Mature male goldfish were exposed to different concentrations of the natural hormone 17 beta -estradiol (El). Two methods of exposure were employed, via ingestion at 0, 1, 10 and 100 ug/g food and via the water at 0, 1 and 10 mug/l. The fish were exposed fur 74-28 days during the spawning period. The males were then paired with an artificially induced, spawning female and their sexual behaviour was observed during a 15 min period. The physiological status of the fish was also examined with respect to GSI. presence of milt and spawning tubercles and the blood plasma concentration of E-2. Despite the relatively short exposure period. exposure to physiological levels of E-2 was shown to severely affect the male goldfish reproductive behaviour and physiology. In conclusion, the results from this study and the ability to interpret the effects on this well-studied species, show that the effects of E-2, and possibly other estrogenic EDCs have severe effects at several vital levels of male goldfish reproduction. The results also suggests that the hormone E-2 can act as an endocrine disrupting chemical (EDC) in the environment.

Benthic ecosystems have come under intense pressure, due to eutrophication-driven oxygen decline and industrial metal contamination. One of the most toxic metals is Cadmium (Cd), which is lethal to many aquatic organisms already at low concentrations. Denitrification by facultative anaerobic microorganisms is an essential process to transform, but also to remove, excess nitrate in eutrophied systems. Cd has been shown to decrease denitrification and sequester free sulfide, which is available when oxygen is scarce and generally inhibits complete denitrification (i.e. N2O to N2). In polluted sediments, an interaction between oxygen and Cd may influence denitrification and this relationship has not been studied. For example, in the Baltic Sea some sediments are double exposed to both Cd and hypoxia. In this study, we examined how the double exposure of Cd and fluctuations in oxygen affects denitrification in Baltic Sea sediment. Results show that oxygen largely regulated N2O and N2 production after 21 days of exposure to Cd (ranging from 0 to 500 μg/L, 5 different treatments, measured by the isotope pairing technique (IPT)). In the high Cd treatment (500 μg/L) the variation in N2 production increased compared to the other treatments. Increases in N2 production are suggested to be an effect of 1) enhanced nitrification that increases NO3 − availability thus stimulating denitrification, and 2) Cd successfully sequestrating sulfide (yielding CdS), which allows for full denitrification to N2. The in situ field sediment contained initially high Cd concentrations in the pore water (∼10 μg/L) and microbial communities might already have been adapted to metal stress, making the effect of low Cd levels negligible. Here we show that high levels of cadmium pollution might increase N2 production and influence nitrogen cycling in marine sediments. © 2019 The Authors

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 multi generational 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 FST (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 FST values, with highest FST 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.

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 mu g 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.

Reproductive behaviour of brown trout (Salmo trutta L.) from an anadromous stock was Studied in a large stream water aquarium. Four adult males and two ovulated females were placed in the aquarium together with eight mature male parr. Four of the parr were exposed during the previous 4 days to two concentrations (0.1 or 1.0 mu g l(-1)) of the pyrethroid pesticide cypermethrin (a disrupter of olfactory receptor function) and four of the parr to the solvent ethanol. The behaviour of all fish was followed for 24 h and then blood and milt was collected. Exposure to the higher concentration of cypermethrin disturbed the reproductive behaviour of the parr. They displayed fewer courting events, spent less time near the nesting females and had lower volumes of strippable milt. They also had significantly lower amounts of 11-ketotestosterone (11-KT) in the blood plasma than the control roup. The higher cypermethrin group also had significantly lower levels of all these variables than the 9 lower cypermethrin group, apart from strippable milt that showed no significant differences between two groups. No significant differences in non-reproductive behaviours were observed between any of the groups. In the control fish, there were significant positive Correlations between (a) the number of courting events and the amount of time spent near the female, (b) blood plasma levels of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) and time spent near the female and (c) plasma levels of 17,20 beta-P and the number of courting events. Further, in control fish, higher plasma levels of 17,20 beta-P were observed in part-interacting with a female compared to those with no female contacts. A priming experiment confirmed a previous study that cypermethrin damages olfactory reception. Parr exposed to cypermethrin had significantly lower blood plasma levels of 17,20 beta-P and 11-KT than control males after exposure to ovarian fluid and urine (known to contain reproductive priming pheromones). When ethanol-exposed males were exposed to ovarian fluid and urine they had significantly higher plasma levels of 17,20 beta-P compared to those exposed to water only.

