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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. © Springer Science+Business Media 2013. Language of Original Document: English

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.

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. Environ. Toxicol. Chem. 2011;30:2353–2362. © 2011 SETAC

The main objective of the current study was to assess the impact of pleasure boat activities on harbour sediment quality in the Stockholm area. Sediment contamination is a growing ecological issue, and there is consequently a need to use sediment bioassays in combination with chemical analysis to determine the impact on the ecosystem. To generate sediment toxicity data relevant for the Baltic Sea, a secondary objective was to further develop and evaluate two well-established bioassays for saltwater, with the macroalga Ceramium tenuicorne and the crustacean Nitocra spinipes, to be useful also for toxicity testing of whole sediment. A major concern has been to minimize any manipulation of the sediments. A third objective was to assess whether a simple leaching procedure could be used to simulate sediment toxicity by comparing results from whole sediment and leachate tests. Surface sediments (0–2 cm) from five different types of pleasure boat harbours were collected. Chemical measurements of boat related compounds, i.e. tin organic substances (tributyltin (TBT), dibutyl tin, and monobutyl tin), polyaromatic hydrocarbons (PAHs), copper, zinc, lead, tin and irgarol were conducted. The sediments were tested for toxic effects using the established Microtox® test as well as the developed sediment tests with C. tenuicorne and N. spinipes. The endpoints are growth inhibition for the algal test and for the crustacean test mortality of larvae and rate of development expressed as the ratio between nauplia and copepodites. Two different procedures have been compared with both organisms, i.e. exposure to whole sediment and to leachate. The duration of both tests is around 1 week. All harbour locations were more or less heavily contaminated with remnants from use of anti-fouling paints. The sediment in a smaller marina (ca. 250 boats) contained the highest levels of TBT (max 1,400 µg/kg dry weight (dw)), whereas the centre of Stockholm City had the highest concentrations of all measured metals (max values Cu 252, Pb 830, Sn 25 and Zn 600 µg/kg dw) as well as high concentrations of total PAH (18 mg/kg dw). All three organisms were well suited to test the toxicity of contaminated sediments and were able to discriminate between more or less polluted sediments. The sediments in the smaller marina were most toxic along with sediments adjacent to slipways and sediments from the centre of Stockholm. No significant difference was found between the two different procedures for the algal tests. The whole sediment test was significantly more toxic to N. spinipes than the test using leachate. Our results show that in spite of prohibition for many years to use tin organic substances and copper, boat activities still contribute with high concentrations of these toxic substances from anti-fouling paints in the surface sediment and that these have effects on organisms normally living in this environment. The present study has also demonstrated an expansion of two well-established toxicity tests for the water phase to be practical also for sediment toxicity testing. This will further increase the usefulness of these test organisms and methods for hazard and risk assessment in a wide range of environments.

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.

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.

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.

In order to give adequate support to risk managers, new risk assessment methods should be developed that are (1) scientifically sound, (2) simplified, and (3) suited for precautionary risk management. In this Perspective we propose that the notion of a precautionary default can be a useful tool in the development of such methods. A precautionary default is a cautious or pessimistic assumption that is used in the absence of adequate information and that should be replaced when such information is obtained. Furthermore, we point out some promising research areas for the development of such indicators, viz. connections between chemical characteristics such as persistence and effect parameters, monitoring of contaminants in polar regions, monitoring of contaminants in breast milk, application of results from (human) toxicology in ecotoxicology and vice versa, (eco) toxicological test systems that are sensitive to effects on reproduction, and the application of bioinformatic methods to complex data, both in genomic research and in ecotoxicology. We conclude that precautionary decision-making does not require less science, but to the contrary it requires more science and improved communication between scientists and risk managers.

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.

A wide variety of chemicals and biological products are used to treat the water and sediment of ponds in semi-intensive and intensive south-east Asian shrimp farming. These products are also often used in shrimp hatcheries and to disinfect equipment for shrimp pond management. In spite of the size and importance of the shrimp farming industry in several south-east Asian countries, documentation of the quality and quantity of chemicals and biological products used during farming is scarce. This paper is a compilation of the literature available on substances used in shrimp farming, and the possible environmental effects of these products are analysed to the extent allowed by the limited information. The role of shrimp farm managers, the chemical industry, governments, inter-governmental organisations and scientists in the development of a sustainable practice is discussed. It is concluded that shrimp farmers should reduce the use of chemicals and biological products because of the risks to the environment, human health and to production, and also, because many chemicals and biological products used in pond management have not been scientifically shown to have a positive effect on production. Clearly, the use of some chemicals, i.e. certain antibiotics, poses a risk of danger towards human health. Some chemicals used in shrimp farming, such as organotin compounds, copper compounds, and other compounds with a high affinity to sediments leave persistent, toxic residues, and are likely to have a negative impact on the environment. However, to assess the reality of these risks, substantial new information about the quantity of chemicals used in marine south-east Asian shrimp farming is needed.

