Environmental risk assessment of perfluoroalkyl substances and halogenated flame retardants released from biosolids-amended soils.

Biosolid application is considered a sustainable management tool as it positively contributes to recycle nutrients and to improve soil properties and fertility. Nevertheless, this waste management technique involves an important input source of emerging organic pollutants in soil. To evaluate the environmental potential risk related to perfluoroalkyl substances (PFASs) and halogenated flame retardants (HFRs) due to the biosolid application to soil, a quantitative ecotoxicological risk assessment was conducted. The analyte concentrations were employed to perform an estimation of the exposure levels to contaminants in the receiving media, defining predicted environmental concentrations (PECs) for terrestrial and aquatic compartments (PECsoil, PECwater, PECsed) and for secondary poisoning via the terrestrial and aquatic food chain (PECoral, predator (T), PECoral, predator (Aq)). The risk characterization ratios (RCRs) were calculated based in the comparison of the PEC values obtained with concentrations with no effect (PNECs) on terrestrial and aquatic ecosystems. Based on the chosen scenarios and experimental conditions, no environmental risk of PFASs and HFRs released from biosolid amended soils to different environmental compartments was detected (RCRsoil, RCRoral, worm, RCRwater, RCRsed and RCRoral, fish were below 1 in all cases). Besides, the potential health risk of PFASs and HFRs to local people who live in the scenario studied and are fed on horticultural crops grown in biosolid amended soil was also below 1, indicating that the risk is not considered significant to human health in the conditions studied. This approach provides a first insight of the risks relative to biosolid amendments to further research based on fieldwork risk assessment.

Effects of dietary 2,2', 4,4'-tetrabromodiphenyl ether (BDE-47) exposure on medaka (Oryzias latipes) swimming behavior.

A diet fortified with 2,2', 4,4'-tetrabromodiphenyl ether (BDE-47: 0, 10, 100, and 1000 ng/g) was dosed to 4-7-day-old post-hatch medaka fish for 40 days to evaluate the effects on the swimming activity of fish using a miniaturized swimming flume. Chlorpyrifos (CF)-exposed fish were selected as the positive control to assess the validity and sensitivity of the behavioral findings. After 20 and 40 days of exposure, the locomotor activity was analyzed for 6 min in a flume section (arena). The CF positive control for each time point were fish exposed to 50 ng CF/ml for 48 h. Swimming patterns, presented as two-dimensional heat maps of fish movement and positioning, were obtained by geostatistical analyses. The heat maps of the control groups at time point 20 revealed visually comparable swimming patterns to those of the BDE-47-treated groups. For the comparative fish positioning analysis, both the arenas were divided into 15 proportional areas. No statistical differences were found between residence times in the areas from the control groups and those from the BDE-47-treated groups. At time point 40, the heat map overall patterns of the control groups differed visually from that of the 100-ng BDE-47/g-treated group, but a comparative analysis of the residence times in the corresponding 15 areas did not reveal consistent differences. The relative distances traveled by the control and treated groups at time points 20 and 40 were also comparable. The heat maps of CF-treated fish at both time points showed contrasting swim patterns with respect to those of the controls. These differential patterns were statistically supported with differences in the residence times for different areas. The relative distances traveled by the CF-treated fish were also significantly shorter. These results confirm the validity of the experimental design and indicate that a dietary BDE-47 exposure does not affect forced swimming in medaka at growing stages.

Bioaccumulation, maternal transfer and effects of dietary 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) exposure on medaka fish (Oryzias latipes) offspring.