Citalopram is an antidepressant drug, which acts by inhibiting the re-uptake of serotonin from the synaptic cleft into the pre-synaptic nerve ending. It is one of the most common drugs used in treatment of depression, it is highly lipophilic and frequently found in sewage treatment plant effluents and surface waters around the world. Citalopram and other selective serotonin re-uptake inhibitors have, at concentrations that occur in nature, been shown to have behavioural as well as physiological effects on fish and other animals. This study is the result of several different experiments, intended to analyse different aspects of behavioural effects of chronic citalopram exposure in fish. Our model species the three-spine stickleback is common in the entire northern hemisphere and is considered to be a good environmental sentinel species. Female three-spine sticklebacks were exposed to 0, 1.5 and 15μg/l nominal concentrations of citalopram for 21 days and subjected to the novel tank (NT) diving test. In the NT test, the fish exposed to 1.5μg/l, but not the 15μg/l fish made a significantly higher number of transitions to the upper half and stayed there for significantly longer time than the fish exposed to 0μg/l. The 15μg/l group, however, displayed a significantly lower number of freeze bouts and a shorter total freezing time. The test for locomotor activity included in the NT test showed that fish treated with 1.5 and 15μg/l displayed a significantly higher swimming activity than control fish both 5-7 and 15-17min after the start of the experiment. In the next experiment we compared fish exposed to 1.5μg/l and 0.15μg/l to pure water controls with regard to shoaling intensity and found no effect of treatment. In the final experiment the propensity of fish treated with 1.5μg/l to approach an unknown object and aggressive behaviour was investigated using the Novel Object test and a mirror test, respectively. The exposed fish ventured close to the unknown object significantly more often and stayed there for significantly longer time than unexposed fish. The aggression test yielded no statistically significant effects. It is concluded that citalopram changes the behaviour of the three-spine stickleback in a way that is likely to have ecological consequences and that it must not be considered an environmentally safe pharmaceutical.

Selective Serotonin Re-uptake Inhibitors (SSRI) are mood-altering, psychotropic drugs commonly used in the treatment of depression and other psychological illnesses. Many of them are poorly degraded in sewage treatment plants and enter the environment unaltered. In laboratory studies, they have been demonstrated to affect a wide range of behaviours in aquatic organisms. In this study we investigated the effect of a three-week exposure to 0.15 and 1.5 μg/l of the SSRI citalopram dissolved in the ambient water on the feeding behaviour in three-spine stickleback (Gasterosteus aculeatus). Feeding, measured as the number of attacks performed on a piece of frozen bloodworms during a 10-min period, was reduced by 30–40% in fish exposed to both 0.15 and 1.5 μg/l citalopram. The effects of the environmentally relevant concentration 0.15 μg/l on feeding, an important fitness characteristic, suggests that the ecological significance of environmental SSRI exposure may be pronounced.

Anthropogenic pollution including metals, petroleum, toxins, nutrients and many others is a growing problem in the marine environment. These are important factors altering the environment and by that the fate of many local populations of marine organisms. The aim of this study was to assess the impact of selected point pollution sources on resident populations of the blue mussel (Mytilus edulis trossulus) in the Baltic Sea using multiple biomarker approach. The study used a nested sampling scheme in which sites from reference (REF) habitats are geographically paired with selected sites from sewage treatment plants (STP) and harbors (HAR). The results showed that mussels from harbors had a higher frequency of histological abnormalities in the digestive gland compared to mussels from sewage effluent affected areas and reference sites. However these mussels together with mussels from STPs had higher lipid content, body mass index (BMI) and gonado-somatic index (GSI) compared to mussels from reference sites. A marked spatial variability was found with a stronger toxicity of ambient environment affecting resident mussel populations in the Gulf of Gdańsk area, while an opposite pattern was found in Tvärminne area. Yet the blue mussels sampled in the Gulf of Gdańsk were characterized by the highest GSI and BMI values compared to Askö and Tvärminne populations. No differences in analyzed biomarker response related to species identity, measured by a species-specific genetic marker, were found indicative of strong genetic introgression in the Baltic Proper.

