Microplastic-mediated transport of PCBs? A depuration study with Daphnia magna.

The role of microplastic (MP) as a carrier of persistent organic pollutants (POPs) to aquatic organisms has been a topic of debate. However, the reverse POP transport can occur if relative contaminant concentrations are higher in the organism than in the microplastic. We evaluated the effect of microplastic on the PCB removal in planktonic animals by exposing the cladoceran Daphnia magna with a high body burden of polychlorinated biphenyls (PCB 18, 40, 128 and 209) to a mixture of microplastic and algae; daphnids exposed to only algae served as the control. As the endpoints, we used PCB body burden, growth, fecundity and elemental composition (%C and %N) of the daphnids. In the daphnids fed with microplastic, PCB 209 was removed more efficiently, while there was no difference for any other congeners and ΣPCBs between the microplastic-exposed and control animals. Also, higher size-specific egg production in the animals carrying PCB and receiving food mixed with microplastics was observed. However, the effects of the microplastic exposure on fecundity were of low biological significance, because the PCB body burden and the microplastic exposure concentrations were greatly exceeding environmentally relevant concentrations.

Deriving in Vivo Bioconcentration Factors of a Mixture of Fragrance Ingredients Using a Single Dietary Exposure and Internal Benchmarking.

Chemicals in mixtures that are hydrophobic with Log KOW > 4 are potentially bioaccumulative. Here, we evaluate an abbreviated and benchmarked in vivo BCF measurement methodology by exposing rainbow trout to a mixture of eight test chemicals found in fragrance substances and three benchmark chemicals (musk xylene (MX), hexachlorobenzene (HCB) and PCB52) via a single contaminated feeding event followed by a 28-day depuration period. Concentrations of HCB and PCB52 in fish did not decline significantly (their apparent depuration rate constants, kT, were close to zero), whereas kT for MX was 0.022 d-1. The test chemicals were eliminated much more rapidly than the benchmark chemicals ( kT > 0.117 d-1). The bioconcentration factors (BCFA) for the test chemicals were in the range of 273 L kg-1 (8-cyclohexadecen-1-one (globanone)) to 1183 L kg-1 (α-pinene); the benchmarked BCFs (BCFG) calculated relative to HCB ranged from 238 L kg-1 (globanone) to 1147 L kg-1 (α-pinene). BCFG were not significantly different from BCFA but had smaller standard errors. BCFs derived here agreed well with values previously measured using the OECD 305 test protocol. We conclude that it will be feasible to derive BCFs of chemicals in mixtures using a single dietary exposure and chemical benchmarking.

Growth Retardation and Altered Isotope Composition As Delayed Effects of PCB Exposure in Daphnia magna.

Trophic magnification factor (TMF) analysis employs stable isotope signatures to derive biomagnification potential for environmental contaminants. This approach relies on species δ(15)N values aligning with their trophic position (TP). This, however, may not always be true, because toxic exposure can alter growth and isotope allocation patterns. Here, effects of PCB exposure (mixture of PCB18, PCB40, PCB128, and PCB209) on δ(15)N and δ(13)C as well as processes driving these effects were explored using the cladoceran Daphnia magna. A two-part experiment assessed effects of toxic exposure during and after exposure; juvenile daphnids were exposed during 3 days (accumulation phase) and then allowed to depurate for 4 days (depuration phase). No effects on survival, growth, carbon and nitrogen content, and stable isotope composition were observed after the accumulation phase, whereas significant changes were detected in adults after the depuration phase. In particular, a significantly lower nitrogen content and a growth inhibition were observed, with a concomitant increase in δ(15)N (+0.1 ‰) and decrease in δ(13)C (-0.1 ‰). Although of low magnitude, these changes followed the predicted direction indicating that sublethal effects of contaminant exposure can lead to overestimation of TP and hence underestimated TMF.

No measurable "cleaning" of polychlorinated biphenyls from Rainbow Trout in a 9 week depuration study with dietary exposure to 40% polyethylene microspheres.

