Selected neonicotinoids and associated risk for aquatic organisms.

Neonicotinoids are one of the newest groups of systemic pesticides, effective on a wide range of invertebrate pests. The success of neonicotinoids can be assessed according to the amount used, for example, in the Czech Republic, which now accounts for 1/3 of the insecticide market. The European Union (EU) has a relatively interesting attitude towards neonicotinoids. Three neonicotinoid substances (imidacloprid, clothianidin and thiamethoxam) were severely restricted in 2013. In 2019, imidacloprid and clothianidin were banned, while thiamethoxam and thiacloprid were banned in 2020. In 2022, another substance, sulfoxaflor, was banned. Therefore, only two neonicotinoid substances (acetamiprid and flupyradifurone) are approved for outdoor use in the EU. Neonicotinoids enter aquatic ecosystems in many ways. In European rivers, neonicotinoids usually occur in nanograms per litre. Due to the low toxicity of neonicotinoids to standard test species, they were not expected to significantly impact the aquatic ecosystem until later studies showed that aquatic invertebrates, especially insects, are much more sensitive to neonicotinoids. In addition to the lethal effects, many studies point to sublethal impacts - reduced reproductive capacity, initiation of downstream drift of organisms, reduced ability to eat, or a change in feeding strategies. Neonicotinoids can affect individuals, populations, and entire ecosystems.

Fitness assessment of Mytilus galloprovincialis Lamarck, 1819 after exposure to herbicide metabolite propachlor ESA.

The lack of data on the chronic effects of chloroacetanilide herbicide metabolites on non-target aquatic organisms creates a gap in knowledge about the comprehensive impacts of excessive and repeated pesticide use. Therefore, this study evaluates the long-term effects of propachlor ethanolic sulfonic acid (PROP-ESA) after 10 (T1) and 20 (T2) days at the environmental level of 3.5 µg.L-1 (E1) and its 10x fold multiply 35 µg.L-1 (E2) on a model organism Mytilus galloprovincialis. To this end, the effects of PROP-ESA usually showed a time- and dose-dependent trend, especially in its amount in soft mussel tissue. The bioconcentration factor increased from T1 to T2 in both exposure groups - from 2.12 to 5.30 in E1 and 2.32 to 5.48 in E2. Biochemical haemolymph profile and haemocyte viability were not affected by PROP-ESA exposure. In addition, the viability of digestive gland (DG) cells decreased only in E2 compared to control and E1 after T1. Moreover, malondialdehyde levels increased in E2 after T1 in gills, and DG, superoxidase dismutase activity and oxidatively modified proteins were not affected by PROP-ESA. Histopathological observation showed several damages to gills (e.g., increased vacuolation, over-production of mucus, loss of cilia) and DG (e.g., growing haemocyte trend infiltrations, alterations of tubules). This study revealed a potential risk of chloroacetanilide herbicide, propachlor, via its primary metabolite in the Bivalve bioindicator species M. galloprovincialis. Furthermore, considering the possibility of the biomagnification effect, the most prominent threat poses the ability of PROP-ESA to be accumulated in edible mussel tissues. Therefore, future research about the toxicity of pesticide metabolites alone or their mixtures is needed to gain comprehensive results about their impacts on living non-target organisms.

Effects of praziquantel on common carp embryos and larvae.

This study aimed to assess the toxicity of praziquantel (anthelmintic drug) in different developmental stages of common carp (Cyprinus carpio) based on mortality, early ontogeny, growth, oxidative stress, antioxidant enzymes, histology and behaviour. Praziquantel at all tested concentrations ranging from 1 to 4 mg/L showed no significant adverse effects on mortality, the early ontogeny and behaviour locomotory (activity, moved distance and velocity) of carp after 35-day exposure. Concentrations of 3 and 4 mg/L caused significantly (P < 0.01) lower growth, total superoxide dismutase and catalase activities compared with controls. Praziquantel is safe for the early life of carp in concentrations ≤ 2 mg/L.