A previous study conducted in our laboratory with growing medaka (Oryzias latipes) showed the capacity of BDE-47 (10-1000ng/g) to bioaccumulate during a 40-day oral exposure. However, the results did not provide evidence for effects during or after the exposure period. In this study, breeding medakas were fed a diet for 40days that contained 1000ng of BDE-47/g. At predefined time points, females (time points 10, 20, 30 and 40), males (time points 30 and 40) and pools of laid eggs (time points 10, 20, 30 and 40) were sampled and collected for: 1) the BDE-47 quantitative analysis in adults in the <24-h-old post-fertilization (hpf) embryos, and in the <24-h-old post-hatch (hph) eleutheroembryos; 2) the evaluation of fecundity, fertility and hatching. Additional pools of embryos collected at time point 40 were evaluated for: 1) the active swimming behavior of the 48hph offspring in the eleutheroembryonic stage; 2) the BDE-47 quantification in the 240hph resultant larvae. BDE-47 accumulated in parents rapidly, and concentrations remained constant at higher levels in males (values within the 50-60ng/g wet weight -w.w.- range) compared with females (70ng/g w.w. range). The BDE-47 concentrations detected in embryos and eleutheroembryos ranged from 200 to 500ng/g w.w. for time points 10-40. Reproductive capacity, hatching and ensuing swim bladder inflation were not affected by parental BDE-47 dietary exposure, nor was the active swimming behavior in eleutheroembryos. The BDE-47 concentration in the 240hph larvae lowered to levels close to those detected in parents. Despite the efficient BDE-47 maternal transfer, these results offered no evidence for BDE-47 effects on fish reproduction or in the early life stages of offspring.

Uptake of perfluoroalkyl substances and halogenated flame retardants by crop plants grown in biosolids-amended soils.

The bioaccumulation behavior of perfluoroalkyl substances (PFASs) and halogenated flame retardants (HFRs) was examined in three horticultural crops and earthworms. Two species, spinach (Spinacia oleracea) and tomato (Solanum lycopersicum L.), were grown in field soil amended with a single application of biosolids (at agronomic rate for nitrogen), to represent the scenario using commercial biosolids as fertilizer, and the third crop, corn (Zea mays) was grown in spiked soil (~50mg PFOS/kg soil, ~5mg Deca-BDE/kg soil and a mixture of both, ~50mg PFOS and ~5mg Deca-BDE/kg soil) to represent a worst-case scenario. To examine the bioaccumulation in soil invertebrates, earthworms (Eisenia andrei) were exposed to the spiked soil where corn had been grown. PFASs and HFRs were detected in the three crops and earthworms. To evaluate the distribution of the compounds in the different plant tissues, transfer factors (TFs) were calculated, with TF values higher for PFASs than PBDEs in all crop plants: from 2 to 9-fold in spinach, 2 to 34-fold in tomato and 11 to 309-fold in corn. Bioaccumulation factor (BAF) values in earthworms were also higher for PFASs (4.06±2.23) than PBDEs (0.02±0.02).

Effects of dietary 2,2', 4,4'-tetrabromodiphenyl ether (BDE-47) exposure in growing medaka fish (Oryzias latipes).

In this research work, we addressed the effects of a diet fortified with BDE-47 (0, 10, 100, 1000ng/g) dosed to 4-7 day-old post-hatch medaka fish for 40 days, followed by an 80-day depuration period. BDE-47 accumulation and overall growth were evaluated throughout the dosing period, and its elimination was quantified over the following 60 days. The histological condition of the thyroid gland, liver and gonads from the 1000ng BDE-47-treated fish were assessed 5 and 70days after exposures finished. The phenotypic males to females ratio was also quantified 70days after treatments finished. Sixty days after the BDE-47 exposures, reproductive capacity (i.e. fecundity, fertility and hatchability) was evaluated in mating groups for a 20-day period. BDE-47 exposure via food from larval through juvenile life stages of medaka fish resulted in steady accumulation with time dose-dependently. This accumulation tendency rapidly decreased after dosing ended. The growth rates showed a significant increase only at the highest concentration 70days after exposures finished. The histological survey did not reveal BDE-47-related alterations in the condition of the potential target organs. However, a morphometrical approach suggested BDE-47-related differences in the thickness of the epithelium that lines thyroid follicles. The reproduction studies showed comparable values for the fecundity, fertility and hatching rates. Dietary BDE-47 dosed for 40days to growing medaka fish did not alter the phenotypic sex ratios at maturity. The dietary approach used herein could not provide conclusive evidence of effects on medaka development and thriving despite the fact that BDE-47 underwent rapid accumulation in whole fish during the 40-day treatment.

Bioaccumulation of emerging organic compounds (perfluoroalkyl substances and halogenated flame retardants) by earthworm in biosolid amended soils.

