Biomarker investigations in adult female perch (Perca fluviatilis) from industrialised areas in northern Sweden in 2003.

Since the new millennium, a notion has developed in certain parts of society that environmental pollutants and their associated effects are under control. The primary objective of this investigation, performed in 2003, was to test whether this was actually the case in an industrialised region in the County of Västernorrland in northern Sweden with well-documented environmental pollution from past and present activities. This was performed by measuring a moderate battery of simple biomarkers in adult female perch at several stations. The point sources included sewage-treatment plants, pulp and paper mills, as well as other industries. The biomarkers included growth, somatic indices, gonad maturation status, gonad pigmentation, fin erosion, skin ulcers, and ethoxyresorufin-O-deethylase (EROD) activity in the liver. The results showed that the environmental pollutants and their associated effects were not under control. In fact, the health of the perch was impaired at all of the polluted stations. Many responses were unspecific with respect to underlying cause, whereas some effects on EROD activity and gonad maturation status were attributed to historical creosote pollution and current kraft pulp mill effluents, respectively. The data presented may also be used as reference values for future investigations of health effects in perch.

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.

An individual-based modeling approach for evaluation of endpoint sensitivity in harpacticoid copepod life-cycle tests and optimization of test design.

In the present study, an individual-based model for Nitocra spinipes was developed and used to optimize the test design of a proposed Organisation for Economic Co-operation and Development test guideline for harpacticoid copepods. The variability between individuals was taken into account, based on measured data, leading to stochastic model output. Virtual experiments were performed with the model to analyze the endpoint sensitivity and the effect of number of replicates and inspection intervals on statistical power. The impact of mortality was evaluated; most sublethal effects could not be determined if the mortality was ≥70%. Most sensitive to mortality was the determination of effects on brood size, for which the statistical power was reduced at 10% mortality. Our simulations show that increasing the number of replicates from 72 to 96 or 144 has little impact on the statistical power, whereas 25 replicates disallow relevant endpoint detection. Furthermore, it was demonstrated that the proposed 1D inspection interval can be shifted to a 3D interval, without losing statistical power. It was demonstrated that developmental endpoints have a higher statistical power than reproductive endpoints in the current test design. The present study highlights the usefulness of individual-based models for optimizing the experimental design. The use of such models in the development of standard test guidelines will lead to a faster and less resource-demanding process.

Evidence of population genetic effects of long-term exposure to contaminated sediments-a multi-endpoint study with copepods.

In the environment, pollution generally acts over long time scales and exerts exposure of multiple toxicants on the organisms living there. Recent findings show that pollution can alter the genetics of populations. However, few of these studies have focused on long-term exposure of mixtures of substances. The relatively short generation time (ca. 4-5 weeks in sediments) of the harpacticoid copepod Attheyella crassa makes it suitable for multigenerational exposure studies. Here, A. crassa copepods were exposed for 60 and 120 days to naturally contaminated sediments (i.e., Svindersviken and Trosa; each in a concentration series including 50% contaminated sediment mixed with 50% control sediment and 100% contaminated sediment), and for 120 days to control sediment spiked with copper. We assayed changes in F(ST) (fixation index), which indicates if there is any population subdivision (i.e., structure) between the samples, expected heterozygosity, percent polymorphic loci, as well as abundance. There was a significant decrease in total abundance after 60 days in both of the 100% naturally contaminated sediments. This abundance bottleneck recovered in the Trosa treatment after 120 days but not in the Svindersviken treatment. After 120 days, there were fewer males in the 100% naturally contaminated sediments compared to the control, possibly caused by smaller size of males resulting in higher surface: body volume ratio in contact with toxic chemicals. In the copper treatment there was a significant decrease in genetic diversity after 120 days, although abundance remained unchanged. Neither of the naturally contaminated sediments (50 and 100%) affected genetic diversity after 120 days but they all had high within treatment F(ST) values, with highest F(ST) in both 100% treatments. This indicates differentiation between the replicates and seems to be a consequence of multi-toxicant exposure, which likely caused selective mortality against highly sensitive genotypes. We further assayed two growth-related measures, i.e., RNA content and cephalothorax length, but none of these endpoints differed between any of the treatments and the control. In conclusion, the results of the present study support the hypothesis that toxicant exposure can reduce genetic diversity and cause population differentiation. Loss of genetic diversity is of great concern since it implies reduced adaptive potential of populations in the face of future environmental change.

Survey of estrogenic and androgenic disruption in Swedish coastal waters by the analysis of bile fluid from perch and biomarkers in the three-spined stickleback.