Psychoactive drugs, such as selective serotonin reuptake inhibitors (SSRI) have been identified in high levels in effluents from Swedish sewage treatment plants (STP) at concentrations high enough to give pharmacological effects in fish. In humans SSRIs are used in the treatment of depression and they have anxiolytic effects. In the present study we exposed Endler guppy (Poecilia wingei) of both sexes to citalopram that showed the highest concentrations of SSRIs in STP effluents and studied reproductive and non-reproductive behaviour. Male courting behaviours were not affected compared to control fish after 14-28 days exposure to 1μgL^-1. In two experiments exposing both sexes to 0.2, 2.3 or 15μgL^-1 for 21 days, fish exposed to the two highest doses showed anxiolytic effects when placed in a novel environment (novel tank diving test, NT). Males were only affected by exposure to 15μgL^-1. They had significantly longer latency to explore the upper half of the aquarium, more visits and longer time spent in the upper half, and showed less bottom freezing behaviour, all markers of anxiolytic behaviour. In females exposure to 2.3 or 15μgL^-1 significantly increased freezing behaviour, while no effects on other behaviour variables were observed. No effects on shoaling behaviour could be discerned. These results show that citalopram have anxiolytic effects on guppy fish and thus affect ecologically relevant behaviours of importance to survival of fish.

Sewage treatment plant effluents contain a complex mixture of pharmaceuticals, personal care products and industrial chemicals, thus exposing aquatic organisms. Still, the consequences of exposure to combinations of different classes of drugs is largely unknown. In this study, we expose adult zebrafish (Danio rerio) males and females to low, environmentally relevant concentrations of the endocrine disrupting chemical 17α-ethinyl estradiol (EE₂) and the selective serotonin re-uptake inhibitor (SSRI) citalopram, alone and in combination, and analyse three non-reproductive behaviours of importance for population fitness.

Two weeks exposure to 0.1 and 0.5 ng/LEE₂ resulted in increased anxiety in males in the scototaxis (light/dark preference) test. Significantly longer latency periods before entering the white zone and fewer visits in the white zone were observed in males exposed to both 0.1 and 0.5 ng/LEE₂ compared to unexposed males. No significant effects of citalopram alone (0.1 and 0.5 µg/L) were observed in the scototaxis test. The combined exposures (0.1 ng/L EE₂ + 0.1 µg/L citalopram and 0.5 ng/L EE₂ + 0.5 µg/L citalopram) resulted in abolishment of the anxiogenic effects of EE₂, with significantly shorter latency period (low dose) and more transitions to white (high and low dose) than in fish exposed to EE₂ alone. No significant effects of either EE₂, citalopramor the combination of the two were observed in females. In the novel tank test, significantly more transitions to the upper half of the tank were observed in males exposed to 0.1 µg/L citalopram alone compared to unexposed males while males exposed to 0.1 ng/lEE₂ had significantly shorter latency period to enter the upper half. Exposure to the combination of the two low concentrations did, however, result in a significantly longer latency and fewer transitions to upper half compared to both control, EE₂- and citalopram-exposed males. These males also spent significantly less time in the upper half than the fish exposed to 0.1 ng/l EE₂ or 0.1 µg/l citalopram alone. No significant effects on novel tank behaviour were observed in females or males exposed to the higher concentrations. In the shoaling test, males exposed to 0.1 µg/L citalopram and females exposed to 0.5 ng/l EE₂ made significantly fewer transitions away from peers while males exposed to 0.1 µg/L citalopram + 0.1 ng/l EE₂ performed significantly more transitions than the fish exposed to 0.1 µg/L citalopram alone.