In the present work, the bioaccumulation behavior of 49 target emerging organic compounds (20 perfluoroalkyl substances, PFASs, and 29 halogenated flame retardants, HFRs) was studied in soil invertebrates (Eisenia andrei). Multi species soil systems (MS·3) were used to assess the fate and the effects associated with the application of four biosolids in agricultural soil on terrestrial soil organisms. Biosolid amendment increased concentrations 1.5-14-fold for PFASs, 1.1-2.4-fold for polybrominated diphenyl ethers, PBDEs, and 1.1-3.6-fold for chlorinated flame retardants, CFRs. Perfluorooctanesulfonate, PFOS, (25%) and BDE-209 (60%) were the predominant PFAS and HFR compounds, respectively, in biosolids-amended soils. Total concentrations (ng/g dry weight) in earthworms from biosolid-amended soils ranged from 9.9 to 101 for PFASs, from 45 to 76 for PBDEs and 0.3-32 for CFRs. Bioaccumulation factors (BAFs) were calculated to evaluate the degree of exposure of pollutants in earthworms. The mean BAF ranged from 2.2 to 198 for PFASs, 0.6-17 for PBDEs and 0.5-20 for CFRs. The relationship of PFAS and PBDE BAFs in earthworms and their log Kow were compared: PFAS BAFs increased while PBDE BAFs declined with increasing log Kow values. The effect of the aging (21 days) on the bioavailability of the pollutants in amended soils was also assessed: the residence time affected differently to the compounds studied.

Effect of soil properties, heavy metals and emerging contaminants in the soil nematodes diversity.

Among soil organisms, nematodes are seen as the most promising candidates for bioindications of soil health. We hypothesized that the soil nematode community structure would differ in three land use areas (agricultural, forest and industrial soils), be modulated by soil parameters (N, P, K, pH, SOM, CaCO3, granulometric fraction, etc.), and strongly affected by high levels of heavy metals (Cd, Pb, Zn, Cr, Ni, Cu, and Hg) and emerging contaminants (pharmaceuticals and personal care products, PPCPs). Although these pollutants did not significantly affect the total number of free-living nematodes, diversity and structure community indices vastly altered. Our data showed that whereas nematodes with r-strategy were tolerant, genera with k-strategy were negatively affected by the selected pollutants. These effects diminished in soils with high levels of heavy metals given their adaptation to the historical pollution in this area, but not to emerging pollutants like PPCPs.

Stage-dependent ethoxyresorufin-O-deethylase (EROD) in vivo activity in medaka (Oryzias latipes) embryos.

Using medaka (Oryzias latipes) embryos, this study aimed to quantitatively characterize the stage-dependent in vivo ethoxyresorufin-O-deethylase (EROD) as indicator of cytochrome P4501A (CYP1A) activity. Embryos were challenged for 24-h to an agonist (ß-naphthoflavone [BNF], 2.5, 5, 10, and 20 µg L(-1)) or to its combination (2.5 µg L(-1)) with an antagonist (α-naphthoflavone [ANF], 25, 50, 100, and 200 µg L(-1)), initiated at four different developmental time points (1, 3, 6, and 9 d post-fertilization [dpf]). Respective induction and competitive inhibition were evaluated over fluorescent images of whole embryo (nonorgan-specific [NOS] EROD activity) and gallbladder (organ-specific [OS] EROD activity). Both flavonoids showed signs of stability in solution. Generally speaking, the mean fluorescence intensity (MFI) values for NOS EROD increased with BNF concentration and exposure challenge. BNF co-exposure with ⩾50 µg ANF L(-1) during the 1-2 and 3-4 dpf challenges lowered NOS EROD to undetectably induced levels. Significant increments in MFIs for OS-EROD were seen from exposures to ⩾2.5 µg BNF L(-1), peaking during the 6-7 dpf challenge regardless of BNF concentration. The simultaneous BNF/ANF incubation showed competitive inhibition for OS EROD activity, although levels were generally detectably induced during all challenges and at all ANF concentrations. The morphometric in vivo gallbladder analysis indicated significant dilation in the 10 dpf-old embryos co-exposed to BNF and 200 µg ANF L(-1). This quantitative approach can be used successfully at 4 dpf at the NOS-EROD or OS-EROD levels, although the NOS-EROD response was sensitive enough to induction or inhibition, even at 2 dpf.

