Fast urbanization and improper waste treatment become question of concern in terms of the environment. It was previously shown that pharmaceuticals can enter water bodies via discharge from a waste water treatment plant‘s (WWTP). Consequently, all water body compartments may be influenced by the contaminated water.

Plenty of studies were done to understand behaviour of pharmaceuticalsand their metabolites (PTMs) in different matrices and their effect on living organisms, but usually they were done under controlled laboratory conditions.

In our study, we aimed to investigate fate of PTMs in the real aquatic environment represented by pond fed mainly with treated wastewater, as a worst case scenario. During a one year study, a complex approach including water, sediment and fish liver monitoring was used in our study to describe the behaviour of PTMs.

As a result, 23 compounds (18 pharmaceuticals and 7 their metabolites) were found in all studied matrices. Seasonal variations in all parameters and concentrations were also investigated.

The partitioning of contaminants between pond compartments was estimated by means of solid water distribution coefficients (Kd) and bioaccumulation factors (BAF) for the livers of fish. Kd values were almost stable throughout the year, which may be a sign of the continuous transport of PTMs between water and sediment. This observation should be taken into account in case of pond sediments application to agriculture fields, because contaminants may be later transported with natural precipitation to the lower soil layers, ground water, plants, etc. Almost all of the studied compounds, with exception of sertraline (BAF of 6200), were not bioaccumulative in fish livers. Further aspect, such as fish health status, reproduction are needed to be investigated.

A pond may be considered as a natural treatment system, where different processes occur, such as photodegradation, interaction between water and the atmosphere, sedimentation processes, biodegradation, etc. The pond removal efficiency was calculated for all PTMs, and favourable conditions for natural pharmaceutical removal were proposed.

Further details can be found in the following publication: Koba, O., Grabicova, K., Cerveny, D., Turek, J., Kolarova, J., Randak, T., Zlabek, V., Grabic, R., 2018. Transport of pharmaceuticals and their metabolites between water and sediments as a further potential exposure for aquatic organisms. Journal of Hazardous Materials 342: 401–407. (IF 2016 = 6,065).

The crayfish are widely accepted as very sensitive and fast bio-indicators of changes in water quality. Studies based on heart beat analysis demonstrated the potential of using crayfish as detector of pollutants. Consequently, several computer-aided systems were developed to use this mechanism. The main complication with applying such a system is the necessity of using metal wires or optical fibres to transmit the signal from sensor which is placed on back of the crayfish to the processing hardware; these attachments restrict system design to one crayfish in one aquarium.

The multidisciplinary collaboration of the two laboratories (Laboratory of Signal and Image Processing and Laboratory of Ethology of Fish and Crayfish) led to an original system for crayfish heart beat monitoring based on completely non-invasive/contactless hardware. The system can determine crayfish heart beat frequency using only the combination of near infra-red (NIR) illuminator and sensitive camera. The heart rate is the only parameter needed in most of the studies using crayfish as bio-indicators. The low-cost system needs no attachment, so more crayfish in one aquarium can be monitored simultaneously and it provides accurate information on heart rate and crayfish need no adaptation to the system. It can be used as equivalent to existing systems to simplify the crayfish studies.

Detailed information on obtained results can be found in publication: Císař, P., Saberioon, M., Kozák, P., Pautsina, A., 2018. Fully contactless system for crayfish heartbeat monitoring: Undisturbed crayfish as bio-indicator. Sensors and Actuators B: Chemical 255: 29-34. (IF 2016 = 5.401; AIS 2016 = 0.786).

Common carp belongs into species with highest worldwide production from aquaculture. Similarly, carp in the Czech Republic is the most cultured fish species. The important position of carp is conditioned by long-term breeding efforts when preservation of specific lines and breeds is crucial for further production of hybrids. Cryobanks with sperm collections are serving as a backup which can be utilized to recover valuable genotypes. Recent progress in reproductive biotechnologies developed an alternative strategy for preservation and recovery of genetic resources in fish. All sexually reproducing organism have germ stem cells, female oogonia and male spermatogonia. Germ stem cells are capable of self-renewal but also to differentiate into eggs and sperm. Researches from Faculty of Fisheries and Protection of Waters developed protocols for cryopreservation and recovery of germ stem cells in common carp under the course of Aquaexcel2020 project funded by the European Union. Developed strategy allows to cryopreserve valuable genetic material using efficient protocols for a virtually indefinite period while ensuring 40-65% post-thaw viability. Both testicular and ovarian tissue was cryopreserved having immense potential especially for female genetic resources because egg cryopreservation in fish is far from facilitation to real application.