The toxicity of nine effluents from Swedish kraft-pulp mills using conventional, elemental chlorine-free (ECF) and totally chlorine-free (TCF) bleaching processes was studied. Effluents represented six different processes: conventional-softwood 30% ClO2; ECF-softwood; TCF-hardwood, for which all the samples were taken before and after secondary treatment; conventional-softwood 8% ClO2; TCF-softwood O3/H2O2; and TCF-softwood H2O2. Comparison of effluents, which had not undergone secondary treatment, suggested that those from mills using conventional bleached pulp were the most toxic, with toxic emission factors (TEF) ≈ 1,000 according to the Microtox® test (AZUR Environmental, Carlsbad, CA, USA). In the acute Nitocra spinipes test, the conventional-softwood 8% ClO2 and the ECF-softwood effluents before secondary treatment were the most toxic, with TEF ≈ 700. In the reproduction test, N. spinipes was two to three times more sensitive than in the lethal test. The species most sensitive to the effluents investigated was the macroalga Ceramium strictum, with TEF values up to 3,000. The conventional effluent using a substitution of 8% ClO2 was the most toxic, and the TCF effluents were the least toxic. Degradation treatment of the ECF and TCF effluents for 28 d in the laboratory did not eliminate acute lethal effects on N. spinipes and the Microtox bacterium.

A bleached sulfate integrated pulp and paper mill producing printing and writing paper from mixed tropical hardwood and bamboo was studied. The mill uses a "conventional bleaching sequence," C-E-H1-H2, with an average molecular chlorine consumption of 50 kg per ton of air-dried pulp (ADP). The content of polychlorinated dibenzofurans (PCDFs) and dibenzo-p-dioxins (PCDDs) in the bleaching filtrate in terms of the nordic toxicity equivalent (N-TEQ) was 33.5, 1.15, 0.56, and 0.014 pg/L for the E, C, H1, and H2 bleaching stages, respectively. The corresponding PCDFs and PCDDs loads in ng/t ADP were in the same ranking, i.e., 670, 69, 11.2, and 0.28, respectively. The congener and isomeric pattern of PCDFs and PCDDs of the bleaching filtrate and the bleached pulp was found to be typical for the chlorine bleaching plant effluent. The obtained dioxin load formed in the mill is in agreement with Western studies for the given multiple chlorine of 0.21-0.23. The load is, however, lower than reported discharges from Scandinavian mills using 1980s bleaching technologies, but substantially higher than the discharges from mills with modern bleaching technologies. Modifications in the bleaching plant to reduce molecular chlorine use are necessary to reduce dioxin formation.http://link.springer-ny.com/link/service/journals/00244/bibs/37n3p303.html

The diagram constructed for selection of sampling methods indicates that, for a systematic error, E ≤ 13%, grab sampling (GS) may be used to characterize effluents with variation coefficient of flow ≤ 120% and of contaminant concentrations ≤ 10%. For the whole studied range of variation coefficient of contaminant concentrations (2–82%), time-proportional compositing (TC) method may be applied with E ≤ 10% for effluents characterized by variation coefficients in flow

A wastewater monitoring program, was incorporated into an industrial process control strategy to minimize process upsets and resulting abnormal discharges. In the present study, the program, which is considered as applicable to a wide range of industrial processes, was applied to the Bai Bang Paper Company (BAPACO), a Vietnamese integrated bleached kraft pulp and paper mill. Through environmental auditing the mill was found to discharge high pollution loads into the environment. A large portion of this discharge was material loss, fiber and spent cooking liquor, associated with accidental discharges and spills. These can be prevented to benefit both the mill economics and the environment. Rapidly assessed pollution parameters (RAPPs) were used to express the losses, settleable solids after 15 min settling (SetS15) for fiber, and conductivity (Cond) for cooking chemical losses. Principal component analysis (PCA) was used on effluent monitoring data to determine periods of ‘normal’ discharges. Average RAPPs during these ‘normal’ periods were used as targets for control.

