2009, Ernst, Bernhard, Höger, Stefan J., O´Brien, Evelyn, Dietrich, Daniel R.

Regular occurrences of the cyanobacterium Planktothrix rubescens have been observed in several lakes that have undergone recent re-oligotrophication, e.g. Lake Ammersee. Planktothrix species are known to produce microcystins, potent phosphatase inhibitors that have been associated with morbidities and mortalities in humans and animals. The aim of this study was to characterise the temporal and spatial abundance and toxicity of P. rubescens in Lake Ammersee.
P. rubescens cell densities and biovolumes were calculated via fluorescence image analyses. P. rubescens was present during the entire observation period from 1999 to 2004, albeit at different cell densities. Maximum biovolumes of 45 cm³ m‾² were observed in May 2001. Filaments were regularly distributed over the entire water column during winter and stratified in distinct metalimnic layers during summer, reaching maximum cell densities of ≤15,000 (winter) and ≤77,000 cells ml‾¹ (summer). The results demonstrate that P. rubescens abundance is strongly influenced by water transparency, i.e. illumination in the metalimnion. Moreover, the P. rubescens abundance appears to result from regular phosphate depletion in the epilimnion. possibly additionally benefiting from high nitrogen loads.
Microcystin (MC) was detectable in 27 and 38 of 54 seston samples via HPLC and Adda-ELISA measurements, respectively. The main microcystin congeners in the seston samples were [Asp³]-MC-RR and [Asp³,Dhb7]-MC-RR. Microcystin concentrations correlated significantly with the respective phycoerythrin (PE)-concentrations. The variation in the MC/PE-ratios was low suggesting that the microcystin production of P. rubescens in Lake Ammersee is consistent and indicates that the appearance of P. rubescens coincides with measurable microcystin levels. Moreover, the observation of pronounced metalimnic oxygen depletions appears to be causally related to recurring high P. rubescens abundance.
In conclusion the results suggest that aquatic organisms such as indigenous fish populations (e.g. coregonids) are regularly confronted with potentially adverse P. rubescens densities, which might provide a possible explanation for the often observed impaired health and growth retardation of coregonid populations in P. rubescens containing pre-alpine lakes.

2006, Ernst, Bernhard, Höger, Stefan J., O'Brien, Evelyn, Dietrich, Daniel R.

The microcystin-producing cyanobacterium Planktothrix is one of the most widespread genera amongst toxin producing cyanobacteria in European lakes. In particular, the metalimnic blooms of Planktothrix rubescens have been associated with growing problems in the professional freshwater fishery as a decrease in yearly yields in the important coregonids fishery often coincides with the appearance of P. rubescens. P. rubescens is a cyanobacterial species known to produce toxic compounds, e.g. microcystins. Although microcystins have been reported to affect fish health, behaviour, development and growth and have also been associated with feral fish kills, there is currently no specific information on the effects of toxic Planktothrix filaments in fish and especially coregonids. Therefore, the aim of this study was to investigate the effects of an environmentally relevant dose of P. rubescens filaments orally applied to coregonids and to discuss the findings in the context of microcystin toxicity previously reported in carp and trout.
A single dose of P. rubescens culture, at a density of 80,000 cells per 120 μl, was applied to coregonids thus corresponding to 0.6 0.9 μg microcystin-LRequiv./kg body weight. Behavioural changes and opercular beat rates, growth, hepatosomatic index, condition and plasma glucose were determined. Liver, kidney, gill and the gastrointestinal tract were assessed histopathologically and immunhistologically. Exposed fish showed behavioural changes, increased opercular beat rates and elevated plasma glucose levels, possibly representing a physiological stress response. Histopathological alterations in liver, gastrointestinal tract and kidney, also immunopositive for microcystin suggested causality of tissue damage and the in situ presence of microcystins.
The observed combination of stress and organ damage may explain the frequently reduced weight and thus the fitness noted in coregonids subjected to regular occurrences of stratified and dispersed P. rubescens blooms, e.g. in lake Ammersee, Bavaria, Germany.

1999, Chou, Ya-Juin, Dietrich, Daniel R.

Nitromusks, musk xylene (MX), musk ketone (MK) and musk moskene (MM) are synthetic fragrances. 4-Amino-MX (4X), 2-amino-MX (2X) and 2-amino-MK (2K) are nitromusk metabolites formed during the sewage treatment process and have been detected in effluent and surface water at concentrations four to 40 times higher than their parent compounds. To date, data to the aquatic toxicity of nitromusk compounds are limited to the parent compounds and the determination of acute and subacute effects in aquatic organisms. No data are available regarding the potential endocrine modulating effects of these compounds and/or their metabolites in aquatic organisms. Therefore, the competitive binding capability of nitromusks and their metabolites to the estrogen receptors (ER) in rainbow trout and xenopus was investigated. No binding of MX, MK and MM to the ER of either species was observed. In contrast, binding to the ER was observed for 4X, 2X and 2K in both species. The IC50 (competitive binding at the ER) of 2X in rainbow trout was 1.3±1.1 mM. In contrast, 4X, 2X and 2K bound to the xenopus ER with an IC50 of 30.8±28.5, 12.9±10.3 and 70.1±88.3 μM, respectively.