Impact of gold mining associated with mercury contamination in soil, biota sediments and tailings in Kenya.

This work considered the environmental impact of artisanal mining gold activity in the Migori-Transmara area (Kenya). From artisanal gold mining, mercury is released to the environment, thus contributing to degradation of soil and water bodies. High mercury contents have been quantified in soil (140 µg kg(-1)), sediment (430 µg kg(-1)) and tailings (8,900 µg kg(-1)), as expected. The results reveal that the mechanism for transporting mercury to the terrestrial ecosystem is associated with wet and dry depositions. Lichens and mosses, used as bioindicators of pollution, are related to the proximity to mining areas. The further the distance from mining areas, the lower the mercury levels. This study also provides risk maps to evaluate potential negative repercussions. We conclude that the Migori-Transmara region can be considered a strongly polluted area with high mercury contents. The technology used to extract gold throughout amalgamation processes causes a high degree of mercury pollution around this gold mining area. Thus, alternative gold extraction methods should be considered to reduce mercury levels that can be released to the environment.

Local deposition of mercury in topsoils around coal-fired power plants: is it always true?

Mercury (Hg) is a toxic element that is emitted to the atmosphere through human activities, mainly fossil fuel combustion. Hg accumulations in soil are associated with atmospheric deposition, while coal-burning power plants remain the most important source of anthropogenic mercury emissions. In this study, we analyzed the Hg concentration in the topsoil of the Kozani-Ptolemais basin where four coal-fired power plants (4,065 MW) run to provide 50 % of electricity in Greece. The study aimed to investigate the extent of soil contamination by Hg using geostatistical techniques to evaluate the presumed Hg enrichment around the four power plants. Hg variability in agricultural soils was evaluated using 276 soil samples from 92 locations covering an area of 1,000 km(2). We were surprised to find a low Hg content in soil (range 1-59 µg kg(-1)) and 50 % of samples with a concentration lower than 6 µg kg(-1). The influence of mercury emissions from the four coal-fired power plants on soil was poor or virtually nil. We associate this effect with low Hg contents in the coal (1.5-24.5 µg kg(-1)) used in the combustion of these power plants (one of the most Hg-poor in the world). Despite anthropic activity in the area, we conclude that Hg content in the agricultural soils of the Kozani-Ptolemais basin is present in low concentrations.

Limitations of waterborne exposure of fish early life stages to BDE-47.

2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) is acknowledged as the most abundant congener of all polybrominated diphenyl ethers (PBDEs). Despite its limited residence in the water column, most ecotoxicological research using fish early life stages (ELS) has focused on its waterborne bioavailability. These studies have been supported either by chemical analysis in solutions or in tissues after ≤ 168 h exposures to relatively high waterborne concentrations with dimethyl sulfoxide (DMSO) as solvent carrier (≤ 0.5%). Using noninvasive physiological and anatomical features in medaka ELS, we investigated the viability of waterborne BDE-47 exposures (100-10,000 µg/L; 1% DMSO) and evaluated the developmental effects in relation to the actual BDE-47 present in water. Embryos were exposed for 10 days under semi-static (24-h renewal) conditions and waterborne BDE-47 concentrations (i.e., dissolved) were quantitated daily and their accumulation in eleutheroembryonic tissues was analyzed 4 days after exposures finished. BDE-47 in solution rapidly decreased after each renewal by >50% in 24h. This was confirmed by discernible precipitation occurring at ≥ 5,000 µg/L on the bottom of the container and attached to the chorionic filaments of eggshell. The fast dissipation from water may explain why, besides the subtle, yet significant effects on post-hatching growth (short length at ≥5000µg/L), no other significant deleterious developmental effects were observed despite the fact that BDE-47 accumulated in tissues in response to BDE-47 treatment. Waterborne BDE-47 exposure was unachievable under traditional semi-static exposure conditions, but was achievable in repeated pulse exposures lasting a few hours whenever the medium was renewed. Hence, this research encourages the use of alternate - more realistic - exposure routes (e.g., particulate matter or sediments) when evaluating early developmental toxicity of BDE-47 or any other PBDE sharing similar properties.