Chronic Toxicity of Primary Metabolites of Chloroacetamide and Glyphosate to Early Life Stages of Marbled Crayfish Procambarus virginalis

Degradation products of herbicides, alone and in combination, may affect non-target aquatic organisms via leaching or runoff from the soil. The effects of 50-day exposure of primary metabolites of chloroacetamide herbicide, acetochlor ESA (AE; 4 µg/L), and glyphosate, aminomethylphosphonic acid (AMPA; 4 µg/L), and their combination (AMPA + AE; 4 + 4 µg/L) on mortality, growth, oxidative stress, antioxidant response, behaviour, and gill histology of early life stages of marbled crayfish (Procambarus virginalis) were investigated. While no treatment effects were observed on cumulative mortality or early ontogeny, growth was significantly lower in all exposed groups compared with the control group. Significant superoxide dismutase activity was observed in exposure groups, and significantly higher glutathione S-transferase activity only in the AMPA + AE group. The gill epithelium in AMPA + AE-exposed crayfish showed swelling as well as numerous unidentified fragments in interlamellar space. Velocity and distance moved in crayfish exposed to metabolites did not differ from controls, but increased activity was observed in the AMPA and AE groups. The study reveals the potential risks of glyphosate and acetochlor herbicide usage through their primary metabolites in the early life stages of marbled crayfish.

Effects of long-term exposure of Mytilus galloprovincialis to thiacloprid: A multibiomarker approach.

Thiacloprid is a neonicotinoid insecticide widely exploited in agriculture and easily mobilized towards aquatic environments by atmospheric agents. However, little information about its toxicological effects on aquatic invertebrate bioindicators is available. In this study, specimens of the mussel Mytilus galloprovincialis were exposed to thiacloprid at environmental (4.5 µg L-1) and 100 times higher than environmental (450 µg L-1) concentrations for 20 days. Thiacloprid affected haemolymph biochemical parameters, cell viability in the digestive gland, antioxidant biomarkers and lipid peroxidation in the digestive gland and gills at environmentally relevant concentrations (4.5 µg L-1). In addition, thiacloprid exposure caused histological damage to the digestive gland and gills. Interestingly, the pesticide was detected at levels equal to 0.14 ng g-1 in the soft tissues of sentinels exposed for 20 days to 450 µg L-1 thiacloprid in seawaterµ. Due to its harmful potential and cumulative effects after long-term exposure of M. galloprovincialis, thiacloprid may pose a potential risk to nontarget aquatic organisms, as well as to human health. This aspect requires further in-depth investigation.

Nitrogen factor of common carp Cyprinus carpio fillets with and without skin.

Consumer protection against food adulteration and misleading labelling is integrated into EU legislation, but accurate analysis of the meat content of farmed freshwater fish products is not possible because of the lack of established nitrogen factors for farmed common carp. The aim of this study was to determine nitrogen factors for farmed common carp Cyprinus carpio. Seven-hundred samples collected in 2018-2019 in three harvest seasons (March/April, Jun/July, and October/November) at seven locations in the Czech Republic were analysed for nitrogen, dry matter, protein, ash, and fat content according to standard ISO methods. The recommended nitrogen factor for fat-free common carp fillet with skin is 3.04 ± 0.13 and, for fillet without skin, 2.95 ± 0.12. Availability of nitrogen factors for common carp can help ensure that consumers are purchasing correctly labelled products.

The effect of chronic exposure to chloridazon and its degradation product chloridazon-desphenyl on signal crayfish Pacifastacus leniusculus.