2007, Ernst, Bernhard, Höger, Stefan J., O'Brien, Evelyn, Dietrich, Daniel R.

Planktothrix rubescens belongs to the most ubiquitous cyanobacterial species in mesotrophic and oligotrophic lakes in the pre-alpine regions. In most of these lakes, coregonids are among the dominant species of the ichthyofauna with great importance for the professional fishery. A possible link between the occurrence of toxic Planktothrix blooms and the recurrent slumps in coregonid yields has been suggested. Indeed, acute toxic effects of microcystins and other cyanobacterial toxins have been shown for various fish species. However, chronic exposure scenarios appear to be more common and thus more environmentally realistic than acute intoxications. The aim of this study was therefore to investigate the physiological stress response and organ pathology in coregonids sub-chronically exposed to ambient water containing low, medium and high P. rubescens densities, known to be typical of pre-alpine lakes. Coregonid hatchlings were exposed in four tanks containing 0 (sham-control) and approximately 1500 (low), 15,000 (medium) and 55,000 (high) P. rubescens cells/ml for up to 28 days. Temperature, oxygen concentration, pH-value, P. rubescens cell density and microcystin concentration were recorded and the fish were observed for behavioural changes and examined for parasite infestations. Gill ventilation rates, general condition factors and mortalities were determined and liver, kidney, gut and gill were assessed histopathologically and immunhistologically.
Depending on the cell density, exposed fish showed behavioural changes, including increased ventilation rates possibly representing a physiological stress response. Susceptibility to ectoparasitic infestation and increased mortality in exposed fish suggested P. rubescens associated effects on fish fitness. Histopathological alterations in liver, gastrointestinal tract and kidney, which were also immunopositive for microcystin suggested causality of tissue damage and the presence of microcystins. In contrast, observed gill pathology appeared to result primarily from mechanical abrasion and irritation due to ectoparasitic infestation. The current exposure experiment confirmed the hypothesis that subchronic and chronic exposure to low cyanobacterial cell densities and hence microcystins can exacerbate physiological stress and sustained pathological alterations in exposed coregonids. The study therefore supports the theory that P. rubescens blooms may be causal to the observed weight reduction and hence fitness of coregonids in pre-alpine lakes such as Lake Ammersee (Germany).

2005, Höger, Birgit, Köllner, Bernd, Dietrich, Daniel R., Hitzfeld, Bettina C.

The detection of residues of various pharmaceuticals in surface waters during the last two decades has prompted concerns about possible adverse effects of this kind of pollution on aquatic organisms. The objective of the present study was to investigate effects of the non-steroidal anti-inflammatory drug diclofenac, one of the pharmaceuticals most prevalent in surface waters, on brown trout (Salmo trutta f. fario), a salmonid species native to German rivers. Brown trout were exposed to 0.5, 5 and 50 μg/L diclofenac for 7, 14 and 21 days, whereby the lowest exposure concentration is comparable with concentrations commonly found in the aquatic environment. Fish exposed to diclofenac displayed significantly reduced haematocrit levels after 7 and 14 days of exposure. After 21 days, trout were examined for histopathological alterations, whereby diclofenac exposure resulted in increased monocyte infiltration in the liver, telangiectasis in gills, and the occurrence of interstitial hyaline droplets, interstitial proteinaceous fluid and mild tubular necrosis in trunk kidney. Concurrent immunohistological analysis revealed an increase of granulocyte numbers in primary gill filaments, as well as granulocyte accumulation and increased major histocompatibility complex (MHC) II expression in kidney, suggestive of an inflammatory process in these organs. Moreover, the ability of diclofenac to hinder the stimulation of prostaglandin E2 synthesis was shown in head kidney macrophages of brown trout in vitro. These findings support the hypothesis that environmental exposure of fish to diclofenac provokes the same mechanism of action in these non-target organisms as previously described for mammalian species and can thus lead to similar (possibly adverse) effects. In general, the present study suggests that exposure of brown trout to diclofenac in concentration ranges commonly found in the environment can result in adverse effects in various organs and possibly compromise the health of affected fish populations.

1999, Chou, Ya-Juin, Dietrich, Daniel R.