Cryopreserved germ cells are thawed and recovered by transplantation into recipients. This technology is not far from surrogacy known in humans to tackle infertility issues. However, there is a difference when germ stem cell transplantation in fish results in self-sustaining system when continuous production of sperm and eggs is achieved. Generally, as a recipient can be chosen different species with more favourable characteristics eg. smaller body size, earlier maturation, resistance to disease – goldfish is fulfilling all mentioned advantages to carp while it is still very ordinary species known to anyone from aquaria or garden ponds.

Sterilization of recipients is next important prerequisite in surrogate reproduction. Sterile recipients ensure no competition between endogenous and exogenous cells after transplantation and produced donor-derived gametes are not contaminated by the gametes of the recipient. Knock-down of gene which is crucial for early germ cell survival is most frequently used methods for sterilization. Recipients are ready for transplantation when swim-up stage is reached. Then, gonadal tissue is digested into single cell suspension. Transplantation into goldfish was performed with glass microcapillary filled with cell suspension. About thousands of cells are injected directly into the body cavity of the recipient. Transplanted cells colonized the gonad of the recipient and started proliferation within few days post-transplantation. Presence of donor-derived germ cells in ⁓50% of the recipients was confirmed three months post-transplantation using molecular analysis. Donor-derived carp sperm after oogonia transplantation has been obtained already from goldfish surrogates. This unique ability is given by the stemness of germ stem cells when transdifferentiation and production of gametes of the opposite sex is further opening possibility to produce eggs and sperm even from a single germ cell donor.

Further information can be found in research articles:

- Franěk, R., Marinović, Z., Lujić, J., Urbányi, B., Fučíková, M., Kašpar, V., Pšenička, M., Horváth, Á. Cryopreservation and transplantation of common carp spermatogonia. PLoS ONE 14: e0205481.
- Franěk, R., Tichopád, T., Steinbach, C., Xie, X., Lujić, J., Marinović, Z., Horváth, Á, Kašpar, V., Pšenička M. Preservation of female genetic resources of common carp through oogonial stem cell manipulation. Cryobiology 87:78-85.

Thousands of pharmaceutically active compounds and their metabolites are present in aquatic environments worldwide at concentrations from ng to μgL-1. These low detected amounts show an impact on non-target organisms as they are designed to act at very low concentrations. However, pollutants persist in aquatic environments as complex mixtures. But how can these mixtures affect aquatic organisms? It remains largely unresolved.

The aim of the study was to determine how differs the response to the exposure of a combination of six pharmaceuticals (citalopram, sertraline, oxazepam, venlafaxine, tramadol, methamphetamine) from their individual exposure effects and how it affects the exploratory and sheltering behaviour as well as life history traits of crayfish. In the absence of shelter, exposed crayfish moved significantly shorter distances and at a lower velocity and showed significantly less activity than that of the control. With available shelter, exposed crayfish moved significantly longer distances, showed higher activity, and spent significantly more time outside the shelter than that of the control. Results suggest that environmentally relevant concentration of the mixture used can alter the behaviour of non-target aquatic organisms even more than detected in an individual exposure to these compounds. It may lead to deterioration of crayfish populations health, their higher vulnerability to predation and consequently into disruption of ecosystem processes. However, there is a need of more studies focused on different combinations of pollutants and their mode of action in the aquatic environment.

Further detailed information can be found in the original article: Hossain, M.S., Kubec, J., Guo, W., Roje, S., Ložek, F., Grabicová, K., Randák, T., Kouba, A., Buřič, M., 2021. A combination of six psychoactive pharmaceuticals at environmental concentrations alter the locomotory behaviour of clonal marbled crayfish. Science of the Total Environment 751: 141383.

