Field cyanobacterial blooms producing retinoid compounds cause teratogenicity in zebrafish embryos

Chemosphere 241 (2020) 125061

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Field cyanobacterial blooms producing retinoid compounds cause teratogenicity in zebrafish embryos Marek Pipal, Jana Priebojova, Tereza Koci, Lucie Blahova, Marie Smutna, Klara Hilscherova* RECETOX Faculty of Science Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic

h i g h l i g h t s
Retinoid-like activity and retinoids occur in field cyanobacterial blooms.
Gross teratogenic effects linked to specific fractions with retinoid-like activity.
ZFET detected teratogenicity at lower level than expected from in vitro assay.
Teratogenicity found at concentrations relevant to the situation in the environment.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 July 2019 Received in revised form 3 October 2019 Accepted 4 October 2019 Available online 6 October 2019

Cyanobacteria routinely release potentially harmful bioactive compounds into the aquatic environment. Several recent studies suggested a potential link between the teratogenicity of effects caused by cyanobacteria and production of retinoids. To investigate this relationship, we analysed the teratogenicity of field-collected cyanobacterial bloom samples by means of an in vivo zebrafish embryo test, an in vitro reporter gene bioassay and by the chemical analysis of retinoids. Extracts of biomass from cyanobacterial blooms with the dominance of Microcystis aeruginosa and Aphanizomenon klebahnii were collected from water bodies in the Czech Republic and showed significant retinoid-like activity in vitro, as well as high degrees of teratogenicity in vivo. Chemical analysis was then used to identify a set of retinoids in ng per gram of dry weight concentration range. Subsequent fractionation and bioassay-based characterization identified two fractions with significant in vitro retinoid-like activity. Moreover, in most of the retinoids eluted from these fractions, teratogenicity with malformations typical for retinoid signalling disruption was observed in zebrafish embryos after exposure to the total extracts and these in vitro effective fractions. The zebrafish embryo test proved to be a sensitive toxicity indicator of the biomass extracts, as the teratogenic effects occurred at even lower concentrations than those expected from the activity detected in vitro. In fact, teratogenicity with retinoid-like activity was detected at concentrations that are commonly found in biomasses and even in bulk water surrounding cyanobacterial blooms. Overall, these results provide evidence of a link between retinoid-like activity, teratogenicity and the retinoids produced by cyanobacterial water blooms in the surrounding environment. © 2019 Elsevier Ltd. All rights reserved.

Handling Editor: David Volz Keywords: Retinoids All-trans retinoic acid Zebrafish Teratogenicity Retinoid-like activity Cyanobacteria

1. Introduction In the last decades, multiple problems associated with anthropogenic water pollution, including eutrophication (i.e. extensive loads of nutrients like phosphorus or nitrogen), were documented in surface water ecosystems (Huisman et al., 2018). Besides contamination of drinking water sources, water eutrophication

* Corresponding author. E-mail address: [email protected] (K. Hilscherova). https://doi.org/10.1016/j.chemosphere.2019.125061 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

poses a constant risk to aquatic biodiversity and leads to cyanobacterial blooms that represent a worldwide threat to surface aquatic ecosystems (Huisman et al., 2018). These blooms can affect dissolved oxygen and pH levels in water and produce a wide spectrum of compounds, which can have adverse impacts on aquatic organisms. Various cyanobacterial metabolites or toxins such as the most studied hepatotoxic microcystins have been
ha et al., 2009). However, many studies revealed the described (Bla toxicity-independece of microcystins or other known cyanotoxins (Jaja-Chimedza et al., 2017; Jonas et al., 2014; Zi et al., 2018) and suggest that there are substances with specific toxicities unrelated

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M. Pipal et al. / Chemosphere 241 (2020) 125061