In conclusion, this study shows that very low concentrations ofEE_2, at or slightly above the predicted noeffect concentration (NOEC), affects anxiety in zebrafish males. Furthermore, citalopram, in spite of marginal effect of its own at such low levels, counteracts the response to EE₂. This study represents an initial effort to understand the effects on water-living organisms of the cocktails of anthropogenic substances contaminating aquatic environments.

The synthetic estrogen 17α-ethinylestradiol (EE2), ubiquitous in the aquatic environment and commonly detected in sewage effluents, interferes with the endocrine system in multiple ways. Exposure during sensitive windows of development causes persistent effects on fertility, reproductive and non-reproductive behavior in mammals and fish. In the present study, three-spined stickleback (Gasterosteus aculeatus) were exposed to nominal 0 and 20 ng/L EE2 from fertilization to 7 weeks post-hatch. After 8 months of remediation in clean water three non-reproductive behaviors, not previously analyzed in developmentally EE2-exposed progeny of wild-caught fish, were evaluated. Chemical analysis revealed that the nominal 0 and 20 ng/L exposure contained 5 and 30 ng/L EE2, respectively. Therefore, the use of control fish from previous experiments was necessary for comparisons. Fish exposed during development showed significant concentration-dependent reduction in anxiety-like behavior in the scototaxis (light/dark preference) test by means of shorter latency to first entrance to the white compartment, more visits in white, and longer total time in white compared to unexposed fish. In the novel tank test, developmental exposure significantly increased the number of transitions to the upper half of the aquaria. Exposure to EE2 during development did not alter shoal cohesion in the shoaling test compared with unexposed fish but fish exposed to 30 ng/L EE2 had significantly longer latency to leave the shoal and fewer transitions away from the shoal compared to fish exposed to 5 ng/L EE2. Skewed sex ratio with more females, sex reversal in genetic males as well as intersex in males was observed after exposure to 30, but not 5 ng/L EE2. In conclusion, EE2 exposure during development in three-spined stickleback resulted in persistent effects on anxiety-like behaviors. These long-term effects from developmental exposure are likely to be of higher relevance for natural populations than are short-term effects from adult exposure.

Blue mussels (Mytilus edulis L.) collected at three sampling sites in each of three geographical regions (South, Middle, North) along the permanent longitudinal South-North salinity gradient of the Baltic Sea, were exposed for 10 days to copper (35 ppb) or 95 octane petrol (0.3%0). During the experiment, they were maintained at the respective sampling site salinity. Scope for growth (SFG) was determined, and biochemical stress markers (protein carbonyl groups, disulfide bond formation, and glutathione transferase (GST), and catalase (CAT) activities) were investigated in gill tissue upon termination of the experiment. Treatment and regional effects for SFG and protein carbonyl groups were all significant for petrol. The largest increase in protein carbonyl groups was observed in the North. Mussels from the southern, more saline (similar to 7%) region had the highest SFG, and displayed the largest SFG decrease in response to treatment, indicating that they had the most energy available for allocation to stress response. They also displayed the least increase in the level of protein carbonyl groups. Mussels from the Northern, less saline (similar to 5%) region had the highest degree of protein carbonyl groups in response to both treatments, and lowest average SFG. Silver stained diagonal gels for samples from one sampling site in South and North, respectively, demonstrated differences in disulfide bond profiles for both stress treatments. There was also a regional difference in the number of protein disulfides observed on diagonal gels. The most diverse protein disulfide response was found in South. No treatment related effects on GST and CAT activities were observed. We suggest that both SFG and protein carbonyl groups show that geographical difference in stress susceptibility, previously established between the North and the Baltic Seas, also apply on a regional scale within the Baltic Sea, along the salinity gradient.

