ZnPT) to the marine amphipod Elasmopus rapax. Apart from conducting non-standard acute toxicity tests, sublethal effects of the chemical to the amphipods were determined using RNA/DNA ratio and total lipid content as biomarkers for cellular growth and energy reserve, respectively. The 96-h median lethal concentrations (LC50) for CuPT and ZnPT were found to be 11.5 and 21.5 ??g/L, respectively. After 7-d waterborne chemical exposure, their RNA/DNA ratios decreased significantly in a concentration-dependent manner, with median effect concentrations (EC50) being estimated at 4.0 and 5.4 ??g/L for CuPT and ZnPT, respectively. However, their lipid content remained constant regardless to the exposure concentrations. As showed in this study, RNA/DNA ratio is a sensitive toxicity endpoint and can be easily adopted as a fitness-related biomarker in chronic toxicity tests and ecotoxicological studies.     

Toxicity of nitromusks in early lifestages of South African clawed frog (Xenopus laevis) and zebrafish (Danio rerio)

Musk xylene (MX), musk ketone (MK) and musk moskene (MM) are synthetic nitro-containing fragrances. Due to their inherent lipophilicity and environmental persistence, they are frequently detected in environmental samples and especially in aquatic ecosystems. Despite this, the current environmental toxicity database of nitromusks is limited. Although nitromusks have been shown to accumulate in aquatic organisms, little is known about their potential developmental effects in the respective aquatic species. To investigate the developmental toxicity of these compounds to amphibians and fish, early lifestages of xenopus (Xenopus laevis) and zebrafish (Danio rerio) were exposed to three nitromusks for 96 h to examine the developmental effects of these compounds in the two species. Nitromusk body concentration measurements were carried out in parallel for correlation with potential developmental effects. No increased mortality, malformation or growth inhibition was observed in either species following 96-h exposure to 400 microg/l MX, MK and MM. However, an approximately 20% reduced viability was observed in xenopus larvae when exposed to 400 microg/l MX, MK and MM for 11 days. Xenopus and zebrafish exposed to 10, 153, 871 and 1637 microg/l 14C-MX for 96 h resulted in whole-body concentrations of 0.7 +/- 0.1, 11.1 +/- 1.1, 38.7 +/- 1.9 and 76.3 +/- 18.3 microg/g, and 4.3 +/- 0.6, 73.3 +/- 11.8, 440.0 +/- 72.7 and 664.0 +/- 47.7 microg/g wet body weight, respectively. Exposure of xenopus larvae to 400 microg/l MX, MK and MM for 11 days, resulted in whole body concentrations (extrapolated from gas chromatographic determinations) of 4700 +/- 5000, 1300 + 300 and 4600 + 4800 microg/g wet weight for MX, MK and MM, respectively. The latter toxicity results, in conjunction with the fact that the concentrations used for the above experiments were between 400- and 10000-fold higher than those detected in the environment, suggest that environmental concentrations of nitromusks are not hazardous for early lifestages of fish and amphibians.     

Combination of single-species laboratory tests for the assessment of the ecotoxicity of p-Benzoquinone

The combination of 15 single-species laboratory tests (6 bacteria, 1 Dinoflagellate, 5 yeasts, 2 Crustacea, and 1 fish) was used for the assessment of the ecotoxicological effects of p-benzoquinone. Marked differences were observed in the susceptibility among the species since the toxicity values were ranged between 0.020 mg/L (Photobacterium phosphoreum) and >700 mg/L (Candida parapsilosis). These results support the philosophy that for assessing toxicity more thoroughly, a battery of several tests carried out with organisms occupying different trophic levels in the environment is required.     