The potential for endocrine disruption close to sewage treatment plant and pulp mill effluent discharge points along the Swedish Baltic Sea coast was explored using a dual survey strategy employing two stationary fish species. The levels of vitellogenin and spiggin as biomarkers of endocrine disruption were determined in juvenile three-spined sticklebacks (Gasterosteus aculeatus L.) together with the sex ratios and the presence of intersex. As an indication of exposure, estrogenic and androgenic substances were analysed by GC-MS in bile from perch (Perca fluviatilis L.). Spiggin and vitellogenin levels in juvenile three-spined sticklebacks were generally low, and, for most sampling sites no deviation in gonad type ratios were observed. No remarkable levels of natural or synthetic estrogens or androgens were observed in bile fluid from perch, while bisphenol A and 4-nonylphenol were detected in perch from both reference sites and exposed sites. Taken together, the results did not indicate estrogenic or androgenic disruption in the investigated waters.

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.

Silica gel as a particulate carrier of poorly water-soluble substances in aquatic toxicity testing.

Aquatic toxicity tests were originally developed for water-soluble substances. However, many substances are hydrophobic and thus poorly water-soluble, resulting in at least two major implications. Firstly, toxicity may not be reached within the range of water solubility of the tested compound(s), which may result in the formation of solids or droplets of the tested substance and consequently an uneven exposure. Secondly, because of multi-phase distribution of the tested substance it may be complicated to keep exposure concentrations constant. To overcome such problems, we have introduced silica gel as a particulate carrier in a toxicity test with the benthic copepod Nitocra spinipes. The main objective of the current study was to evaluate whether a controlled exposure could be achieved with the help of silica gel for testing single poorly water-soluble substances. A secondary objective was to evaluate whether an equilibrium mass balance model could predict internal concentrations that were consistent with the toxicity data and measured internal concentrations of two model hydrophobic substances, i.e., the polybrominated diphenyl ethers BDE-47 and BDE-99. Larval N. spinipes were exposed for 6 days to BDE-47 and BDE-99, respectively, in the silica gel test system and, for comparative reasons, in a similar and more traditional semi-static water test system. Via single initial amounts of the model substances administered on the silica gel, effects on both larval development and mortality resulted in higher and more concentration-related toxicity than in the water test system. We conclude that the silica gel test system enables a more controlled exposure of poorly water-soluble substances than the traditional water test system since the concentration-response relationship becomes distinct and there is no carrier solvent present during testing. Also, the single amount of added substance given in the silica gel test system limits the artefacts (e.g., increased chemical load in test system) that a semi-static renewal may introduce when testing substances that partition to non-water phases. However, measured and modelled internal concentrations did not match toxicity, which may indicate that chemical equilibrium was not reached during the test. Further experiments are thus needed to explain the processes behind the observed positive effects of silica gel and a kinetic model would likely also be more appropriate to describe the concentrations and distributions in the two test systems.

A multilevel approach to predict toxicity in copepod populations: assessment of growth, genetics, and population structure.

One of the goals of environmental risk assessment (ERA) is to understand effects of toxicant exposure on individual organisms and populations. We hypothesized that toxicant exposure can reduce genetic diversity and alter genotype composition, which may ultimately lead to a reduction in the average fitness of the exposed population. To test this hypothesis, we exposed a copepod, Nitocra psammophila, to a toxic reference compound and assayed resulting alterations in genetic structure, i.e. expected heterozygosity and percent polymorphic loci, as well as other population- and fitness-related measures, i.e. population abundance, demographic structure and juvenile growth. The copepods were exposed to 0.11-1.1 microg of the pentabromo-substituted diphenyl ether (BDE-47) mg(-1) freeze-dried algae for 24 days (i.e. >1 generation). There was no significant decline in total population abundance. However, there were significant alterations in population structure, manifested as diminished proportion of nauplii and increased proportion of copepodites. In addition, individual RNA content in copepodites decreased significantly in exposed individuals, indicating declined growth. Finally, in the exposed populations, heterozygosity was lower and genotype composition was altered compared to the controls. These results therefore confirm the hypothesized reduction in overall genetic variability resulting from toxicant exposure. Multilevel approaches, such as the one used in the present study, may help unravel subtle effects on the population level, thus increasing the predictive capacity of future ERA.

Ten challenges for improved ecotoxicological testing in environmental risk assessment.