Persistent hydrophobic chemicals sorbed to plastic can be transferred to fish and other aquatic organisms upon ingestion. However, ingestion of plastic could also lead to enhanced elimination of these chemicals if the plastic is less contaminated than the fish. Here, we attempted to measure the influence of ingestion of uncontaminated polyethylene microspheres on the depuration rates of polychlorinated biphenyls (PCBs) in an in vivo fish feeding experiment. Rainbow trout were given feed contaminated with PCBs for two consecutive days, then clean feed for three days to allow for egestion of the contaminated food. A control group of fish were then fed ordinary food pellets and a treatment group were fed pellets that additionally contained 40% by weight polyethylene microspheres. Condition factors and growth rates in both groups were similar, indicating no negative effect of the plastic microspheres on the nutritional status of the fish. Fish were sampled after zero, three, six and nine weeks, homogenized, solvent-extracted and analyzed by GC/MS. PCB concentrations declined in both groups at a rate consistent with growth dilution. There was no significant difference in the elimination rate constants between the control and treatment group, indicating that ingestion of uncontaminated plastic did not cause a measurable enhancement of depuration of PCBs by the fish in this study.

Effects of an oil spill in a harbor assessed using biomarkers of exposure in eelpout.

Oil spills occur commonly, and chemical compounds originating from oil spills are widespread in the aquatic environment. In order to monitor effects of a bunker oil spill on the aquatic environment, biomarker responses were measured in eelpout (Zoarces viviparus) sampled along a gradient in Göteborg harbor where the oil spill occurred and at a reference site, 2 weeks after the oil spill. Eelpout were also exposed to the bunker oil in a laboratory study to validate field data. The results show that eelpout from the Göteborg harbor are influenced by contaminants, especially polycyclic aromatic hydrocarbons (PAHs), also during "normal" conditions. The bunker oil spill strongly enhanced the biomarker responses. Results show elevated ethoxyresorufin-O-deethylase (EROD) activities in all exposed sites, but, closest to the oil spill, the EROD activity was partly inhibited, possibly by PAHs. Elevated DNA adduct levels were also observed after the bunker oil spill. Chemical analyses of bile revealed high concentrations of PAH metabolites in the eelpout exposed to the oil, and the same PAH metabolite profile was evident both in eelpout sampled in the harbor and in the eelpout exposed to the bunker oil in the laboratory study.

Sucralose induces biochemical responses in Daphnia magna.

The intense artificial sweetener sucralose has no bioconcentration properties, and no adverse acute toxic effects have been observed in standard ecotoxicity tests, suggesting negligible environmental risk. However, significant feeding and behavioural alterations have been reported in non-standard tests using aquatic crustaceans, indicating possible sublethal effects. We hypothesized that these effects are related to alterations in acetylcholinesterase (AChE) and oxidative status in the exposed animals and investigated changes in AChE and oxidative biomarkers (oxygen radical absorbing capacity, ORAC, and lipid peroxidation, TBARS) in the crustacean Daphnia magna exposed to sucralose (0.0001-5 mg L(-1)). The sucralose concentration was a significant positive predictor for ORAC, TBARS and AChE in the daphnids. Moreover, the AChE response was linked to both oxidative biomarkers, with positive and negative relationships for TBARS and ORAC, respectively. These joint responses support our hypothesis and suggest that exposure to sucralose may induce neurological and oxidative mechanisms with potentially important consequences for animal behaviour and physiology.

A benchmarking method to measure dietary absorption efficiency of chemicals by fish.

Understanding the dietary absorption efficiency of chemicals in the gastrointestinal tract of fish is important from both a scientific and a regulatory point of view. However, reported fish absorption efficiencies for well-studied chemicals are highly variable. In the present study, the authors developed and exploited an internal chemical benchmarking method that has the potential to reduce uncertainty and variability and, thus, to improve the precision of measurements of fish absorption efficiency. The authors applied the benchmarking method to measure the gross absorption efficiency for 15 chemicals with a wide range of physicochemical properties and structures. They selected 2,2',5,6'-tetrachlorobiphenyl (PCB53) and decabromodiphenyl ethane as absorbable and nonabsorbable benchmarks, respectively. Quantities of chemicals determined in fish were benchmarked to the fraction of PCB53 recovered in fish, and quantities of chemicals determined in feces were benchmarked to the fraction of decabromodiphenyl ethane recovered in feces. The performance of the benchmarking procedure was evaluated based on the recovery of the test chemicals and precision of absorption efficiency from repeated tests. Benchmarking did not improve the precision of the measurements; after benchmarking, however, the median recovery for 15 chemicals was 106%, and variability of recoveries was reduced compared with before benchmarking, suggesting that benchmarking could account for incomplete extraction of chemical in fish and incomplete collection of feces from different tests.