Musk xylene (MX), musk ketone (MK) and musk moskene (MM) are synthetic nitro-containing fragrances. Due to their inherent lipophilicity and environmental persistence, they are frequently detected in environmental samples and especially in aquatic ecosystems. Despite this, the current environmental toxicity database of nitromusks is limited. Although nitromusks have been shown to accumulate in aquatic organisms, little is known about their potential developmental effects in the respective aquatic species. To investigate the developmental toxicity of these compounds to amphibians and fish, early lifestages of xenopus (Xenopus laevis) and zebrafish (Danio rerio) were exposed to three nitromusks for 96 h to examine the developmental effects of these compounds in the two species. Nitromusk body concentration measurements were carried out in parallel for correlation with potential developmental effects. No increased mortality, malformation or growth inhibition was observed in either species following 96-h exposure to 400 μg/l MX, MK and MM. However, an approximately 20% reduced viability was observed in xenopus larvae when exposed to 400 μg/l MX, MK and MM for 11 days. Xenopus and zebrafish exposed to 10, 153, 871 and 1637 μg/l 14C-MX for 96 h resulted in whole-body concentrations of 0.7±0.1, 11.1±1.1, 38.7±1.9 and 76.3±18.3 μg/g, and 4.3±0.6, 73.3±11.8, 440.0±72.7 and 664.0±47.7 μg/g wet body weight, respectively. Exposure of xenopus larvae to 400 μg/l MX, MK and MM for 11 days, resulted in whole body concentrations (extrapolated from gas chromatographic determinations) of 4700±5000, 1300±300 and 4600±4800 μg/g wet weight for MX, MK and MM, respectively. The latter toxicity results, in conjunction with the fact that the concentrations used for the above experiments were between 400- and 10 000-fold higher than those detected in the environment, suggest that environmental concentrations of nitromusks are not hazardous for early lifestages of fish and amphibians.

2006, Höger, Birgit, Taylor, Sean, Hitzfeld, Bettina C., Dietrich, Daniel R., Heuvel, Michael R. van den

Rainbow trout (Oncorhynchus mykiss) were exposed to 1.5 and 15% v/v secondary treated sewage effluent for 32 weeks in flow-through mesocosms. The exposure encompassed the full period of reproductive development for rainbow trout. Trout did not show any evidence of a dose-dependent change in growth. Fish exposed to 15% effluent were the only group to show mortality (5%) over the duration of the experiment. Trout at the highest effluent concentration had significantly higher liver size than reference water fish. Both male and female trout in the 15% exposure group also exhibited significantly higher gonad weight than the reference group. In female trout, this gonad size increase could be explained by higher egg numbers. Female and male trout both displayed a significant increase in plasma 17β-estradiol levels after exposure to 15% effluent, while neither sex had dose-dependent differences in plasma testosterone. Male trout displayed elevated vitellogenin levels and reduced plasma 11-ketotestosterone concentration after exposure to 15% effluent. Chemical examination of steroidal compounds, including both estrogens and androgens, in the wastewater revealed that only estrone was detectable at a mean concentration of 4.5 ng/L. It is assumed that the effects observed in trout exposed to 15% effluent were consistent with stimulation of reproductive development due to very low levels of estrogens. Overall, long-term exposure to treated sewage effluent containing low levels of estrogen did not have significant negative implications for reproductive development in rainbow trout.

2003, Ellis, Rosanne J., Heuvel, Michael R. van den, Bandelj, Emil, Smith, Murray A., McCarthy, Lynda H., Stuthridge, Trevor R., Dietrich, Daniel R.

The androgenic potential of a New Zealand pulp and paper mill effluent was measured by applying a combination of in vitro and in vivo bioassays with mosquitofish (Gambusia affinis) and goldfish (Carassius auratus). The in vivo method assessed the rate of gonopodial development (masculinization) and alterations from normal reproductive behavior in adult female mosquitofish exposed for 21 d to untreated or secondary-treated pulp mill effluent. A second in vivo mosquitofish exposure tested the effect of glass-fiber (type C) filtration of secondary-treated effluent on rates of expression of the same endpoints. Extractable organics analyses of effluents and extracts thereof were conducted. Mosquitofish demonstrated significant masculinization on exposure to either treated or untreated effluent; the frequency of gonopodial development was reduced with effluent secondary-treatment. Male mating behavior was observed in the masculinized adult females. Glass-fiber (type F) filtration of the treated effluent eliminated the masculinizing effect, suggesting that the bioactive compounds were associated with the suspended solids. The in vitro method measured the binding of compounds within a treated thermomechanical/bleached kraft effluent extract to androgen receptors contained in goldfish testis cytosol. Exposure to extracts of either the particulate (glass-fiber filtered) or the dissolved organic fraction of the effluent produced significant binding (as indicated by the displacement of radiolabeled testosterone) to the androgen receptor in goldfish gonadal tissue. Thus, the dissolved organics extract of the treated effluent contained compounds androgenic to goldfish in vitro but not to mosquitofish in vivo. The combined in vitro and in vivo data suggest that the effluent in question could exert effects on the reproductive physiology of fishes through an androgenic mechanism. The androgenic compounds androstenedione and testosterone were not detected in the extracts used for the in vitro component of this study.