Baltic Sea blue mussels Mytilus edulis) were experimentally exposed to the genotoxic model substance benzo[a]pyrene (B[a]P) to study DNA adduct formation. The specific aims were (a) to examine where in the mussels the DNA adducts were formed, in gills or digestive glands (b) to study the dose-response relationship between B[a]P exposure and DNA adduct formations and (c) to examine the persistence of the formed adducts. A Scope for growth (SFG) study was also run to compare physiological responses of the mussels with the degree of DNA adduct formation. In an initial dose-response experiment, the mussels were exposed to 0.5, 50, and 100 mug/l of tritium labelled B[a]P under semi-static conditions for 4 days, and thereafter the bioaccumulation of B[a]P and DNA adduct formation in different tissues was determined using liquid scintillation counting and P-32-postlabelling analysis. respectively. In a following exposure-depuration experiment, mussels were exposed to 17 mug/l of radiolabelled B[a]P under semi-static conditions for 6 days. B[a]P accumulation and DNA adduct formation were determined during the exposure, and B[a]P elimination and persistence of DNA adducts were studied during 28 days of depuration in uncontaminated water, The results revealed large tissue differences in DNA adduct formation. DNA adduct levels were not elevated in the digestive gland of the mussels at any exposure concentration (0-100 mug/l). even though the highest B[a]P tissue concentrations were found in the digestive gland (1.0+/-0.1 mg B[a]P/g tissue dry wt at 100 mug/l, mean+/-SE. n = 12). DNA adducts were on the other hand formed in the gills, with the highest levels found in mussels exposed to 50 and 100 mug B[a]P/l. and a dose dependent increase in adduct levels (from 1.6 to 5.9 nmol adducts/mol nucleotides) from 0 to 50 mug B[a]P/l, In gills, DNA adduct levels increased with time during the 6-day exposure period in the exposure-depuration experiment, and then persisted for at least 2 weeks after exposure cessation while B[a]P tissue levels exhibited a rapid decrease (half-life of 8 days). No significant differences were observed in SFG between the control and exposed groups. Since DNA adducts exhibited a relatively high persistence in gills compared to B[a]P tissue concentrations, they seem to be a more integrated measure of genotoxic exposure than only chemical analysis of the contaminant bioaccumulation. The results also suggest that if using analysis of DNA adducts in H. edulis for monitoring purposes. analysis of gills in addition to the more commonly used digestive gland should be taken into consideration.

The world's largest brackish water sea area, the Baltic Sea, is considered to be one of the most polluted seas of the world. Many new pollutants are constantly entering the environment, such as brominated flame-retardants (BFRs). BFRs represent a group of compounds that structurally resemble hydrophobic organic contaminants, but only scarce data about their toxicity to marine organism exist. Thus, the purpose of this study was to analyze long-term in vivo cytotoxicity and genotoxicity of hexabromocyclodododecane (HBCDD) to exposed marine invertebrates using a suite of cytogenetic biomarkers. This included a set of nuclear and nucleolar characteristics and the micronucleus test. The use of those parameters reflects different mechanisms of nuclear activity regulation in cells (as a parameter of cytotoxicity) and measures subcellular processes. The induction of nuclear abnormalities (like the formation of micronuclei) was also employed here as a parameter of genotoxicity. In order to reflect the proliferative and metabolic activity of the cells the number of argylophillic nucleolar organiser regions (NORs) in interphase cells was scored. Over a period of 50 days an in vivo exposure experiment with a clam Macoma balthica and different concentrations of HBCDD (nominal concentrations of 0, 100 and 250 μg/l) with three replicates each was performed. Gill cells were used as “sentinel systems” considering specificity in metabolism, repair mechanisms, adaptative response and cell proliferation. A significant increase in nuclear and nucleolar abnormalities and in the frequency of dead cells was observed during the duration of the experiment with the highest peak occurring 10 days after exposure for nuclear abnormalities and 20–30 days after exposure for malfunction of ribosomal genes (NORs) (GLM analyses and Spearman correlation, p < 0.05). Thus, the induction of micronuclei and other nuclear abnormalities reflected the toxic potential of HBCDD to marine invertebrates while an increase in the number of NOR may also reflect adaptive responses of the system as enhanced induction of proliferative regeneration of the gill tissue