to eutrophication, such as endocrine disruptive or teratogenic properties (Jaja-Chimedza et al., 2015; Jonas et al., 2014). Many fish, amphibian and crustacean species are endangered or close to extinction (Halliday, 1998; Heatwole and Wilkinson, 2013). There is a great concern about amphibian and fish decline and there are reported teratogenic effects in frogs in nature (Gardiner and Hoppe, 1999; Huisman et al., 2018; Zi et al., 2018). It has been suggested that these effects, including limb, bone or skin deformations, may be associated with the disruption of retinoid signalling since they are similar to those of the known teratogen, retinoic acid (RA) (Gardiner et al., 2003; Gardiner and Hoppe, 1999). Several studies have shown that cyanobacteria can release retinoid compounds into surface waters. Retinoic acid and its analogues were detected in lakes containing massive cyanobacterial water blooms and in the biomass and exudates of several cyanobacterial species (Jav urek et al., 2015; Sehnal et al., 2019; Wu et al., 2013, 2012). Retinoids play a crucial role in the regulation of vertebrate development, with RA acting as the active compound. Some RA analogues are metabolized to active RA. Several bioactive isomers of RA have been described, such as all-trans retinoic acid (ATRA), 9cis retinoic acid (9cis-RA) or 13-cis retinoic acid (13cis-RA), that interact with two major groups of nuclear retinoid receptors Retinoic Acid Receptors (RARs) and Retinoid-X Receptors (RXRs) (Oliveira et al., 2013). These receptors form a dimer that binds to DNA and directly influences downstream pathways such as the expression of homeobox genes that are crucial in early development and highly conserved across vertebrate species (Duester, 2008). Therefore, possible teratogenicity caused by compounds from cyanobacterial blooms with retinoid-like activity is of significant concern for vertebrates, including humans. In addition to the identified retinoids in the environment, retinoid-like activity was detected by in vitro biodetection tools in extracts and exudates of several cyanobacterial species cultured in
et al., the laboratory (Jonas et al., 2014; Kaya et al., 2011; Priebojova 2018). As documented in recent studies, retinoid content and retinoid-like activity are linked and are a common feature of cyanobacteria species in both laboratory cultures and environmental
et al., 2018; Sehnal et al., 2019). Retinoids blooms (Priebojova produced by cyanobacteria could play a role in previously observed malformations, including edema, bent tails, undeveloped eyes and neural tube deformities, in zebrafish embryos exposed to extracts of cyanobacteria Microcystis aeruginosa, Anabaena flos-aquae, Cylindrospermopsis raciborskii, Aphanizomenon ovalisporum, Planktothrix agardhii, Aphanizomenon flos-aquae and others (Acs et al.,
et al., 2006; Ghazali et al., 2013; Berry et al., 2009; Burýskova 2009; Oberemm et al., 1999). In recent studies, teratogenicity along with retinoid-like activity in vitro were also reported after the exposure of fish and amphibians to samples from some laboratorycultured cyanobacteria species (Jonas et al., 2015; Smutn
a et al., 2017) which could potentially contain retinoid-like compounds, although they were not measured explicitly. The above-listed evidence suggests a link between retinoids, retinoid-like activity and teratogenicity with cyanobacteria as the source. Cyanobacteria produce very complex mixtures containing thousands of compounds, which makes the identification of the responsible agents difficult. Nevertheless, retinoids are among the compounds produced by cyanobacteria with significant biological
et al., 2018; Sychrova
et al., 2016). Among the activity (Priebojova produced retinoids are retinoic acids (RA) and their analogues and precursors like retinal (RAL) (Wu et al., 2013). Although retinoid compounds were detected in lakes, ponds and reservoirs with developed cyanobacterial blooms (Jav urek et al., 2015; Sehnal et al., 2019; Wu et al., 2013, 2012), so far only in vitro retinoid-like activity was observed, in addition to the several retinoids analysed in lab
et al., oratory cyanobacterial cultures (Kaya et al., 2011; Priebojova