Volcanic mercury in Pinus canariensis.

Mercury (Hg) is a toxic element that is emitted to the atmosphere by both human activities and natural processes. Volcanic emissions are considered a natural source of mercury in the environment. In some cases, tree ring records taken close to volcanoes and their relation to volcanic activity over time are contradictory. In 1949, the Hoyo Negro volcano (La Palma-Canary Islands) produced significant pyroclastic flows that damaged the nearby stand of Pinus canariensis. Recently, 60 years after the eruption, we assessed mercury concentrations in the stem of a pine which survived volcano formation, located at a distance of 50 m from the crater. We show that Hg content in a wound caused by pyroclastic impacts (22.3 µg kg(-1)) is an order of magnitude higher than the Hg concentrations measured in the xylem before and after the eruption (2.3 µg kg(-1)). Thus, mercury emissions originating from the eruption remained only as a mark-in pyroclastic wounds-and can be considered a sporadic and very high mercury input that did not affect the overall Hg input in the xylem. In addition, mercury contents recorded in the phloem (9.5 µg kg(-1)) and bark (6.0 µg kg(-1)) suggest that mercury shifts towards non-living tissues of the pine, an aspect that can be related to detoxification in volcanism-adapted species.

Effects of individual and a mixture of pharmaceuticals and personal-care products on cytotoxicity, EROD activity and ROS production in a rainbow trout gonadal cell line (RTG-2).

The presence of pharmaceuticals and personal-care products (PPCPs) in aquatic environments is of concern. Although measured concentrations of individual substances are low, little consideration has been given to the likely chronic nature of the exposures or to the potential for mixture effects. The purpose of the present study was to use the RTG-2 rainbow trout cell line to analyse sub-lethal and cytotoxic effects of PPCPs present in a wastewater-treatment-plant (WWTP) effluents and their mixtures. Interactions with cytochrome P450 1A enzyme, oxidative stress, cellular senescence and cell viability were assessed using 7-ethoxyresorufin-o-deethylase (EROD), reactive oxygen species (ROS), ß-galactosidase (ß-gal) and neutral red (NR) uptake assays, respectively. Not all of the compounds that were tested exhibited significant effects. The lowest-observed-effect concentrations and half maximal effective concentrations (EC50 ) were within the range 0.15 to 784.47 µg l(-1) . Clear dose-response curves were found for cells exposed to different mixtures of PPCPs. The lowest-observed-effect concentrations and concentrations causing EC50 were within the range 0.05 to 54.61 µg l(-1) . Four out the seven tested mixtures induced EROD activity. ROS production was detected in two mixtures. The ß-gal inhibition response was observed in six out the seven tested mixtures and occurred at a higher concentration than was observed for EROD induction activity or ROS generation. The present study clearly shows that the stress response through which cells mount a homeostatic response to toxicants can be potentially used for an initial, rapid and cost-effective assessment of the complex mixtures of PPCP that present in WWTP effluents are difficult and expensive to analyse chemically.

Source identification of soil mercury in the Spanish islands.

This study spatially analysed the relation between mercury (Hg) content in soil and Hg in rock fragment for the purpose of assessing natural soil Hg contribution compared with Hg from human inputs. We present the Hg content of 318 soil and rock fragment samples from 11 islands distributed into two Spanish archipelagos (the volcanic Canary Islands [Canaries] and the Mediterranean Balearic [Balearic] islands). Assumedly both are located far enough away from continental Hg sources to be able to minimise the effects of diffuse pollution. Physical and chemical soil properties were also specified for the samples. Hg contents were significantly greater in the Balearic limestone soils (61 µg kg(-1)) than in the volcanic soils of the Canaries (33 µg kg(-1)). Hg levels were also greater in topsoil than in rocky fragments, especially on the Balearics. The soil-to-rock ratios varied between 1 and 30. Interestingly, the highest topsoil-to-rock Hg ratio (>16 ×) was found in the vicinity of a coal-fired power plant in Majorca, whereas no similar areas in the Canary archipelago were identified.