The effects of chloridazon (Ch) and its metabolite chloridazon-desphenyl (Ch-D) at the environmentally relevant concentrations of 0.45 µg/L and 2.7 µg/L on signal crayfish Pacifastacus leniusculus were assessed in a 30-day exposure followed by a 15-day depuration period. Locomotion, biochemical haemolymph profile, oxidative and antioxidant parameters, and histopathology were evaluated. Crayfish exposed to Ch at 0.45 µg/L and 2.7 µg/L showed significantly (p < 0.01) higher CAT activity and GSH level in hepatopancreas and gill compared to controls. The concentration of Ch at 2.7 µg/L was associated with significantly (p < 0.01) higher levels of GLU, LACT, ALT, AST in haemolymph compared to controls. Chloridazon-desphenyl exposure at both tested concentrations caused significantly higher (p < 0.01) GLU, LACT, ALT, AST, NH3, and Ca in haemolymph; lipid peroxidation (TBARS) levels in hepatopancreas; and CAT activity and GSH level in hepatopancreas and gill. Alterations of structure including focal dilatation of tubules, increased number of fibrillar cells, and haemocyte infiltration in the interstitium were observed with 2.7 µg/L Ch and with both Ch-D exposures. Locomotion patterns did not vary significantly among groups. A 15-day recovery period was insufficient to restore normal physiological parameters in exposed groups. Chloridazon and its metabolite Ch-D exerts harmful effects on crayfish.

Nutritional evaluation of elongate mudskipper Pseudapocryptes elongatus (Cuvier, 1816) from Diamond Harbor, West Bengal, India.

The mudskipper Pseudapocryptes elongatus (Cuvier, 1816), (Gobiidae) are caught throughout the year and they are considered as most edible mudskipper species in Sundarbans. They are targeted by special gears and sold live in large quantities in the local markets of Diamond Harbor, Namkhana and Kakdweep. The adult P. elongatus (14-18 cm total length) specimens were purchased from the Diamond Harbor landing center for evaluation of its biochemical components such as Protein, carbohydrate, lipid, amino acid, fatty acid, vitamin, and minerals. The percentage composition of protein was high (27.34%) followed by the carbohydrate (4.57%) and lipid (2.72%). The amino acids both essential (47.72%) and non-essential (42.91%) was found adequate. Among fatty acids, polyunsaturated fatty acid (PUFA) was found the maximum (31.55%) followed by saturated fatty acid (SFA; 27.66%) and monounsaturated fatty acid (MUFA; 19.55%). Vitamin A (112.5 IU/100 g) and Ca2+ (62.2 mg/100 g) were found maximum levels. The present finding supports that P. elongatus is valuable for human consumption due to its high-quality protein with well-balanced amino acids.

Acute effects of neonicotinoid insecticides on Mytilus galloprovincialis: A case study with the active compound thiacloprid and the commercial formulation calypso 480 SC.

Pesticides can enter aquatic environments potentially affecting non-target organisms. Unfortunately, the effects of such substances are still poorly understood. This study investigated the effects of the active neonicotinoid substance thiacloprid (TH) and the commercial product Calypso 480 SC (CA) (active compound 40.4% TH) on Mytilus galloprovincialis after short-term exposure to sublethal concentrations. Mussels were tested for seven days to 0, 1, 5 and 10 mg L-1 TH and 0, 10, 50 and 100 mg L-1 CA. For this purpose, several parameters, such as cell viability of haemocytes and digestive cells, biochemical haemolymph features, superoxide dismutase (SOD) and catalase (CAT) enzymatic activity of gills and digestive gland, as well as histology of such tissues were analysed. The sublethal concentrations of both substances lead to abatement or completely stopping the byssal fibres creation. Biochemical analysis of haemolymph showed significant changes (P < 0.01) in electrolytes ions (Cl-, K+, Na+, Ca2+, S-phosphor), lactate dehydrogenase (LDH) enzyme activity and glucose concentration following exposure to both substances. The TH-exposed mussels showed significant imbalance (P < 0.05) in CAT activity in digestive gland and gills. CA caused significant decrease (P < 0.05) in SOD activity in gills and in CAT activity in both tissues. Results of histological analyses showed severe damage in both digestive gland and gills in a time- and concentration-dependent manner. This study provides useful information about the acute toxicity of a neonicotinoid compound and a commercial insecticide on mussels. Nevertheless, considering that neonicotinoids are still widely used and that mussels are very important species for marine environment and human consumption, further researches are needed to better comprehend the potential risk posed by such compounds to aquatic non-target species.

Effects of chloridazon on early life stages of marbled crayfish.