Ash from incinerated waste consists mainly of a complex mixture of metals and other inorganic elements and should be classified based on its inherent hazardous effects according to EUs Waste Framework Directive. In a previous study, we classified eight eluates from ash materials from Swedish incineration plants, both chemically and ecotoxicologically (using bacteria, algae, crustacean and fish). Based on measured concentrations in the eluates together with literature acute toxicity data on the crustacean Nitocra spinipes we identified six elements (i.e. Zn, Cu, Pb, Al, K and Ca) potentially responsible for the observed ecotoxicity. However, comparing the used test methods with N. spinipes, the acute test was relatively insensitive to the eluates, whereas the (sub)chronic test (i.e. a partial life cycle test, investigating larval development ratio) was very sensitive. The overall aim of this follow-up study was to verify if the pinpointed elements could be responsible for the observed (sub)chronic toxicity of the eluates. Individual effect levels (i.e. NOEC values) for these six elements were therefore generated using the (sub)chronic test. Our results show that for six of the eight eluates, the observed ecotoxicity can be explained by individual elements not classified as ecotoxic (Al, K and Ca) according to chemical legislation. These elements will not be considered using summation models on elements classified as ecotoxic in solid material for the classification of H-14, but will have significant implications using ecotoxicological test methods for this purpose.

Abnormalities of a gill cover bone, the operculum, were found in perch Perca fluviatilis from an area affected by pulp mill effluents in the Gulf of Bothnia, Baltic Sea, Sweden. One of the abnormalities was expressed as a shortening of the distal part of the operculum, and was termed opercular deformity. This deformity was quantified by taking two measurements of the operculum. The second abnormality was manifested as craterous formations on the operculum. Both abnormalities were studied using a sample of opercula collected in the pulp mill effluent area between the years 1982 and 1989. The maximum prevalences of the abnormalities, 34% for the craterous formation and 20% for the opercular deformity, were found in 1983 and 1984, respectively. In a reference area the prevalences were 1% and 1.4%, respectively. After 1984, the prevalences decreased together with prevalences of other fish diseases and deformities in the same area. This coincided with a reduction in concentrations of effluent. The observed high prevalence of the opercular deformity and the craterous formations were attributed to the effects of the pulp mill effluent. In addition, an analysis of the biochemical composition opercula from perch caught in another pulp mill effluent area showed a dose-dependent alteration of several constituents, indicating a relationship with exposure to the effluent. This study shows the potential of opercular abnormalities to monitor effects of pulp mill effluents, and also, it is suggested, for retrospective studies on large samples of opercula used for age determination in ecological studies of fish.

During a 2-year experimental period female baltic salmon (Salmo salar) were fed pellets impregnated with oil extracted from Baltic herring (Clupea harengus). This extract contained lipid-soluble xenobiotics present in Baltic herring, which constitute a major part of the natural diet of Baltic salmon. The fish were examined at the time of ovulation in November each year. After 2 years of feeding, the load of polychlorinated dibenzo-paradioxins and furans in the exposed group was about twice that in the control group, but still low compared with concentrations in feral Baltic salmon. In spite of the relatively low exposure level, several vital biochemical functions were disturbed in the treated fish. Organic skeletal variables were affected indicating that the bone metabolism had been altered. Furthermore, the activities of enzymes involved in steroid biosynthesis were affected, which could lead to disturbances in reproductive functions. Splenocytes from exposed fish sampled in November 1990 showed a reduced mitogenic response, indicating that their immune system was suppressed. Feeding the salmon with pollutant-impregnated pellets also resulted in an induction of the hepatic ethoxyresorufin-O-deethylase (EROD) activity after only 6 weeks of exposure. Likewise, morphological abnormalities, i.e. hypertrophic hepatocytes and various stages of hepatic degeneration, were already apparent after 6 weeks of exposure. However, no EROD induction or morphological responses were recorded at the second and third sampling event, i.e. after one and 2 years of exposure, respectively. this could indicate that some physiological functions may adapt to a restricted xenobiotic load.