et al., 2016) and field samples (Jav 2018; Sychrova urek et al., 2015; Sehnal et al., 2019), and unfortunately the relationship to the effects observed in vivo is still unknown. Alternatively, retinoid-like activity together with in vivo effects were analysed in laboratory cultures for several cyanobacterial species (Jonas et al., 2015, 2014) with limited relevance to the environment and also lacked the detection of retinoid compounds. We investigate the hypothesis that compounds with retinoidlike activity play a crucial role in teratogenic effects of samples from environmental cyanobacterial blooms. It is the first study to examine the link between the in vitro retinoid-like activity of environmental cyanobacterial bloom biomasses, and the presence of specific retinoids and teratogenic effects in vivo. We aim to quantify retinoid content and evaluate the relevance of in vitro retinoid-like activity towards teratogenic effects in whole organisms. We therefore conducted a detailed investigation of the extracts of two cyanobacterial bloom biomasses with different species composition with high retinoid-like activity in vitro, sampled from surface water reservoirs in the Czech Republic. This study investigates the relationship of the in vitro RAR-mediated bioactivity detected using a reporter gene assay, and the presence of retinoids in the samples with in vivo teratogenicity in zebrafish embryos. We also fractionate the biomass extracts to separate and characterize the potential bioactive compounds and fractions. The obtained information is of high environmental relevance since we have worked with complex field biomasses and mixtures of compounds they contain. 2. Materials and methods 2.1. Sampling and preparation of extracts Cyanobacterial biomasses were sampled in summer 2012 and 2013 from different water bodies in the Czech Republic dominated by developed water blooms. The two biomasses investigated in this study have a different taxonomic composition. The first sample from Vranov reservoir, locality Bítov, was dominated by Microcystis aeruginosa (100%, 1.23  107cells per mL; further referred as “Ma”) and the second sample collected from reservoir Nova Ruda was dominated by Aphanizomenon klebahnii (92%, 2.46  106 cells per mL; further referred to as “Ak”). The biomasses were sampled by a plankton net (20 mm mesh) and details about the sampling sites and conditions are listed in Supplementary Information (Table S1). Biomasses were freeze-dried, extracted into 100% methanol and processed as previously optimized and described (Jav urek et al., 2015). The extractions were performed in glass tubes in parallel in a small volume (200 mg dw per 5 mL) by sonication (2  2 min). The samples were kept on ice during sonication, the prepared extracts were then centrifuged (3000g, 5 min, room temperature) and the supernatant was collected. The biomasses were then reextracted by 1 mL methanol, re-centrifuged and then the supernatants were pooled for individual biomasses. The resulting extracts were concentrated under nitrogen to a final concentration of 400 g dw L1 and then centrifuged again in plastic tubes (11 000g, 15 min, room temperature) to remove suspended particles prior to further use (with no effect of the centrifugation on bioactivity in vitro, data not shown). The final extracts were kept in amber glass vials in 100% methanol at 20  C. 2.2. In vitro bioassay The in vitro retinoid-like activities of the biomass extracts were measured on murine embryonic carcinoma cell line P19/A15 with endogenous expression of retinoic acid receptor (RAR) stably transfected with the reporter luciferase gene under the control of

M. Pipal et al. / Chemosphere 241 (2020) 125061

retinoic acid-responsive element (pRAREb2-TK-luc plasmid) as
et al., 2016). previously described (Nov
ak et al., 2007; Sychrova Briefly, luminescence was measured after 24 h exposure to four dilutions of each sample. The tested concentrations were 0.25, 0.5, 1 and 2 g dw L1. The exposures were done in triplicate and independently repeated three times. With each experiment, an all-trans retinoic acid (ATRA) standard was tested in a series of dilutions (concentration range 0.45e1000 nM, equivalent to 135e300 000 ng L1) for a calibration curve of the retinoid-like response. Total retinoid-like activity was then expressed as the equivalent concentration of the ATRA standard (REQ in vitro, ng ATRA g dw1 biomass) causing the same effect as the tested sample (non-linear regression of ATRA calibration curve).