A team of Czech scientists discovered the first cave crayfish in New Guinea and in the Southern Hemisphere. Cherax acherontis Patoka, Bláha and Kouba, 2017 is the first cave crayfish found outside North America. This is a unique discovery that contributes to the knowledge of evolutionary processes of unrelated species and their adaptation to the extreme environment, such as the caves.

Researchers Jiří Patoka from the Czech University of Life Sciences Prague and Martin Bláha and Antonín Kouba from the University of South Bohemia in České Budějovice have been working with the New Guinean crayfishes for several years, and among others, they already scientifically described two other species. The moment they came to know from local guide about endemic crayfish inhabiting a submerged river flowing through karst caves in the mountains of the Papua Province, they decided to arrange an expedition to discover and explore this enigmatic cave dweller.

Cherax acherontis is a crayfish endemic to the submerged river Yumugima in Hagepma/Jugurama cave in the New Guinea Highlands, Jayawijaya Regency, Papua Province, Indonesia. Its Latin named is derived from Acheron, one of the rivers that ran through the underworld of ancient Greek mythology. Crayfish has reduced eyes due to lack of light, and its body has lost almost all pigmentation. The other features are similar to North American cave crayfish species and corroborate convergent evolution when unrelated species develop similarly due to the environmental pressure.

Further details can be found in the following publication: Patoka, J., Bláha, M., Kouba, A., 2017. Cherax acherontis (Decapoda: Parastacidae), the first cave crayfish from the Southern Hemisphere (Papua Province, Indonesia). Zootaxa 4363: 137-144. (IF 2016 = 0.972)

Short video from the expedition (in Czech) available here.

Carp is the most important fish in the Czech Republic with share of 85– 90% on total fish production. Worldwide carp annual production over 4 million tons ranks this species on the fourth the most cultured fish. Still, carp breeding is far beyond the methods used in other fish species (Atlantic salmon, rainbow trout, gilthead seabream and European seabass). The most used method for increasing fish performance is selective breeding. It is based on selecting fish with the best phenotypic value of a given trait, such as growth, resistance to diseases, yield of edible parts, for subsequent artificial reproduction. If the selected trait is sufficiently heritable, the offspring of such fish will show higher performance of a given trait in comparison with the unselected stock. In this way, the desired traits can be continually improved in the next generations as genetic gain is cumulative. However, if we want to increase the yield of edible parts (processed body, filets), the phenotypic value of such traits cannot be ascertained other than by killing and processing of fish. As an alternative, selection based on the performance of siblings is commonly used. However, it requires the killing and processing of part of the fish from a given stock and, above all, the knowledge of the pedigree of each individual in the stock. Under the pond conditions it can be achieved only by methods of molecular biology, and this is disproportionately expensive. For such traits, it is therefore necessary to look for another reliable selection method of suitable candidates.

Scientists from the Faculty of Fisheries and Protection of Waters at the University of South Bohemia in České Budějovice have been involving together with Klatovy fishery corp. in a European project focused on the boosting of European aquaculture in the six main fish species to advanced level through selective breeding. Czech scientists and their foreign colleagues have focused on edible body parts yield in common carp. Carp headless carcass forms 58 – 62% of live fish weight and we assume that the selection might increase the percentage up to 10%. During the fish slaughtering, edible parts percentages were determined as well as thickness of the abdominal wall and the depth of the abdominal cavity by ultrasound. Besides, the muscle fat content was recorded by fatmeter and various lengths, contents and ratios were detected from pictures of fish. These measurements can be done on live fish. Recorded traits were combined with multiple linear regression and their values ​​compared with the real yield of headless carcass and fillets. It was found that the yield of edible parts can be predicted with high accuracy on live fish. In addition, ratio between the thickness of the abdominal muscle and the depth of the body cavity is in direct proportion to the edible parts yield.

All traits are also highly heritable. Thus, yield of edible body parts can be indirectly increased by selecting for values calculated based on ultrasound measurements. There is a theoretical possibility to increase filets yield up to 0.7% per generation. From a practical point of view, this non-invasive method of improving the performance of slaughtering traits could be effectively utilized under the operational conditions of Czech fishery after training the professional staff. However, it is necessary to say that the use of indirect selection has to be verified on other carp breeds and after the realized selection. Both partners would like to continue in the research even after the end of FISHBOOST project. Detailed information can be found in the original article: Prchal, M., Bugeon, J., Vandeputte, M., Kause, A., Vergnet, A., Zhao, J., Gela, A., Haffray, P., Kocour, M., 2018. Potential for genetic improvement of the main slaughter yields in common carp with in vivo morphological predictors. Frontiers in Genetics 9, 283.