Effects of municipal solid waste compost and mineral fertilizer amendments on soil properties and heavy metals distribution in maize plants (Zea mays L.).

Soil amendments based on crop nutrient requirements are considered a beneficial management practice. A greenhouse experiment with maize seeds (Zea mays L.) was conducted to assess the inputs of metals to agricultural land from soil amendments. Maize seeds were exposed to a municipal solid waste (MSW) compost (50 Mg ha(-1)) and NPK fertilizer (33 g plant(-1)) amendments considering N plant requirement until the harvesting stage with the following objectives: (1) determine the accumulation of total and available metals in soil and (2) know the uptake and ability of translocation of metals from roots to different plant parts, and their effect on biomass production. The results showed that MSW compost increased Cu, Pb and Zn in soil, while NPK fertilizer increased Cd and Ni, but decreased Hg concentration in soil. The root system acted as a barrier for Cr, Ni, Pb and Hg, so metal uptake and translocation were lower in aerial plant parts. Biomass production was significantly enhanced in both MSW and NPK fertilizer-amended soils (17%), but also provoked slight increases of metals and their bioavailability in soil. The highest metal concentrations were observed in roots, but there were no significant differences between plants growing in amended soil and the control soil. Important differences were found for aerial plant parts as regards metal accumulation, whereas metal levels in grains were negligible in all the treatments.

Environmental risk assessment of ivermectin: A case study.

The veterinary parasiticide ivermectin was selected as a case study compound within the project ERAPharm (Environmental Risk Assessment of Pharmaceuticals). Based on experimental data generated within ERAPharm and additional literature data, an environmental risk assessment (ERA) was performed mainly according to international and European guidelines. For the environmental compartments surface water, sediment, and dung, a risk was indicated at all levels of the tiered assessment approach. Only for soil was no risk indicated after the lower tier assessment. However, the use of effects data from additional 2-species and multispecies studies resulted in a risk indication for collembolans. Although previously performed ERAs for ivermectin revealed no concern for the aquatic compartment, and transient effects on dung-insect populations were not considered as relevant, the present ERA clearly demonstrates unacceptable risks for all investigated environmental compartments and hence suggests the necessity of reassessing ivermectin-containing products. Based on this case study, several gaps in the existing guidelines for ERA of pharmaceuticals were shown and improvements have been suggested. The action limit at the start of the ERA, for example, is not protective for substances such as ivermectin when used on intensively reared animals. Furthermore, initial predicted environmental concentrations (PECs) of ivermectin in soil were estimated to be lower than refined PECs, indicating that the currently used tiered approach for exposure assessment is not appropriate for substances with potential for accumulation in soil. In addition, guidance is lacking for the assessment of effects at higher tiers of the ERA, e.g., for field studies or a tiered effects assessment in the dung compartment.

Use of a novel battery of bioassays for the biological characterisation of hazardous wastes.

Four toxicity bioassays were used for the biological characterisation of nine hazardous wastes and extracts. This evaluation included conventional and novel bioassays, and combined in vivo and in vitro tests in order to facilitate the effect characterisation. This test battery incorporated different relevant taxonomic groups for the aquatic compartment and covered several ecotoxicological endpoints. The toxicity bioassays used for this characterisation were the acute immobilisation daphnia test, an acute toxicity test with larvae of Xenopus laevis, an in vitro test with the fish cell line RTG-2 comprising endpoints for cellular defence and viability, and finally the DR-CALUX assay to detect dioxin-like compounds. The aim of this study is to contribute to the development of a cost-effective battery of toxicity tests for the acute screening of hazardous and toxic wastes for the aquatic compartment. For this objective, the correlations between toxicity data derived from all bioassay were studied using a multivariate analysis, including the Principal Component Analysis. The results showed that Daphnia and Xenopus were effective assays to detect toxicity and they could be incorporated to a screening test battery. On the other hand, the toxicity results with the in vitro test RTG-2 showed that this test could be a good alternative to in vivo tests, demonstrating an acceptable sensitivity for toxicity detection and contributing other advantages as reducing assays cost and animal testing. Finally DR-CALUX test implemented the tests-batteries in the screening of hazardous wastes when there is a suspicious that dioxin-like compounds are presented in the samples.