2.3. Chemical analysis and fractionation The fractionation method was developed on an Agilent (1200 series) LC/DAD instrument. A Waters X-Bridge C18 column (5 mm, 4.6  100 mm) was used for the separation with an increasing methanol gradient. Each sample was separated into twelve fractions based on polarity. The mobile phase consisted of water (A) and methanol (B). The following gradient at a flow rate of 1 mL min1 was used for elution: 0e5 min 25%e65% B, 5e8 min 65%e 100% B, 8e36 min 100% B. After each run, the column was reequilibrated to 25% B for ca. 10 min. The temperature of the column was maintained at 23  C. Biomass samples were injected at 100 ml in 50% methanol (concentration of 200 g dw L1). Fractions were collected continuously during the 36 min run of the separation: Fraction 1 was collected for the first 2 min of the separation, Fractions 2e6 were collected for 2.5 min each, Fractions 7 and 8 for 2 min each, Fractions 9 and 10 again for 2.5 min each, Fraction 11 for 6 min and Fraction 12 for 6.5 min. The same fractions from several runs were pooled, and chromatograms of pooled runs were verified to show the same retention times of individual peaks. The pooled samples were evaporated under nitrogen to achieve a final concentration 400 g dw L1 in 100% methanol. Individual fractions containing compounds separated by their polarity were tested by in vitro assays to identify the most effective fractions for further characterization of the active compounds and in vivo effects. For targeted analysis, seven compounds were analysed by LC
et al. (2018). Both MS/MS as previously reported by Priebojova raw extracts and their fractions were analysed for all-trans retinoic acid (ATRA), 9-cis retinoic acid (9cis-RA), 13-cis retinoic acid (13cisRA), retinal (RAL), all-trans 4-keto retinoic acid (4keto-ATRA), alltrans 5,6-epoxy retinoic acid (5,6epoxy-ATRA) and 4-keto retinal (4keto-retinal). The stereoisomers 9cis-RA and 13cis-RA co-eluted at the same time and had the same parent and fragment ions, therefore they are reported as a sum (Table 1). The separation was conducted on a Waters Acquity UPLC (Waters, Milford, MA, USA) using a Waters Acquity UPLC BEH C18 column (1.7 mm, 2.1  100 mm) and VanGuard C-18 guard column (Waters, Milford, MA, USA). Acetonitrile and water with the addition of 0.1% (v/v) formic acid were used as the mobile phase in gradient elution. Detection was performed on a Waters Xevo TQ-S mass spectrometer (Waters, Milford, MA, USA) in positive ESI mode. Quantification of analytes was based on external deuterium labelled retinoid standards of ATRA-d5 (for ATRA and 9cis/13cis-RA), 4keto-retinald3 (for 4keto-retinal, 4keto-ATRA and 5,6epoxy-ATRA) and retinald5 (for retinal). Microcystins (MC) were also analysed in the biomass extracts and fractions using an LC-MS/MS method as previously described (Jav urek et al., 2015). Quantification of analytes was based on external standards of MC-RR, MC-YR and MC-LR.

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2.4. In vivo bioassay (zFET) A zebrafish (Danio rerio) embryo toxicity test (zFET) was used for the detection of bioactivity in vivo. Wild-type zebrafish juveniles (<3 months) were purchased from a local supplier and were raised in standard laboratory conditions for at least 6 months prior to collection of embryos for experiments (Lawrence, 2007; OECD, 2013). The fishes were maintained in aquaria containing local tap -1 water (CaCO3 100e180 mg L-1, NO 3 10e25 mg L , pH 7.0e7.2) at 26 ± 1  C with a 14:10 h light:dark photoperiod and fed a mixture of dried food twice daily (Tubifex, EasyFish; Spirulina, SERA; Gammarus, Dajana; Vipan flake food, SERA) and live brine shrimp (Artemia salina) once daily. Mesh traps with artificial plants were placed in the aquaria the evening before spawning and were collected within 1 h of the start of the light period the following day. The collected embryos were rinsed with distilled water and immediately placed in standard fish medium (ISO 7346, 1996). The embryos were then examined under a stereomicroscope to identify fertilised eggs which were selected and used in experiments. Tests were conducted in glass Petri dishes in triplicate (60 mm diameter; 10 mL per dish per 20 embryos). All exposures were static, starting 3e4 h post fertilization (hpf), latest at epiboly developmental stage (Kimmel et al., 1995), and terminated at 120 hpf. The exposed embryos were incubated at 27 ± 1  C in the dark with no photoperiod in order to account for the photosensitivity of retinoids. Dissolved oxygen and pH were monitored during the test. Oxygen was measured by a probe (Inolab IDS Multi 9420, FDO®925, WTW) for a prolonged time (ca. 10 min) in the exposure wells after equilibrium was reached while avoiding any stirring or manipulation that would compromise the oxygen levels. No significant changes were detected during the exposure, with pH maintained at 8.1 ± 0.4 and oxygen saturation above 80% (26 ± 1  C) in all treatments and test solutions. The ATRA standard and the two field-collected biomass extracts and their fractions were tested in a dose-response manner. Experiments with the ATRA standard used a concentration series of 0.4, 0.8, 1.2, 1.6, 2, 4, 6 and 10 mg L1. Field biomass samples were tested at extract concentrations of 0.05, 0.1, 0.2, 0.3, 0.6 and 1 g dw L1. The biomass extract fractions with detected retinoid-like activity were further tested in zFET to address the range of effective concentrations: Ma Fraction 5 at 0.1, 0.2 and 0.6 g dw L1; Ak Fraction 4 at 0.6 g dw L1 and Ak Fraction 5 at 0.2, 0.3, 0.6 g dw L1. These concentration ranges were based on the results of a preliminary screening test. The fractions of individual extracts with no retinoid-like activity in vitro were pooled and tested together as one sample at a concentration of 0.6 g dw L1; the Ma Fraction 4 was also pooled with the non-active fractions for the zFET assay since there was no detectable effect on development in the limit test (Ma F4 concentration 0.6 g dw L1). Fish medium (ISO 7346, 1996) was used as a negative control. Stock solutions of ATRA and the biomass extracts were prepared in 100% methanol and stored at 20  C, at concentrations of 1 g L1 for ATRA and 400 g dw L1 for the extracts and their fractions. Prior to exposure, every vial was ultra-sonicated in a water bath for at least 1 min or until fully dissolved. Afterwards, an appropriate amount of stock solution extracted in methanol was added into the amber glass vial for the highest tested concentration and the methanol was evaporated under a stream of nitrogen gas. After complete evaporation of methanol, the fish medium was added to the vial and the dried sample was re-dissolved and ultra-sonicated for at least 1 min or until no suspension or particles were visible. Subsequent serial dilutions were made from the highest concentration in the fish medium. Embryos were monitored daily under a stereomicroscope, dead embryos were removed and all deviations from normal