Water reuse and aquaculture: Pharmaceutical bioaccumulation by fish during tertiary treatment in a wastewater stabilization pond.

With increasing demand for aquaculture products, water reuse is likely to increase for aquaculture around the world. We determined the bioaccumulation of selected pharmaceuticals from several therapeutic classes by two important aquaculture fish species (common carp – Cyprinus carpio and pikeperch – Sander lucioperca) which were restocked to a wastewater stabilization pond for six months. Forty and nineteen from 66 selected pharmaceuticals and their metabolites were positively found in water and sediment samples. Fourteen pharmaceuticals and their metabolites were detected in at least one fish tissue. We observed differences for fish species and organ specific bioaccumulation factors among studied pharmaceuticals. Though muscle tissues consistently accumulated lower levels of the target analytes, several substances were elevated in the brain, liver and kidney tissues (e.g., antidepressant sertraline) of both species. Low residual concentrations of these target analytes in aquaculture products (fish fillets) suggest that these ponds (which commonly served as a tertiary step for wastewater treatment and can be used as the worst-case scenario) are promising to support the water-food nexus in aquaculture.

Further details can be found in the following publication: Grabicová, K., Grabic, R., Fedorova, G., Vojs Staňová, A., Bláha, M., Randák, T., Brooks, B.W., Žlábek, V., 2020. Water reuse and aquaculture: Pharmaceutical bioaccumulation by fish during tertiary treatment in a wastewater stabilization pond. Environmental Pollution 267: 115593. (IF 2019 = 6.792; AIS 2019 = 1.229).

Psychoactive pharmaceuticals are receiving increased attention because modulation of biological targets in the central nervous system can lead to altered behaviour of exposed organisms (e.g. predators-prey relationships, social traits, feeding rate, reproduction or migration strategies).

Ten Czech rivers with diverse flows and contribution of communal pollution was selected for examination of psychoactive pharmaceuticals occurrence. We used several common sampling approaches – grab water samples, passive samples, the whole-body homogenate of juvenile fish and selected adult fish tissues. We found statistically significant relationship between grab water and passive samples while no relationship was observed for passive samples and tissue specific pharmaceuticals accumulation. Highly bioaccumulative antidepressant sertraline was found in all adult fish brain samples except of fish from Vltava River in National Park Šumava. 

We could further conclude that instream dilution of effluent plays more important role than size of pollution source.

Further details can be found in the following publication: Grabicová, K., Grabic, R., Fedorova, G., Kolářová, J., Turek, J., Brooks, B.W., Randák, T., 2020. Psychoactive pharmaceuticals in aquatic systems: A comparative assessment of environmental monitoring approaches for water and fish. Environmental Pollution 261, 114150.

The rate of spread of invasive species into new localities is alarming. It is increasingly common that several such species meet at the same locality. These species put severe pressure on local populations of native species. Due to their ability to effectively utilize food resources, changes in the food webs could possibly result in collapses of entire ecosystems.

In this study, we focused on a locality with two, respectively three co-occurring non-native crayfish species – marbled crayfish, spiny-cheek crayfish, and red swamp crayfish. These species belong to important invasive species in Europe. Using stable isotopes analysis, we investigated their trophic niches at the studied locality. We found that the trophic niches of spiny-cheek crayfish and marbled crayfish are reduced in the presence of red swamp crayfish and that this species preys on the formerly mentioned. Thus, we had the opportunity to get a deeper insight into their trophic relationships.

Detailed information can be found in: Veselý, L., Ruokonen, T.J., Weiperth, A., Kubec, J., Szajbert, B., Guo, W., Ercoli, F., Bláha, M., Buřič, M., Hämäläinen, H., Kouba, A., 2021. Trophic niches of three sympatric invasive crayfish of EU concern. Hydrobiologia 848: 727–737.