Internal benchmarking improves precision and reduces animal requirements for determination of fish bioconcentration factors.

The enactment of new chemical regulations has generated a large need for the measurement of the fish bioconcentration factor (BCF). Past experience shows that the BCF determination lacks precision, requires large numbers of fish, and is costly. A new protocol was tested that shortens the experiment from up to 12 weeks for existing protocols to 2 weeks and reduces the number of fish by a factor of 5, while introducing internal benchmarking for the BCF determination. Rainbow trout were simultaneously exposed to 11 chemicals. The BCFs were quantified using one of the test chemicals, musk xylene, as a benchmark. These were compared with BCFs measured in a parallel experiment based on the OECD 305 guideline. The agreement was <20% for five chemicals and between 20%-25% for two further, while two chemicals lay outside the BCF operating window of the experiment and one was lost due to analytical difficulties. This agreement is better than that observed in a BCF Gold Standard Database. Internal benchmarking allows the improvement of the precision of BCF determination in parallel to large reduction in costs and fish requirements.

Measuring bioconcentration factors in fish using exposure to multiple chemicals and internal benchmarking to correct for growth dilution.

Modern chemical legislation requires measuring the bioconcentration factor (BCF) of large numbers of chemicals in fish. The BCF must be corrected for growth dilution, because fish growth rates vary between laboratories. Two hypotheses were tested: (1) that BCFs of multiple chemicals can be measured simultaneously in one experiment, and (2) that internal benchmarking using a conservative test substance in the chemical mixture can be used to correct for growth dilution. Bioconcentration experiments were conducted following major elements of the OECD 305 guideline. Fish were simultaneously exposed to 11 chemicals selected to cover a range of BCFs and susceptibility to biotransformation. A method was developed to calculate the growth-corrected elimination rate constant from the concentration ratio of the analyte and a benchmarking chemical for which growth dilution dominated other elimination mechanisms. This method was applied to the experimental data using hexachlorobenzene as the benchmarking chemical. The growth dilution correction lowered the apparent elimination rate constants by between 5% and a factor of four for eight chemicals, while for two chemicals the growth-corrected elimination rate constant was not significantly different from zero. The benchmarking method reduced the uncertainty in the elimination rate constant compared to the existing method for growth dilution correction. The BCFs from exposing fish to 10 chemicals at once were consistent with BCF values from single-chemical exposures from the literature, supporting hypothesis 1.

In-vivo passive sampling to measure elimination kinetics in bioaccumulation tests.

The application of in-tissue passive sampling to quantify chemical kinetics in fish bioconcentration experiments was investigated. A passive sampler consisting of an acupuncture needle covered with a PDMS tube was developed together with a method for its deployment in rainbow trout. The time to steady state for chemical uptake into the passive sampler was >1d, so it was employed as a kinetically limited sampler with a deployment time of 2 h. The passive sampler was employed in parallel with the established whole tissue extraction method to study the elimination kinetics of 10 diverse chemicals in rainbow trout. 4-n-nonylphenol and 2,4,6-tri-tert-butylphenol were close to or below the limit of quantification in the sampler. For chlorpyrifos, musk xylene, hexachlorobenzene, 2,5-dichlorobiphenyl and p,p'-DDT, the elimination rate constants determined with the passive sampler method and the established method agreed within 18%. Poorer agreement (35%) was observed for 2,3,4-trichloroanisole and p-diisopropylbenzene because fewer data were obtained with the passive sampling method due to its lower sensitivity. The work shows that in-tissue passive sampling can be employed to measure contaminant elimination kinetics in fish. This opens up the possibility of studying contaminant kinetics in individual fish, thereby reducing the fish requirements and analytical costs for the determination of bioconcentration factors.