4 Table 1 Total retinoid-like activity and concentration of retinoids and microcystins in cyanobacterial extracts. In vitro retinoid-like activity determined in assay using P19/A15 cell line is presented as all-trans retinoic acid equivalent (REQ in vitro) ± standard deviation (N ¼ 3). The concentrations of detected compounds are expressed in ng per g of dry weight of biomass. REQchem are calculated from results of chemical analyses and expressed as ATRA equivalent
et al., 2018) and the detected concentrations of individual compounds in samples. REQ explained shows the percentage of in vitro activity (REQ in vitro) that can be based on potencies of individual compounds (REP, Priebojova explained by detected compounds (REQchem).

Retinoids [ng g dw1]

Extract Ma Microcystis aeruginosa (100%) REP Whole Fraction 4 Fraction 5 Fraction non-RA

2850±505 697±91 1640±229 n.i. LOD

16

LOD LOQ

REQ chem [ng ATRA g dw1] ATRA

9cis/13cis-RAa

1.00 296 n.d. 227 n.d.

retinal

4keto-ATRA

5,6epoxy-ATRA

0.33

0.02

0.36

0.73

0.11

n.d. n.d. n.d. n.d.

20e80 n.d. 459 777

383 329 n.d. n.d.

119 n.d. 52 n.d.

12e40 n.d. 12e40 59

12 120

20 40

20 80

20 40

12 40

12 40

ATRA

9cis/13cis-RA

retinal

4keto- ATRA

5,6epoxy-ATRA

4keto- retinal

848 n.d. 791 n.d.

216 n.d. 178 n.d.

142 n.d. 437 n.d.

2136 1840 n.d. n.d.

222 n.d. 83 n.d.

n.d. 116 129 n.d.

12 120

12 40

20 40

20 40

20 40

20 40

REQ explained [%]

Microcystins [mg g dw1]

4keto-retinal

560 117 450 21

18 17 17 n.i.

MC YR

MC RR

MC LR

18 28 n.d. 19

92 64 n.d. n.d.

120 n.d. n.d. 163

0.04 0.12

0.002 0.006

0.02 0.06

MC YR

MC RR

MC LR

n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d.

0.04 0.12

0.002 0.006

0.02 0.06

Extract Ak Aphanizomenon klebahnii (92%)

Whole Fraction 4 Fraction 5 Fraction non-RA

2869±467 1027±131 1601±165 n.i. LOD

16

LOD LOQ

n.i.- no induction, n.d.- not detected, LaD- Limit of Detection, LOQ- Limit of Quantification. a 9-cis RA and 13-cis RA co-eluted and had the same parent and fragment ions, therefore they are reported as a sum.

1844 668 918 n.d.

64 65 57 n.i.