New regulations, in particular the new European chemicals legislation (REACH), will increase the demands on environmental risk assessment (ERA). The requirements on efficient ecotoxicological testing systems are summarized, and 10 major issues for the improvement of ERA practices are discussed, namely: (1) the choice of representative test species, (2) the development of test systems that are relevant for ecosystems in different parts of the world, (3) the inclusion of sensitive life stages in test systems, (4) the inclusion of endpoints on genetic variation in populations, (5) using mechanistic understanding of toxic effects to develop more informative and efficient test systems, (6) studying disruption in invertebrate endocrine mechanisms, that may differ radically from those we know from vertebrates, (7) developing standardized methodologies for testing of poorly water-soluble substances, (8) taking ethical considerations into account, in particular by reducing the use of vertebrates in ecotoxicological tests, (9) using a systematic (statistical) approach in combination with mechanistic knowledge to combine tests efficiently into testing systems, and (10) developing ERA so that it provides the information needed for precautionary decision-making.

The juvenile three-spined stickleback (Gasterosteus aculeatus L.) as a model organism for endocrine disruption. I. Sexual differentiation.

Juvenile three-spined stickleback (Gasterosteus aculeatus L.) is introduced as a unique model organism for both androgenic and oestrogenic endocrine action. Intersex is often used as an indicator for disruption of sexual differentiation in fish exposed to different kinds of effluents from human activities. In wild fish it has exclusively been reported in terms of feminisation due to xenoestrogens in the environment. The assumption that the intersex individuals are feminised genetic males can only be proven by genetic sex identification of the intersexual individuals. Intersex and gonadal sex reversal were induced in three-spined sticklebacks by treatment with natural and synthetic steroid hormones. Juvenile sticklebacks were exposed to three nominal concentrations of 17 beta-oestradiol (E2); i.e. 0.01, 1.0 and 10.0 microg/L; which were administered to the water either continuously from hatching to the end of the experiment (39-58 days post hatch), during the first 2 weeks after hatching only, from 14 days after hatching onwards, or during the chorionated embryo stage until hatching. Other groups were exposed to 17 alpha-ethinylestradiol (EE2) at 0.05 microg/L and 17 alpha-methyltestosterone (MT) at 1.0 microg/L (nominal concentrations). MT was applied continuously, during the first 2 weeks post hatch only, or from 14 days after hatching onwards. Gonad histology was examined and the genetic sex was identified with male sex-linked PCR markers. Treatment with oestrogens caused feminisation at the two highest E2 concentrations and with EE2. Exposure to E2 before hatching had no effect. Intersexual individuals from oestrogen treatments were genetic males. The genetic sex marker identified apparent total reversal of the gonad type of genetic males. Treatment with MT did not reveal a clear picture, since intersex was observed in both genetic females and males. MT also caused severe testis abnormalities, mainly the development of large branched cavities with unidentified origin. The process of sex differentiation is most sensitive to the influence of external steroids during the first 2 weeks after hatching. A lower incidence of intersex could also be induced in sticklebacks exposed from 14 days after hatching by E2 treatment, but not with MT. The combination of gonad histopathology with genetic sex identification in juvenile sticklebacks is suggested as a tool for detecting endocrine disruption in laboratory studies, and might become very useful in field surveys.

The juvenile three-spined stickleback (Gasterosteus aculeatus L.) as a model organism for endocrine disruption II--kidney hypertrophy, vitellogenin and spiggin induction.

This study investigated the suitability of juvenile three-spined sticklebacks, Gasterosteus aculeatus L., for detecting both androgen- and oestrogen-induced endocrine disruption. The investigated endpoints were kidney hypertrophy and the induction of the protein markers spiggin and vitellogenin. Juveniles were exposed to steroid hormones 17 beta-oestradiol (E2: nominal 0.01, 1.0 and 10 microg/L), 17 alpha-ethinylestradiol (EE2: nominal 0.05 microg/L) and 17 alpha-methyltestosterone (MT: nominal 1.0 microg/L) from the day of hatching until the termination of the experiments between 39 and 58 days after hatching. E2 (10 microg/L) and MT were applied during different time windows: (a) 14 days after hatching only and (b) continuously with start 14 days after hatching. Kidney hypertrophy is an androgen-dependent secondary sexual character in adult male sticklebacks and corresponds to the production of the glue protein spiggin during the breeding season. The kidneys were hypertrophied and spiggin levels were elevated in juvenile sticklebacks after treatment with MT. Paradoxically, slightly elevated spiggin levels and kidney hypertrophy were observed also in fish treated with high dose E2. Levels of vitellogenin, the oestrogen-inducible yolk precursor protein, were elevated in juvenile sticklebacks after E2 medium and high dose and EE2 treatment. The tested endpoints are suitable for the study of endocrine disruption in juvenile sticklebacks, a fish species that is easy to handle in laboratory and relevant for temperate geographical regions.