Sucralose - an ecotoxicological challenger?

The non-calorie sweetener sucralose - sucrose containing three chlorine atoms - is intensively sweet and has become a popular substitute for sugar. Its widespread use, exceptional stability in combination with high water solubility have thus resulted in contamination of recipient waters. Earlier studies on sucralose in aquatic organisms indicate low bioaccumulation potential and negligible acute/chronic toxicity, but the close structural resemblance with sucrose in combination with the importance of sugar in nature, warrant a more detailed ecotoxicological assessment. The aim of this investigation was therefore to study behavioural and physiological effects of sucralose in crustaceans. Our results show that both physiology and locomotion behaviour were affected by exposure to sucralose. In Daphnia magna, the behavioural response was manifested as altered swimming height and increased swimming speed, whereas in gammarids the time to reach food and shelter was prolonged. Regardless if these behavioural responses were initiated via traditional toxic mechanisms or stimulatory effects, they should be considered as a warning, since exposed organisms may diverge from normal behaviour, which ultimately can have ecological consequences.

A flow-through passive dosing system for continuously supplying aqueous solutions of hydrophobic chemicals to bioconcentration and aquatic toxicity tests.

A continuous supply of water with defined stable concentrations of hydrophobic chemicals is a requirement in a range of laboratory tests such as the OECD 305 protocol for determining the bioconcentration factor in fish. Satisfying this requirement continues to be a challenge, particularly for hydrophobic chemicals. Here we present a novel solution based on equilibrium passive dosing. It employs a commercially available unit consisting of ~16000 polydimethylsiloxane (PDMS) tubes connected to two manifolds. The chemicals are loaded into the unit by repeatedly perfusing it with a methanol solution of the substances that is progressively diluted with water. Thereafter the unit is perfused with water and the chemicals partition from the unit into the water. The system was tested with nine chemicals with logK(OW) ranging from 4.1 to 6.3. The aqueous concentrations generated were shown to be largely independent of the water flow rate, and the unit to unit reproducibility was within a factor of ~2. In continuous flow experiments the aqueous concentrations of most of the study chemicals remained constant over 8d. A model was assembled that allows prediction of the operating characteristics of the system from the logK(OW) or PDMS/water partition coefficient of the chemical. The system is a simple, safe, predictable and flexible tool that generates stable aqueous concentrations of hydrophobic chemicals.

Triclosan in individual human milk samples from Australia.

Triclosan is a chlorinated phenol ether that has been in widespread use as a broad-spectrum antibacterial agent for four decades. When compared to the limited international data available on human body burden of triclosan, results from a pooled blood study suggested that triclosan concentrations in Australia were a factor two higher than observed in Sweden. This study determined triclosan levels in individual human milk samples (n=151) collected between 2002 and 2005 from primiparous Australian mothers. It provided the first report of population triclosan levels and individual variation in Australia and gave a measure of infant exposure via breast feeding. The distribution of triclosan concentration was positively skewed, with 7.2% of the samples below the LOQ, 66% with a concentration of less than or equal to 1.0 ng g(-1)fresh weight and the remaining samples above 1 ng g(-1) reaching a maximum concentration of 19 ng g(-1)fresh weight. The mean and median triclosan concentrations were 1.3±2.7 ng g(-1)f.w. and 0.26 ng g(-1)f.w., respectively. The results of this study showed high variability in triclosan concentrations between individuals and no correlations with maternal age (p=0.094), maternal weight (p=0.971) or infant age at the time of sample collection (p=0.621). A large number of samples contained low or non-quantifiable concentrations of triclosan and so, in Australia, ubiquitous background exposure due to environmental sources is low. This means that body burden can be influenced by an individual's use of triclosan containing product. Given that triclosan containing product use is continuing, it is important that monitoring in both humans and the environment is continued and that triclosan containing products are adequately labeled so that an individual can choose to avoid exposure.

Equilibrium sampling of environmental pollutants in fish: comparison with lipid-normalized concentrations and homogenization effects on chemical activity.