The effects of chloridazon exposure at concentrations of 2.7 µg/L (maximal real environmental concentration in the Czech Republic), 27 µg/L, 135 µg/L and 270 µg/L on early life stages of marbled crayfish (Procambarus virginalis) were evaluated. Significantly higher glutathione S-transferase activity and reduced glutathione level was observed at all tested concentrations of chloridazon compared with the control. Chloridazon in concentrations 27, 135 and 270 µg/L caused delay ontogenetic development and slower growth. Histopathological changes in hepathopancreas were found in two highest tested concentrations (135 µg/L and 270 µg/L). Crayfish behaviour was not altered in control vs. exposed animals, while the activity parameters tend to decline with increasing chloridazon concentrations.

Effects of metazachlor and its major metabolite metazachlor OA on early life stages of marbled crayfish.

The effects of the herbicide metazachlor and its major metabolite metazachlor OA at two concentrations, including environmentally relevant concentrations of metazachlor (0.0115 µmol/l and 0.0790 µmol/l) and metazachlor OA (0.0117 µmol/l and 0.0805 µmol/l), respectively, were evaluated on early ontogeny, growth, behaviour, oxidative stress, antioxidant enzyme levels, histology, and mortality of marbled crayfish Procambarus virginalis. Both tested concentrations of metazachlor and metazachlor OA were associated with significantly lower growth and delayed ontogenetic development compared to controls. Exposure of metazachlor at 0.0115 µmol/l and metazachlor OA at 0.0117 µmol/l and 0.0805 µmol/l resulted in significantly lower activity of total superoxide dismutase (SOD), catalase (CAT), glutathione s-transferase (GST), glutathione reductase (GR), and reduced glutathione (GSH) compared with control and resulted in gill anomalies ranging from wall thinning to focal disintegration of branchial structure. Metazachlor at the environmentally relevant concentration of 0.0790 µmol/l was associated with significant alterations of crayfish distance moved and walking speed. The potential risk associated with metazachlor use in agriculture related to effects on non-target aquatic organisms.

Single and combined effects of thiacloprid concentration, exposure duration, and water temperature on marbled crayfish Procambarus virginalis.

The increasing utilization of chemicals and ongoing climate change have a negative impact on aquatic ecosystems. The present study examined combined effects of water temperature, chemical concentration, and duration of exposure to the neonicotinoid thiacloprid on marbled crayfish Procambarus virginalis. Crayfish were exposed to thiacloprid at the environmental concentration of 4.50 µg L-1 and 10% 96LC50 to marbled crayfish, 64.64 µg L-1, at water temperature of 17 and 23 °C for 28 days followed by a 28 day depuration period. No crayfish died during the experiment. Both thiacloprid concentrations at 23 °C showed a synergistic effect with temperature on the biochemical indicators in haemolymph compared to those at 17 °C. Both concentrations of thiacloprid at both temperatures were associated with significant differences from thiacloprid-free controls (P < 0.01) in haemolymph glucose, ammonia, calcium, inorganic phosphate, and lactate; haemolymph enzymes aspartate aminotransferase, alanine aminotransferase, creatine kinase, and alkaline phosphatase; antioxidant biomarkers superoxide dismutase, catalase, glutathione S-transferase, and reduced glutathione in hepatopancreas, muscle, and gill, and showed lipid peroxidation in hepatopancreas and muscle. Histological analyses revealed structural changes and damage to gill and hepatopancreas of exposed crayfish.

Assessing the effects of neonicotinoid insecticide on the bivalve mollusc Mytilus galloprovincialis.