Bioaccumulation assessment via an adapted multi-species soil system (MS.3) and its application using cadmium.

This paper presents an experimental design for quantifying the transfer of chemicals at low trophic levels of terrestrial ecosystems. The soil microcosms, MS.3(foodchain) (food chain multi-specie soil system) covered the transfer from soil to earthworms (Eisenia fetida) and from soil to plant (Triticum aestivum), then to phytophagus (Rhopalosiphum padi) and finally predator (Chrysoperla carnea) species. Cadmium was used as model pollutant. Cadmium accumulation in foliar invertebrates was related to the species biology. A significant transfer of this metal through the minimized food chain was found for all species, but not a biomagnification in the predator species. Results pointed out the relevance of foliar invertebrates and their trophic relationships as additional exposure routes for assessing secondary poisoning in predators. Hence, MS.3(foodchain), could be applied for terrestrial environmental risk assessment when potential bioaccumulation could be expected.

Ecological impact of repeated applications of chlorpyrifos on zooplankton community in mesocosms under Mediterranean conditions.

The effects of the insecticide chlorpyrifos were studied in plankton-dominated mesocosms under Mediterranean conditions. Chlorpyrifos was applied four times at 1 week intervals at nominal concentrations of 0.033, 0.1, 0.33, and 1 microg/l simulating repeated agricultural applications. The lowest 7 days time weighted averaged concentrations (TWAC) during the 28 days exposure period were calculated using the FOCUS equation to express the no observed effect concentration (NOEC) values. At population level the lowest NOEC calculated was 0.012 microg/l (treatment concentration 0.033 microg/l). The most affected taxon was Cladocera (Daphnia group galeata) followed by Copepoda (cyclopoids and nauplii). No effects were observed on phytoplankton (chlorophyll-a biomass) at any treatment level. The smallest NOEC(community) calculated by means of multivariate techniques was 0.1 microg/l when expressed in terms of the nominal treatment level and 0.074 microg/l when based on the lowest 7 days TWA concentration during the 28 days application period. Indirect effects on zooplankton populations were observed due to shifts in competition and predation between populations. Compared with previous micro/mesocosm experiments simulating a single application exposure regime, results from our study revealed a lower threshold level for the most sensitive measurement endpoint (difference a factor of three (in terms of nominal treatment level), more severe indirect effects and longer recovery periods of the affected populations (> 13 weeks in the test systems treated with 1 microg/l). These differences could be attributed to the repeated pulse exposure scenario approach designed for our studies together with the particular climatic conditions involving our Mediterranean mesocosms (i.e., temperature, cladocerans life history, and algae blooms).

Responses of zooplankton in lufenuron-stressed experimental ditches in the presence or absence of uncontaminated refuges.

Outdoor experimental ditches were used to evaluate the influence of untreated refuges on the recovery of zooplankton communities following treatment with the fast-dissipating insecticide lufenuron. Each experimental ditch was divided into three sections of the same surface area. The treatments differed in the proportion of ditch (0, 33, 67, and 100% of the surface area) to which the insecticide was applied at the same nominal treatment (3 mug/L). During the first week postapplication, a barrier was placed between treated and untreated ditch sections. The untreated sections were included to provide a source of organisms for recovery of affected zooplankton populations in the treated sections of the ditch after the removal of the barrier. Cyclopoida were the most affected by lufenuron treatment, followed by Daphnia gr. galeata. These and other direct effects of treatment on larvae of the phantom midge Chaoborus spp. resulted in clear indirect effects on populations of Calanoida, Ceriodaphnia, and Rotifera. Overall, faster recovery of the zooplankton community was observed in the treated sections of ditches that were sprayed for a smaller proportion of their surface area. Nevertheless, individual zooplankton populations showed considerable differences in rate of recovery. Cyclopoida showed a relatively slow rate of recovery even in the partially treated ditches. Daphnia gr. galeata recovered more rapidly in treated ditch sections in the presence of unsprayed ditch sections, illustrating the potential influence of unexposed refuges. Furthermore, the presence of refuges most likely dampened the magnitude and duration of indirect effects in the ditches treated with lufenuron.