Within the EU, ash should be classified by its inherent hazardous effects under criterion H-14 (ecotoxic) in the Directive on waste (2008/98/EC). Today, however, there are no harmonized quantitative criterions for such a classification, but it is stated that biological test systems can be used. In this study seven ash materials were leached and characterized, both biologically and chemically. The objectives were to evaluate if (a) clear concentration-response relationships could be achieved for the selected toxicity tests (bacteria, algae, crustacean and fish), (b) some test(s) are generally more sensitive and (c) the toxic responses were consistent with the chemical analyzes. Interestingly, our results indicate that high concentrations of non-hazardous components (Ca, K) influenced the toxicity of almost all ash eluates, whereas hazardous components (e.g. Zn, Pb) only influenced the toxicity of the eluates ranked as most hazardous. If considering both hazardous and non-hazardous substances, the observed toxic responses were relatively consistent with the chemical analyzes. Our results further showed that the (sub)chronic tests were much more sensitive than the acute tests. However, the use of extrapolation factors to compensate for using the less sensitive acute tests will likely lead to either over- or underestimations of toxicity. Our recommendation is therefore that classification of waste according to H-14 should be based on (sub)chronic test data. Finally, given that treatment of the eluates prior to toxicity testing has a major significance on the concentration and speciation of released substances, further studies are needed in order to propose a relevant testing scheme.

In this chapter, we will evaluate how and if a 3R based approach can be applied in testing of ecotoxicity of chemicals. The 3R approach (reduce-refine-replace) is a strategy to reduce or totally abolish the use of experimental animals in favour of alternative methods. We review the current status of alternatives in aquatic ecotoxicology and how well they perform in comparison with current in vivo methods. We will conclude that theoretically can alternative methods and approaches replace animal based testing but the way to reach this goal is long. A strong development of more sophisticated alternative methods is needed focusing on specific and physiologically/toxicologically relevant ­endpoints. We underline the importance to gain more information on toxic mechanisms of chemicals. New exciting biochemical techniques are waiting around the corner, e.g. in the genomics area and they need to be integrated in future test paradigms.

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.

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.

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.

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.

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.

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.

Between January 1999 and December 2001, the European Community project COMPREHEND was performed. The overall aim of COMPREHEND was to assess endocrine disruption in the aquatic environment in Europe, consequent to effluent discharge, with emphasis on estrogenic activity. COMPREHEND demonstrated the widespread occurrence of estrogenic effluents across Europe and presented evidence of impacts on a range of wild fish species. Using a variety of bioassays in combination with chemical analytical methods, estrogenic steroids of human origin from domestic wastewater effluents were identified as the most pervasive problem, although alkylphenols may be important estrogenic components of some industrial effluents. New tools have been developed for the identification of estrogenic effluents, and recommendations are made for the improvement of existing techniques. We have shown that individual fish within natural populations may be feminized to varying degrees, but it has not been possible to show, using traditional fish population parameters, that the survival of fish populations is threatened. However, laboratory-based fish life-cycle studies demonstrate the sensitivity of fish to estrogen (and androgen) exposure and how this might lead to complex (and potentially damaging) genetic changes at the population level. New approaches to this problem, utilizing recent advances made in the field of molecular and population genetics, are recommended. Finally, a study of estrogenic and androgenic activity of waste waters during the treatment process has shown that some of the existing wastewater treatment technologies have the potential to eliminate or minimize the hormonal activity of the final effluent.

In recent years, reports have described endocrine-disruptive effects of environmental oestrogens in fish, but little is known about similar effects in crustaceans. The objective of the present study was therefore to examine whether the oestrogens 17-beta-oestradiol, 17-alpha-ethynylestradiol and diethylstilbestrol (DES), could affect mortality, larval development rate, fecundity and sex ratio in the sexually reproducing harpacticoid copepod Nitocra spinipes. Newly released nauplii (24-h old) were exposed to 1/1,000, 1/100 and 1/10 (nominal concentrations) of each oestrogen's 96 h-LC50 value foror = 18 days at 22 +/- 1 degrees C. The percentage of gravid females and the number of developed copepodites were both reduced at 0.03 mg l(-1) DES, although the latter response was not significant. None of the other two oestrogens induced any measurable effects. Since the only observed significant response appeared at a DES concentration no more than 10 times below the 96 h-LC50 value, there is no evidence of endocrine-disruptive activity in N. spinipes exposed to oestrogens.