In the present work, the marine invertebrate Mytilus galloprovincialis was used as model organism to evaluate the toxic effects of the neonicotinoid Calypso 480 SC (CAL) following 20 days of exposure to sub-lethal concentrations of 7.77 mg L-1 (0.1% 96 h-LC50) and 77.70 mg L-1 (1% 96 h-LC50), and a recovery period of 10 days in uncontaminated seawater. Results revealed that exposure to both concentrations of CAL increased significantly mortality rate in the cells of haemolymph and digestive gland, while digestive gland cells were no longer able to regulate cell volume. Exposure significantly reduced haemolymph parameters (Cl-, Na+), affected the enzymatic activities of superoxide dismutase of digestive gland and catalase of gill, and caused also histopathological alterations in digestive gland and gills. Main histological damages detected in mussels were lipofuscin accumulation, focal points of necrosis, mucous overproduction and infiltrative inflammations. Interestingly, alterations persisted after the recovery period in CAL-free water, especially for haemocyte parameters (K+, Na+, Ca2+, lactate dehydrogenase, glucose). A slight recovery of histological conditions was detected. These findings suggested that sub-chronic exposure to the neonicotinoid insecticide caused significant alterations in both cell and tissue parameters of M. galloprovincialis. Considering the ecologically and commercially important role of mussels in coastal waters, a potential risk posed by neonicotinoids to this essential aquatic resource can be highlighted.

Effects of S-metolachlor and its degradation product metolachlor OA on marbled crayfish (Procambarus virginalis).

Increasing production of energy crops in Europe, mainly maize and rapeseed, has altered patterns of pesticide use in recent decades. The long-term effects of S-metolachlor (S-M) and of its metabolite metolachlor OA (M-OA) at the environmentally relevant concentration of 4.2 µg L-1 and at 42 µg L-1 (ten-fold concentration) on marbled crayfish (Procambarus virginalis) were evaluated in a 28-day exposure and after a subsequent 28-day recovery period. Indicators assessed were behaviour; biochemical haemolymph profile; oxidative and antioxidant parameters of gill, hepatopancreas, and muscle; and histology of hepatopancreas and gill. Results showed biochemical haemolymph profile (lactate, alanine aminotransferase, aspartate aminotransferase, inorganic phosphate), lipid peroxidation in hepatopancreas, and antioxidant parameters (catalase, reduced glutathione, glutathione S-transferase) of hepatopancreas and gill of crayfish exposed to S-M and M-OA to significantly differ from controls (P < 0.01). Antioxidant biomarker levels remained different from controls after a 28-day recovery period. Differences in behaviour including speed of movement and velocity, and histopathological damage to gill and hepatopancreas were associated with S-M and M-OA exposure and persisted after 28 days in S-M- and M-OA-free water. Results suggest harmful effects of low concentrations of S-M and its metabolite M-OA on non-target organisms and provide information for assessing their effects at environmentally relevant concentrations.

Acute exposure of common yabby (Cherax destructor) to the neonicotinoid pesticide.

Calypso 480 SC (CAL) is an insecticide containing the active substance thiacloprid (480g/L) belonging to the family of neonicotinoids. Crayfish represent an important component of aquatic ecosystems and are considered sensitive indicators of water pollution. The aim of this study was to investigate the effects of acute exposure of CAL on common yabby crayfish (Cherax destructor) at concentrations of 0.1, 0.5, 1, 5, 10, 25, and 50mg/L. Crayfish behavior and mortality were monitored daily, and the lethal concentrations (LC0, LC50, LC100) of CAL were evaluated at 24h, 48h, 72h, and 96h. Three concentrations of CAL (0.1, 1, 10mg/L) were selected for assessment of oxidative stress (lipid peroxidation) and antioxidant parameters superoxide dismutase, catalase, glutathione S-transferase and reduced glutathione in crayfish hepatopancreas, muscle, and gill tissue at the conclusion of the acute test. Differences in behavior of exposed crayfish from controls were observed at concentration≥5mg/L of CAL. The 96h LC50 value for the common yabby was 7.7mg/L for Calypso 480 SC. The acute exposure showed reduction of the level lipid peroxidation in hepatopancreas in all experimental groups compared with control. The results of antioxidant activity showed a significant change on glutathione S-transferase in tissue of hepatopancreas while no differences were observed on the others antioxidant parameters in crayfish's tissues. The study provides valuable information on effects of neonicotinoid insecticides on a non-target organism, the crayfish, an indicator of pollution and an important element of stable aquatic ecosystems.

Effects of s-metolachlor on early life stages of marbled crayfish.