Influence of pH, light cycle, and temperature on ecotoxicity of four sulfonylurea herbicides towards Lemna gibba

In chemical regulation, e.g. the EU Water Framework Directive, REACH, or the Pesticide Directive, standardized ecotoxicological tests are applied to evaluate and rank the hazard of compounds and for deriving environmental quality standards (EQS). Standardized test methods prescribe fixed testing conditions e.g. specific temperature, pH, light intensity etc. However, environmental conditions under which the organisms live are rarely identical to the standard conditions. Thus, the ecotoxicity of compounds found in standard test is not only a function of the compounds inherent physico-chemical properties but is also affected by test conditions. It is therefore important to study the effect of changes in test conditions in order to get reliable input ecotoxicity data for assessing the potential risk posed by a compound. The objective of this study was to investigate the implications of changing test conditions on the toxicity of four sulfonylurea herbicides (SUs). The toxicity of the four SUs towards Lemna gibba was investigated at three pH levels (6, 7.5 and 9), at two temperatures (15 and 24 °C) and two light regimes (continuous and 12:12 h light:dark cycle) The EC50 increased twofold to tenfold for the four SUs when pH was increased from 6 to 9. Decreasing the temperature from 24 to 15 °C or introducing a dark:light cycle did not cause any trends in changes in toxicity. The results show that test conditions can have an effect on the toxicity and this should be considered when the standard test results are used for derivation of EQS.     

Impact of cadmium on the ecdysteroids production in <Emphasis Type="Italic">Gammarus fossarum</Emphasis>

Gammarus fossarum is an important test organism which is currently used as a bio-indicator as well as in ecotoxicological tests. Nevertheless, data on ecdysteroids in endocrine toxicity test are not yet available for these species, despite its crucial role in molting and reproduction. In the present paper, ecdysteroids concentrations were studied during the molt cycle (in females) and embryonic development in G. fossarum (Crustacea, Amphipoda) in order to propose an ecdysteroids toxicity test. Ecdysteroids levels in G. fossarum showed a single peak during premolt at stage Dl-D2. In embryos, ecdysteroids levels progressively increased over stages 3 and 4, with peak levels at stage 4. A Cadmium toxicity test was proposed to examine if the molting and embryogenesis disturbances previously observed after cadmium exposure (Geffard et al. 2010) could be attributed to changes in ecdysteroids titers. Exposure to the different cadmium concentrations (3; 9; 300; 900 µg/l) increased ecdysteroids secretion by Y-organs in vitro, but it had no significant effect on exposed embryos (in vivo). Based on previous findings, we are led to conclude that the molting impairments in cadmium-exposed females of G. fossarum is connected to the changes in ecdysteroids concentrations.     

The effect of water hardness on the toxicity of uranium to a tropical freshwater alga Chlorella sp

Uranium (U) derived from mining activities is of potential ecotoxicological concern to freshwater biota in tropical northern Australia. Few data are available on the effects of water hardness (Ca and/or Mg), which is elevated in U mine wastewaters, on the toxicity and bioavailability of U to freshwater biota, particularly algae. This study determined the effect of water hardness (8, 40, 100 and 400 mg CaCO(3) x l(-1), added as calcium (Ca) and magnesium (Mg) sulphate) on the toxicity (72 h growth rate inhibition) of U to the unicellular green alga, Chlorella sp., in synthetic freshwater, at constant pH (7.0) and alkalinity (8 mg CaCO(3) x l(-1)), similar in chemical composition to sandy coastal streams in tropical northern Australia. A 50-fold increase in water hardness resulted in a 5-fold decrease (P0.05) differences in the predicted speciation (% distribution) of U amongst the four water hardness levels. The reduction in U toxicity with increasing water hardness was most likely due to competition between U and Ca and/or Mg for binding sites on the algal cell surface. The minimum detectable effect concentrations of U were approximately 3 and 24 times higher (at 8 and 400 mg CaCO(3)x l(-1) hardness, respectively) than the national interim U guideline value (0.5 micro g x l(-1)) for protecting aquatic ecosystems. Overall, the results reinforce the need for a more flexible U guideline based on a hardness-dependent algorithm, which may allow environmental managers to relax the national guideline for U on a site-specific basis.     