The diagram constructed for selection of sampling methods indicates that, for a systematic error, E≤13%, grab sampling (GS) may be used to characterize effluents with variation coefficient of flow ≤120% and of contaminant concentrations ≤10%. For the whole studied range of variation coefficient of contaminant concentrations (2–82%), time-proportional compositing (TC) method may be applied with E≤10% for effluents characterized by variation coefficients in flow

The ongoing conflict between the highlanders and the lowlanders at Mae Soi, Jomthong, Chiang Mai, was explored to identify its causes. The study shows that perceptions about the conflict by both groups are at variance. Lowlanders perceive the conflict to be environmental, i.e. due to deforestation, water reduction, and contamination. Highlanders attribute social, political and economic causes to the conflict. The study also finds a high dependency on the forest for fuel wood, fodder and building materials, and a low dependency on the forest for food products. It can be concluded that environmental effects of the agriculture practices in the hill areas have reduced both the forested area and its quality. Water reduction has occurred but it is not possible to single out one particular group as responsible for this change. Cash crops introduced among the hill tribes to replace opium poppy cultivation need pesticides (both organochlorines and organophosphates are used), but pesticide concentrations found in downstream river water in this study are estimated to be within safe limits for drinking purposes. The conflict is both environmental and socioeconomic. Resolving the conflict thus requires a broader perspective than resolving environmental problems alone.

Chemical analyses of organochlorine compounds in effluents, sediment, and biota samples were done for the Bai Bang Paper Company (BAPACO), a bleached kraft pulp and paper mill in Vietnam. High chlorine consumption in the bleaching process resulted in a high specific discharge of adsorbable organic halogens (AOX), 4.7 kg/ton of air dried pulp (ADP), and polychlorinated phenolic compounds (31 g/ton ADP) in the effluent. The extractable organochlorine content in the accumulated fiber sludge of the sedimentation tank (70–100 μg/g) was more than 100 times higher than the sediment from the receiving river. Chlorinated compounds including dioxins were detected at low levels in sediment and rice, crab, mollusc and fish samples. Significant amounts of the chlorinated persistent compounds are probably transported by the Red River to the river mouth and coastal area. Heavy metal content was high in sediment and mollusc samples from the receiving rice field and fish pond. There may be a possible risk for human intoxication of the metals through the aquatic food chain via the mollusc but not via the rice.

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.

Fourhorn sculpin (Myoxocephalus quadricornis) were exposed for one year to two concentrations (0.1 and 1 vol %) of an artificial heavy metal-containing effluent from a sulfide ore smelter. At the end of the experiment, the fish were examined for alterations in vertebral structures, for otolith asymmetry and for physiological disturbances. Fourhorn sculpin exposed to the effluent displayed ‘black tail’, spinal curvature and increased frequency of vertebral deformities, particularly in the tail region. Comparison of weight differences between right and left otoliths revealed increased weight asymmetry among exposed fish. The effluent also caused a dose-dependent elevation of the ascorbic acid content in the liver and a strong inhibition of the enzyme δ-aminolevulinic acid dehydratase in the red blood cells. In addition, the effluent gave rise to an increased plasma potassium level and a decreased plasma chloride level. These physiological alterations indicate that the complex metal-containing effluent has profound effects on metabolic processes and ion balance. The results also confirm effects previously shown in field studies on fish living in the vicinity of the sulfide ore smelter.

In a laboratory investigation, fourhorn sculpin (Myoxocephalus quadricornis) were exposed for 5-9 months to waste water from pine and birch lines from a bleached kraft pulp plant. The bleached kraft mill effluents (BKME) affected both the carbohydrate metabolism and the xenobiotic metabolism. Thus, an elevated muscle glycogen content in fish exposed to effluent from the pine pulp line suggests a metabolic imbalance. A liver enlargement and a strong elevation of the hepatic cytochrome P-450-dependent ethoxyresorufin-O-deethylase activity after exposure to the bleached pine pulp effluents indicate the presence of cytochrome P-450-inducing agents in the BKME. The results also demonstrate that many physiological test parameters may be used as good indicators of sublethal disturbances in fish to BKME exposure.

Adult minnows, Phoxinus phoxinus, were orally dosed with PCB (Clophen A50) for 40 days and were studied for 300 days for long-term sublethal effects. Clophen A50 was found to have a growth promoting effect and demonstrated several effects on reproduction. Swimming performance, studied by the rotatory-flow technique, was not affected at the concentrations that gave effects on growth and reproduction. The clearance of accumulated PCB from the body was slow.

Fourhorn sculpins were exposed to different bleach plant effluents. The relationship between organic compounds, analysed by gaschromatography in the fat of the sculpins, and the spinal deformations observed in the sculpins have been investigated. A classification in sculpins with and without spinal deformations could be observed using the chromatographic data.