Influence of nutrient levels on uptake and effects of mercury, cadmium, and lead in water spinach.

In Southeast Asia the aquatic macrophyte water spinach (Ipomoea aquatica Forsk.) is a popular vegetable that is cultivated in freshwater courses. These often serve as recipients for domestic and other sorts of wastewater that often contain a variety of pollutants, such as heavy metals. In addition, fertilizers are frequently used where water spinach is cultivated commercially for the food market. To estimate the importance of ambient nutrient concentrations for accumulation of mercury (Hg), cadmium (Cd), and lead (Pb) in water spinach, plants were exposed to nutrient solutions of different strength and with varying metal concentrations. Metal-induced toxic effects, which might possibly affect the yield of the plants, were also studied. The lower the nutrient strength in the medium was, the higher the metal concentrations that accumulated in the different plant parts and the lower the metal concentration in the medium at which metal-induced toxic effects occurred. Accordingly, internal metal concentrations in the plants were correlated to toxic effects. Plants exposed to metals retained a major proportion of the metals in the roots, which had a higher tolerance than shoots for high internal metal concentrations.

Inhibition of larval development of the marine copepod Acartia tonsa by four synthetic musk substances.

A nitro musk (musk ketone) and three polycyclic musks (Tonalide, Galaxolide and Celestolide) were tested for acute and subchronic effects on a marine crustacean, the calanoid copepod Acartia tonsa. Sublethal effects on A. tonsa larvae were investigated with a rapid and cost effective bioassay, which is based on the easily detectable morphological change from the last nauplius to the first copepodite stage during copepod larval development. The inhibition of larval development after 5 days exposure was a very sensitive endpoint, with 5-d-EC(50)-values as low as 0.026 mg/l (Tonalide), 0.059 mg/l (Galaxolide), 0.066 mg/l (musk ketone) and 0.160 mg/l (Celestolide), respectively. These values were generally more than one order of magnitude below the 48-h-LC(50)-values found for adults, which were 0.47 mg/l (Galaxolide), 0.71 mg/l (Celestolide), 1.32 mg/l (musk ketone) and 2.5 mg/l (Tonalide). Since the synthetic musks strongly inhibited larval development in A. tonsa at low nominal concentrations, they should be considered as very toxic. The larval development test with A. tonsa is able to provide important aquatic toxicity data for the evaluation of synthetic musks, for which there is little published ecotoxicological information available regarding Crustacea. It is suggested that subchronic and chronic copepod toxicity tests should be used more frequently for risk assessment of environmental pollutants.

Accumulation of heavy metals in water spinach (Ipomoea aquatica) cultivated in the Bangkok region, Thailand.

The aquatic plant water spinach (Ipomoea aquatica), either wild or cultivated, is found throughout Southeast Asia and is a widely consumed vegetable in the region. Many of the waters where I. aquatica grows serve as recipients for domestic and other types of wastewater. Because these waters contain not only nutrients, but often also a wide variety of pollutants such as heavy metals from various human activities, many people risk intoxication. To estimate the accumulation of lead (Pb), cadmium (Cd), total mercury (total Hg), and methylmercury in I. aquatica and the potential hazard to human health via consumption, nine sites for cultivation of I. aquatica in the greater Bangkok region of Thailand were sampled. At seven of the sites, I. aquatica was cultivated for the local food market. The concentrations of methylmercury, total Hg, Pb, and Cd in I. aquatica were 0.8 to 221, 12 to 2,590, 40 to 530, and < or = 10 to 123 microg/kg dry weight, respectively. At all sites at least one element showed relatively high concentrations and no reference site could be established. From threshold values for highest tolerable intake of these metals by humans and information about consumption of I. aquatica among local people, Pb and Cd concentrations in I. aquatica do not seem to be a direct threat to human health. However, concentrations of Hg were very high at some sites, and were higher in leaves (highest mean value: 1,440 microg/kg dry wt) than in stems (highest mean value: 422 microg/kg dry wt). This might be a threat, especially to children and fetuses, because Hg in I. aquatica was composed of methylmercury, partly or totally, at most sites to 11% or less and at one site from 50 to 100%. At the latter site, I. aquatica was not cultivated for the food market. Because other food sources, such as fish, may have high concentrations of methylmercury, these results indicate a need for monitoring of Hg, especially methylmercury, in different foodstuffs in the region.