Towards a more representative in vitro method for fish ecotoxicology: morphological and biochemical characterisation of three-dimensional spheroidal hepatocytes

The use of fish primary cells and cell lines offer an in vitro alternative for assessment of chemical toxicity and the evaluation of environmental samples in ecotoxicology. However, their uses are not without limitations such as short culture periods and loss of functionality, particularly with primary tissue. While three-dimensional (spheroid) technology is now established for in vitro mammalian toxicity studies, to date it has not been considered for environmental applications in a model aquatic species. In this study we report development of a reproducible six-well plate, gyratory-mediated method for rainbow trout (Oncorhynchus mykiss) hepatocyte spheroid culture and compare their functional and biochemical status with two-dimensional (2D) monolayer hepatocytes. Primary liver spheroid formation was divided into two stages, immature (1–5 days) and mature (≥6 days) according to size, shape and changes in functional and biochemical parameters (protein, glucose, albumin and lactate dehydrogenase). Mature spheroids retained the morphological characteristics (smooth outer surface, tight cell–cell contacts) previously described for mammalian spheroids as demonstrated by light and scanning electron microscopy. Glucose production and albumin synthesis were significantly higher in mature spheroids when compared to conventional 2D monolayer cultures (P < 0.01) and increased as spheroids matured (P < 0.01). Basal lactate dehydrogenase (LDH) leakage significantly decreased during spheroid formation and was significantly lower than 2D cultures (P < 0.01). It is therefore suggested that mature spheroids can maintain a high degree of functional, biochemical and morphological status over-time in culture that is superior to conventional 2D models and can provide realistic organotypic responses in vitro. Trout spheroids that take ~6–8 days to reach maturity would be suitable for use in acute toxicological tests and since it is possible to culture individual spheroids for over a month, there is potential for this work to lead towards in vitro bioaccumulation alternatives and to conduct high throughput screens of chronic exposure. This is an important step forward for developing alternative in vitro tools in future fish ecotoxicological studies.     

Ecotoxicological monitoring of remediation in a coke oven soil

Assessment of contaminated sites is usually based on chemical analyses of hazardous compounds in soil. This is enough either to assess the environmental hazard of contaminated soil nor to evaluate the efficiency of applied remediation techniques. Information on the bioavailability of complex mixtures of xenobiotics and degradation products cannot be provided by chemical analytical data, but results from bioassays can integrate the effects of pollutants in mixtures. In the preservation of human health and environmental quality, it is important to evaluate ecotoxicological effects of contaminated industrial soils to complement the techniques of analytical chemistry. The monitoring of a coke oven soil and the evaluation of the landfarming treatment technique, conducted on-site in a pilot scale installation, was done by a battery of ecotoxicological tests: acute, chronic, and genotoxicity tests. Contaminated soil samples revealed toxic effects for different species mainly due to high concentrations of polycyclic aromatic hydrocarbons (PAHs). After landfarming treatment, soil samples presented significant reduction in toxicity, confirming the effectiveness of the landfarming process pointed out by a significant reduction in low molecular weight PAH concentrations. Comparative analysis of the different ecotoxicological tests allowed the establishment and validation of a more suitable procedure for the monitoring of PAH contaminated soils.     

Ecotoxicological effects at contaminated sites

Contamination sites pose significant environmental hazards for terrestrial and aquatic ecosystems. They are important sources of pollution and may result in ecotoxicological effects on terrestrial, groundwater and aquatic ecosystems. At severely contaminated sites, acute effects occur, but the core problem lies in long-term chronic effects. Ecotoxicological effects occur at all levels of biological organization, from the molecular to the ecosystem level. Not only certain organisms may be affected, but the ecosystems as a whole, both terrestrial and aquatic, in its function and structure. Contaminants at large contaminated sites often share critical properties such as high acute and/or chronic toxicity, high environmental persistence, often high mobility leading to contamination of groundwater, and high lipophilicity leading to bioaccumulation in food webs. Contaminants present at polluted sites occur as mixtures, therefore interactions between individual compounds are of importance. The bioavailability is a key factor for ecotoxicological effects of contaminants. This is demonstrated by a case study on organotins. Organotins belong to the most toxic pollutants known so far for aquatic life. Widespread contamination of harbor sediments occurs globally due to the ongoing use of organotins in antifouling paints in large ships. In lake sediments, tributyl- and triphenyltin are very persistent and bioavailable to biota even after a long time. Bioavailability of these compounds is dependent on pH and organic matter. Organotins are accumulated in sediments, but remobilization occurs when contaminated sediments are disturbed and dredged. A key question in dealing with contaminated sites is the assessment and evaluation of the toxicity of contaminants to the environment. Usually, established OECD tests and whole effluent toxicity tests are performed for an ecotoxicological evaluation and for hazard assessment. However, these assays are often expensive, laborious and sometimes not sensitive enough. As a consequence, we have used fast and reliable in vitro systems such as fish cell lines for the evaluation of sediments and landfill leachates contaminated by polychlorinated hydrocarbons (PAH). Determination of cytotoxicity as a measure for acute toxicity, and induction of cytochrome P4501A (CYP1A) as a biomarker of exposure and toxicity were found to be important measures, which can be used for hazard and risk assessment. We have developed a concept for the ecotoxicological evaluation of PAH contamination based on induction equivalents, which can be applied for aquatic and terrestrial ecosystems. One of the key question and present gaps, however, includes the long-term chronic ecotoxicological effects on soil and aquatic biota, which are largely unknown.     