The effects of s-metolachlor chronic exposure at concentrations of 1.1 µg/L (maximal real environmental concentration in the Czech Republic), 11 µg/L (environmental relevant concentration) and 110 µg/L on early life stages of marbled crayfish (Procambarus virginalis) was evaluated under laboratory conditions. All s-metolachlor exposures resulted in higher mortality, delay ontogenetic development with accompanied slower growth and excited behaviour (increase of total distance moved and walking speed). Significantly lower superoxide dismutase, catalase, glutathione S-transferase activity and reduced glutathione level was observed at two higher tested concentrations (11 and 110 µg/L) of s-metolachlor compared with the control. S-metolachlor in concentrations 110 µg/L showed alteration of the tubular system of hepatopancreas including focal disintegration of tubular epithelium and notable reduction in epithelial cells number, especially B-cells. In conclusion, potential risk associated with using of s-metolachlor in agriculture, due to effects on non-target aquatic organisms as documented on early life stages of marbled crayfish in this study, should be taken into account.

Chronic toxicity of metolachlor OA on growth, ontogenetic development, antioxidant biomarkers and histopathology of early life stages of marbled crayfish.

The metolachlor OA is a metabolite of herbicide metolachlor and s-metolachlor. The objective of the present study was to assess the effect metolachlor OA on early life stages of marbled crayfish (Procambarus virginalis). The early life stages of marbled crayfish were exposed for 45 days to three concentrations of metolachlor OA: 4.2 µg/L (environmentally relevant concentration, E1), 42 µg/L (E2) and 420 µg/L (E3) under laboratory conditions. The effects were assessed on the basis of mortality, growth, ontogenetic development, behaviour, oxidative stress, antioxidant biomarkers and histopathology. Metolachlor OA caused significantly lower growth, superoxide dismutase, catalase and glutathione s-transferase activity in all tested concentrations. Metolachlor OA in higher concentrations (42 and 420 µg/L) resulted in significantly delayed ontogenetic development, lower reduced glutathione level and lipid peroxidation. Metolachlor OA has not significant effect on behaviour (activity, total distance moved and walking speed). Histological examination revealed alteration of hepatopancreas and gills in crayfish exposed to two higher tested concentrations. Hepatopancreas reflected histomorphological structural changes of individual cell types. Changes of gills included focal hemocytic infiltration together with enlargement of intralamellar space packed with granular substance. In conclusion, chronic metolachlor OA exposure affected growth, ontogenetic development, and the antioxidant system and caused pathological changes in hepatopancreas and gills of early life stages of marbled crayfish.

Do progestins contribute to (anti-)androgenic activities in aquatic environments?

Unknown compounds with (anti-)androgenic activities enter the aquatic environment via municipal wastewater treatment plants (WWTPs). Progestins are well-known environmental contaminants capable of interfering with androgen receptor (AR) signaling pathway. The aim of the present study was to determine if 15 selected progestins have potential to contribute to (anti-)androgenic activities in municipal wastewaters and the respective recipient surface waters. AR-specific Chemically Activated LUciferase gene eXpression bioassay in agonistic (AR-CALUX) and antagonistic (anti-AR-CALUX) modes and liquid chromatography tandem atmospheric pressure chemical ionization/atmospheric photoionization with hybrid quadrupole/orbital trap mass spectrometry operated in high resolution product scan mode (LC-APCI/APPI-HRPS) methods were used to assess (anti-)androgenic activity and to detect the target compounds, respectively. The contribution of progestins to (anti-)androgenic activities was evaluated by means of a biologically and chemically derived toxicity equivalent approach. Androgenic (0.08-59 ng/L dihydrotestosterone equivalents - DHT EQs) and anti-androgenic (2.4-26 µg/L flutamide equivalents - FLU EQs) activities and progestins (0.19-75 ng/L) were detected in selected aquatic environments. Progestins displayed androgenic potencies (0.01-0.22 fold of dihydrotestosterone) and strong anti-androgenic potencies (9-62 fold of flutamide). Although they accounted to some extent for androgenic (0.3-29%) and anti-androgenic (4.6-27%) activities in influents, the progestins' contribution to (anti-)androgenic activities was negligible (≤2.1%) in effluents and surface waters. We also tested joint effect of equimolar mixtures of target compounds and the results indicate that compounds interact in an additive manner. Even if progestins possess relatively strong (anti-)androgenic activities, when considering their low concentrations (sub-ng/L to ng/L) it seems unlikely that they would be the drivers of (anti-)androgenic effects in Czech aquatic environments.