Equilibrium sampling of organic pollutants into the silicone polydimethylsiloxane (PDMS) has recently been applied in biological tissues including fish. Pollutant concentrations in PDMS can then be multiplied with lipid/PDMS distribution coefficients (D(Lipid,PDMS) ) to obtain concentrations in fish lipids. In the present study, PDMS thin films were used for equilibrium sampling of polychlorinated biphenyls (PCBs) in intact tissue of two eels and one salmon. A classical exhaustive extraction technique to determine lipid-normalized PCB concentrations, which assigns the body burden of the chemical to the lipid fraction of the fish, was additionally applied. Lipid-based PCB concentrations obtained by equilibrium sampling were 85 to 106% (Norwegian Atlantic salmon), 108 to 128% (Baltic Sea eel), and 51 to 83% (Finnish lake eel) of those determined using total extraction. This supports the validity of the equilibrium sampling technique, while at the same time confirming that the fugacity capacity of these lipid-rich tissues for PCBs was dominated by the lipid fraction. Equilibrium sampling was also applied to homogenates of the same fish tissues. The PCB concentrations in the PDMS were 1.2 to 2.0 times higher in the homogenates (statistically significant in 18 of 21 cases, p < 0.05), indicating that homogenization increased the chemical activity of the PCBs and decreased the fugacity capacity of the tissue. This observation has implications for equilibrium sampling and partition coefficients determined using tissue homogenates.

Determination of cyclic volatile methylsiloxanes in biota with a purge and trap method.

The three cyclic volatile methylsiloxanes (cVMS), octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5), and dodecamethylcyclohexasiloxane (D6), are recently identified environmental contaminants. Methods for the trace analysis of these chemicals in environmental matrices are required. A purge and trap method to prepare highly purified sample extracts with a low risk of sample contamination is presented. Without prior homogenization, the sample is heated in water, and the cVMS are purged from the slurry and trapped on an Isolute ENV+ cartridge. They are subsequently eluted with n-hexane and analyzed with GC/MS. The method was tested for eight different matrices including ragworms, muscle tissue from lean and lipid-rich fish, cod liver, and seal blubber. Analyte recoveries were consistent within and between matrices, averaging 79%, 68%, and 56% for D4, D5, and D6, respectively. Good control of blank levels resulted in limits of quantification of 1.5, 0.6, and 0.6 ng/g wet weight. The repeatability was 12% (D5) and 15% (D6) at concentrations 9 and 2 times above the LOQ. The method was applied to analyze cVMS in fish from Swedish lakes, demonstrating that contamination in fish as a result of long-range atmospheric transport is low as compared to contamination from local sources.

Characterization of additional sewage treatment technologies: ecotoxicological effects and levels of selected pharmaceuticals, hormones and endocrine disruptors.

In the present study, two conventional (with and without sand filter) and four additional (moving bed biofilm reactor, ozone, moving bed biofilm reactor combined with ozone and a membrane bio reactor) treatment technologies were operated in small-scale at Hammarby Sjöstad sewage treatment plant, Stockholm, Sweden. The effluents were tested with five short-term (≤ 7 days exposure) ecotoxicological tests, and analyzed for a number of target analytes, comprising pharmaceuticals, natural hormones and industrial chemicals. Overall, the tested effluents generated few adverse effects at lower concentrations (< 50% sewage effluent), and no major differences were observed between any of the treatments. The effluent treated with the moving bed biofilm reactor resulted in the lowest effects in the ecotoxicological tests. The most efficient treatment technology with regard to the pharmaceutical residues was the ozone treatment, which however caused negative effects in some of the ecotoxicological tests.