Application of in-situ bioassays with macrophytes in aquatic mesocosm studies

Aquatic mesocosm studies assess ecotoxicological effects of chemicals by using small artificial ponds as models of lentic ecosystems. In this study, methods of controlled insertion of macrophytes within an outdoor mesocosm study were explored. Although analytically confirmed concentrations of the model herbicide terbuthylazine were high enough to expect direct effects on phytoplankton, functional parameters and dominant taxa abundance indicated only minor and transient effects. In-situ assays with Lemna minor, Myriophyllum spicatum, Potamogeton lucens and Chara globularis revealed adverse effects at concentrations in accordance with literature data. Complex interactions such as nutrient limitation and competition were possible reasons for the observed growth promotion at the lower concentration of about 5 μg/l terbuthylazine. The approach of macrophyte in-situ bioassays within a mesocosm study proved to be applicable. Presumed advantages are simultaneous acquisition of toxicity data for several species of aquatic plants under more realistic conditions compared to laboratory tests and inclusion of macrophytes as important structural and functional components in mesocosms while limiting their domination of the model ecosystem.     

The role of CYP1A inhibition in the embryotoxic interactions between hypoxia and polycyclic aromatic hydrocarbons (PAHs) and PAH mixtures in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>)

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants with elevated concentrations in waters that may also experience hypoxia. Previous research has shown interactions between hypoxia and some PAHs (fluoranthene, α-naphthoflavone) but no interaction with others (benzo[a]pyrene (BaP), β-naphthoflavone). Here we examine how hypoxia (7.4% oxygen, ~35% of normoxia) affects the embryotoxicity of PAHs that act through different mechanisms and the role that CYP1A inhibition may play in these interactions. About 500 μg/l BaP and 1–200 μg/l benzo[k]fluoranthene (BkF) interacted synergistically with hypoxia to induce pericardial edema in developing zebrafish (Danio rerio). Hypoxia protected from the embryotoxicity of pyrene (PY) and had no effect on the toxicity of polychlorinated biphenyl-126. Despite previous reports of other CYP1A inhibitors interacting with hypoxia, up to 2,000 μg/l dibenzothiophene, 2-aminoanthracene (AA), and carbazole (CB) all failed to induce embryotoxicity under normoxic or hypoxic conditions. The toxicity of PAH mixtures—including binary mixtures of BaP/AA and BaP/CB and two environmentally relevant, complex mixtures—were exacerbated severely by hypoxia to induce or worsen pericardial edema and cause mortality. The interactions between hypoxia and BkF and PY were closely mimicked by morpholino knockdown of CYP1A, indicating a potential role for metabolism of these compounds in their toxicity. Our results indicate that various PAHs may exhibit synergistic, antagonistic or additive toxicity with hypoxia. The enhanced toxicity of environmental mixtures of PAHs under hypoxia suggests that risk assessments that do not take into account potential interactions with hypoxia may underestimate the threat of PAHs to fish in contaminated sites.