The ion balance in the flounder ( Ptatichthys flesus L.) was studied after 4 and 9 wk of exposure to sublethal cadmium levels (5–500 μg Cd/l) in brackish water. The cadmium exposure had no effect on the major blood plasma electrolytes, sodium and chloride, indicating an intact osmoregulation. In contrast, cadmium seriously affected the regulation of other ions. Potassium and calcium concentrations showed a strong and dose-dependent depression in blood plasma, whereas the plasma concentrations of inorganic phosphate and magnesium were significantly elevated. It is suggested that these ion disturbances might be associated with cadmium-induced pathological changes in ion-regulating tissues. The reduced concentrations of plasma calcium and potassium might be responsible for neuromuscular disturbances, such as hyperexcitability, spasms and tetanic body contractions, observed in some flounders exposed to the highest cadmium concentration (500 μg Cd/l). In spite of the pronounced disturbance of the calcium metabolism, the spinal columns of the cadmiumexposed flounders did not show any signs of demineralization, fractures or other deformations. This might be due to the fact that the flounder probably has an acellular bone tissue, which only to a small degree seems to be affected by the disturbed calcium balance. It is suggested that fish species with acellular bone tissue run a minor risk of suffering from skeletal damage after cadmium exposure than fish species with an active cellular bone tissue.

The effects of tetrachloro-1,2-benzoquinone (TCQ), a component in bleached kraft mill effluents (BKME), on vertebral and physiological parameters were investigated in juvenile fourhorn sculpin, Myoxocephalus quadricornis L. After about 4.5 months of exposure to 0.1 and 0.5 mg TCQ/liter in 7% salinity brackish water, the fish demonstrated vertebral deformities, aberrant mechanical properties of the vertebrae, effects on white and red blood cell counts, enhanced activity of delta-aminolevulinic acid dehydratase in erythrocytes, and increased levels of ascorbic acid in the liver. The effects are discussed in relation to those previously observed in fish exposed to complex BKME in the field and the laboratory.

The harpacticoid copepod Nitocra spinipes has been tested for acute toxicity of 12 metal chlorides in brackish water. Their order of toxicity, expressed as 96 h LC50, was in good agreement with other investigations performed in freshwater and seawater. The 96 h LC50-values were of intermediate levels compared to these two environments. The organochlorines p,p′-DDE and p,p′-DDE methyl sulphone were tested for effects on reproduction and mortality during two weeks, and it was found that p,p′-DDE was the most toxic. It is concluded from the investigation that N. spinipes is a suitable toxicity test organism in brackish water.

In a laboratory investigation fourhorn sculpin (Myoxocephalus quadricornis) were exposed for 5-9 months to wastewater from pine and birch lines from a bleached kraft pulp plant. This long-term exposure to bleached kraft mill effluent (BKME) affected the hematology, the ion balance, and the vertebral structure. Decreased values for hematocrit and hemoglobin and a reduced red blood cell count, as well as increased levels of methemoglobin, indicated disturbances in the red blood cell status. The white blood cell picture was not significantly affected by the BKME exposure. Decreased levels of potassium and chloride ions in the blood plasma in some of the exposed fish suggest an impaired ability to maintain ion homeostasis. Elevated frequencies of vertebral deformations in fish exposed to BKME confirm previous observations of vertebral damage in feral fourhorn sculpin caught in the receiving body of water of the same bleached kraft pulp industry. Many of the parameters analyzed in this investigation may be used as health indicators in future laboratory and field studies on fish exposed to BKME.

The growth of the Ephemeroptera species Heptagenia fuscogrisea Retzius, Leptophlebia marginata L. and Ephemerella mucronata Bengtsson was studied from the middle of September 1967 to the middle of May 1968 in the River Ricklean in northern Sweden. Sampling was performed also during periods with ice cover. Growth was estimated from four different body measures and proceeded fairly steady through all the winter for the three species. There were no indications of changed body proportions during the investigation period.

The bioaccumulation potential in bleaks ( Alburnus alburnus , Pisces) and acute toxic and reproductive effects on Nitocra spinipes (Crustacea), of 4,5,6-trichloroguaiacol (I), tetrachloroguaiacol (II) and tetrachlorocatechol (III) was investigated. Continuous flow tests with I and II (10 μg/l) gave a rapid bi-phasic uptake in the fish with I and II reaching a level of 4 μg/g fresh weight after 14 d. An equally rapid excretion was determined with the detection limit of I and II being reached after 10 days in pure water. The 96-h LC50 values for I, II and III to N. spinipes was determined to 5.2, 3.9 and 3.3 mg/l, respectively, in static tests. The fecundity of N. spinipes was reduced to 50 % of the control values at 37±6 and 54±4 μ g/l (II) per liter in static and continuous flow tests, respectively.