Serial mixed-mode cation- and anion-exchange solid-phase extraction for separation of basic, neutral and acidic pharmaceuticals in wastewater and analysis by high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

A novel solid-phase extraction (SPE) method is presented whereby 15 basic, neutral and acidic pharmaceuticals in wastewater were simultaneously extracted and subsequently separated into different fractions. This was achieved using mixed-mode cation- and anion-exchange SPE (Oasis MCX and MAX) in series. Analysis was performed by high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC/QTOF-MS). A fast separation was achieved, with all compounds eluting within 6min, narrow chromatographic peaks, with a peak base width of 6s on average, and a high mass accuracy of quantified wastewater sample ions, with average mass errors in absolute value of 0.7mDa or 2.7ppm. The recovery of the SPE method in the analysis of sewage treatment plant (STP) influent and effluent wastewater was on average 80% and the ion suppression 30%. For less demanding samples Oasis MCX used alone may be an alternative method, although for STP influent waters containing high loads of organic compounds the clean-up achieved using only Oasis MCX was insufficient, leading to unreliable quantitation. Furthermore, serial SPE separation according to molecular charge added an additional degree of analyte confirmation. For quantitation, an approach combining external standard calibration curves, isotopically labelled surrogate standards and single-point standard addition was used. The applicability of the method was demonstrated in the analysis of influent and effluent wastewater from an STP, using small sample volumes (25-50mL). The effluent wastewater had been subjected to three different treatments; activated sludge, activated sludge followed by ozonation, and a membrane bioreactor (MBR). Ozone treatment proved superior in removal of the analysed pharmaceuticals, while the MBR provided higher removal efficiencies than the activated sludge process.

The influence of age and gender on triclosan concentrations in Australian human blood serum.

The bactericide triclosan has found wide-spread use in e.g. soaps, deodorants and toothpastes. Recent in vitro and in vivo studies indicate that triclosan might exert adverse effects in humans. Triclosan has previously been shown to be present in human plasma and milk at concentrations that are well correlated to the use of personal care products containing triclosan. In this study we investigated the influence of age, gender, and the region of residence on triclosan concentrations in pooled samples of Australian human blood serum. The results showed no influence of region of residence on the concentrations of triclosan. There was a small but significant influence of age and gender on the serum triclosan concentrations, which were higher in males than in females, and highest in the group of 31-45 year old males and females. However, overall there was a lack of pronounced differences in the triclosan concentrations within the dataset, which suggests that the exposure to triclosan among different groups of the Australian population is relatively homogenous. A selection of the dataset was compared with previous measurements of triclosan concentrations in human plasma from Sweden, where the use of triclosan is expected to be low due to consumer advisories. The triclosan concentrations were a factor of 2 higher in Australian serum than in Swedish plasma.

Development and reproduction of the freshwater harpacticoid copepod Attheyella crassa for assessing sediment-associated toxicity.

Both freshwater and marine sediments are sinks for many anthropogenic substances. This may pose a risk to benthic and epibenthic organisms and it is crucial that toxicity tests that are available for environmental risk assessment can identify potentially adverse effects of sediment-associated substances on benthic organisms, such as harpacticoid copepods. While marine harpacticoids have been protected via a number of acute and chronic sediment tests, the freshwater harpacticoid copepod community has so far been neglected in such activities. The main aim of the present study was therefore to (a) find a suitable freshwater harpacticoid copepod, (b) establish robust laboratory mass cultures and (c) develop a chronic test for assessment of sediment-associated toxicity using spiked sediments. After several cultivation trials with a number of potential test species, the choice fell on the benthic freshwater harpacticoid copepod Attheyella crassa, a species that possesses many of the characteristic features identified as prerequisites for toxicity test organisms, e.g. it has a sexual reproduction, it is relatively easy to grow and keep in mass cultures in the laboratory, and it has a small body size. Owing to the relatively long generation time of freshwater harpacticoids (in relation to many marine harpacticoids), it was decided that the test should be separated into a development part (21 days) and a reproduction part (14 days) running in parallel. As a reference substance we used the fungicide tebuconazole, which is currently subject to risk assessment and which partitions to soil and sediment. Clear concentration-related responses were observed for all endpoints analyzed. Nauplia body length was the most sensitive endpoint with a measured time weighted LOEC(water) of 20microg/L. The corresponding LOEC(water) for larval mortality and offspring production was 65 and 62microg/L, respectively. In conclusion, A. crassa is an ecologically relevant test species for freshwater ecosystems and particularly for the cold, oligotrophic and often acidic lakes of Northern Europe. Regardless of the relatively long generation time of this species, our results clearly show that sediment-associated toxicity related to development and sexual reproduction can be assessed within 2-3 weeks exposure with the developed bioassay.