Biochemical and histological effects of sub-chronic exposure to atrazine in crayfish Cherax destructor.

Atrazine (ATR) is a triazine herbicide banned in the European Union. It remains one of the most widely used herbicides in other parts of the world. Considering the scarcity of data on its possible harm to the environment and to human health, we assessed sub-chronic effects of a 14-day exposure at the environmentally relevant concentration of 6.86 µg/L and at 10% of the 96hLC50 (1.21 mg/L) in crayfish Cherax destructor and their recovery in a 14-day period in ATR-free water. Indicators assessed were behavior; hemolymph biochemical profile; oxidative and antioxidant parameters in gill, hepatopancreas, and muscle; and histology of gill and hepatopancreas. Crayfish exposed to the environmental concentration showed significant differences (P < 0.01) from controls in biochemical parameters of hemolymph (lactate, alkaline phosphatase) and activity of superoxide dismutase, as well as in histology of gill tissue. The higher concentration led to low motor activity, differences in biochemical profile of hemolymph (lactate, alkaline phosphatase, ammonia, glucose), antioxidant biomarkers (superoxide dismutase, catalase, glutathione reductase, glutathione S-transferase, reduced glutathione), as well as gill and hepatopancreas histology. Some observed effects persisted after 14-days recovery in ATR-free water. The results provide evidence that environmental concentrations of ATR produce negative effects on freshwater crayfish.

Two synthetic progestins and natural progesterone are responsible for most of the progestagenic activities in municipal wastewater treatment plant effluents in the Czech and Slovak republics.

Vast numbers of xenobiotics are known still to be present in treated municipal wastewater treatment plant (WWTP) effluents. Some of these possess endocrine-disrupting potency and pose risks for exposed aquatic animals. We searched for 17 potential environmental contaminants having affinity to the progesterone receptor. Relative potency values of these progesterone receptor-active chemicals were obtained. On the basis of relative potencies and measured environmental concentrations, the contribution of progestins to measured progestagenic activities was evaluated. Wastewaters (influent and effluent) and surrounding surface waters (upstream and downstream) at six municipal WWTPs were screened using instrumental chemical analysis and in vitro reporter gene bioassay. We showed the presence of target compounds and (anti-)progestagenic activities in municipal wastewater and surface water. Nine and seven progestins were identified in influent and effluent wastewaters, respectively. Only two compounds, progesterone and medroxyprogesterone were found in surface waters. Progestagenic agonistic activities in influents were partially masked by strong anti-progestagenic activities that were detected in all influents and ranged from 2.63 to 83 ng/L of mifepristone equivalents (EQs). Progestagenic activities were detected in all effluents and ranged from 0.06 to 0.47 ng/L of reference compound ORG 2058 EQs (a synthetic progestin equivalents), thus indicating incomplete removal of progestins during wastewater treatment processing. This activity poses a continuing risk for the aquatic environment. By contrast, anti-progestagenic activities showed better removal efficiency in WWTPs compared to progestagenic agonistic activities. Anti-progestagenic activities were found in only three of six effluents and ranged from 0.26 to 2.1 ng/L mifepristone EQs. We explained most of the progestagenic activity in municipal WWTP effluents by the presence of synthetic progestins and progesterone, which contributed 65-96% of such activity in samples where no antagonistic activity was found. The progestins medroxyprogesterone acetate, megestrol acetate and progesterone contributed most to the progestagenic activity detected in municipal effluents. Anti-progestagenic activities were found in some municipal effluents, but no causative agents were revealed because two analysed selective progesterone receptor modulators (SPRMs) with anti-progestagenic activities, mifepristone and ulipristal acetate, were not present in the effluents.