M. Pipal et al. / Chemosphere 241 (2020) 125061

REQ in vitro [ng ATRA g dw1]

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development were recorded. At the end of the experiment, all embryos were anaesthetized using MS-222, fixed in 2.5% methylcellulose and evaluated and photographed individually under the stereomicroscope (OLYMPUS, 90 magnification). Each malformation was recorded as present or not present, and their frequency of occurrence in each respective experiment was calculated. Individual malformations were not evaluated in highly deformed embryos with severe edemas. These were classified as embryos with complex deformation since counting of individual defects might be misleading. The frequency of embryos with any deformation in individual treatments (referred to as “combined malformations”) was used for the calculation of EC values regardless of the type of malformation. 2.5. Statistical analysis The effects of extracts and their fractions in the P19/A15 bioassay were expressed as the Retinoid EQuivalent concentration of the ATRA standard that would cause the same effect (REQ in vitro; ng of ATRA per g of biomass dry weight, ng ATRA g dw1). REQs in vitro were calculated by relating the EC20 value of the ATRA calibration to the concentration of the tested sample that induced the same response. EC20 values were calculated from nonlinear logarithmic regression of dose-response curves (GraphPad Prism 6,
et al., GraphPad Software, USA) as previously described (Priebojova 2018). The contribution of detected retinoids to total REQs was also calculated and expressed as a combined sum of individual

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compound contributions (REQ chem, Table 1). The detected concentration of each compound was multiplied by its relative potency (REP, Table 1) compared to ATRA. These relative potencies were previously experimentally derived using the same in vitro P19/A15
et al., 2018). assay used in this study (Priebojova The REQ chem was derived according to the concentration addition concept by summing the contribution of the detected compounds. A mass-balance comparison of this REQ chem based on the chemical analyses and the REQ derived from the in vitro bioassay was conducted to determine how much of the total activity detected in the bioassay (REQ in vitro) could be explained by the analysed compounds (% REQ Explained, Table 1). The mortality and malformation frequencies in experiments with zebrafish embryos were expressed as a percentage of affected embryos, and each dish with 20 embryos was treated as an individual replicate (N ¼ 9). Only surviving embryos were included in the calculations of malformation frequency. Dose-response curves, EC values and Confidence Intervals (CI) were calculated in GraphPad Prism 6 using non-linear regression (variable slope). LOEC and NOEC values were evaluated by Fisher’s exact test using Statistica 13 software (StatSoft, USA). 3. Results 3.1. In vitro assays The in vitro bioassays showed significant total retinoid-like activity in both of the studied extracts of biomasses from

Fig. 1. Retinoid-like activity of samples Ma (A) and Ak (B) and their fractions determined by P19/A15 in vitro assay. Four concentrations were measured for each sample (N¼3) and activity is expressed as percents of maximal induction caused by calibration standard ATRA.

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cyanobacterial water blooms dominated by different species. The Ma sample showed total retinoid-like activity of 2850 ± 505 ng of ATRA equivalent per gram of biomass dry weight (ng ATRA g dw1) and the Ak sample showed a similar level of activity of 2869 ± 467 ng ATRA g dw1 (Table 1). The fractionation divided each extract into twelve fractions, which were tested for in vitro retinoid-like activity. A significant RAR-mediated response was detected by the in vitro bioassay in two fractions of both extracts. REQs of 697 ± 91 and 1640 ± 229 ng ATRA g dw1 were detected in Fractions 4 and 5 of the Ma extract, respectively (Table 1). Similarly, Fractions 4 and 5 of the Ak biomass extract contained REQs of 1027 ± 131 ng ATRA g dw1 and 1601 ± 165 ng ATRA g dw1, respectively. No other fraction of either extract showed detectable retinoid-like activity up to the highest tested concentration of 2 g dw L 1 during the in vitro screening (Fig. 1, Table 1). Based on these results and the distribution of retinoid-like activity among the fractions, we then pooled together the in vitro non-active fractions to simplify further testing. Hereafter, ‘Fraction non-RA’ refers to a sample of pooled Fractions 1e3 and 6e12 of the respective extracts with each fraction at the declared concentration. This pooled sample was further used in all subsequent assays along with the other samples to determine the final in vitro retinoid-like activity, in vivo teratogenicity, as well as for chemical analysis (Table 1).

3.2. Chemical analysis The analysed retinoids were detected in the ng per gram of biomass dry weight (ng g dw1) range in the Ma and Ak extracts (Table 1). We analysed a set of compounds that could contribute to the total retinoid-like activity, including all-trans retinoic acid (ATRA), 9/13-cis retinoic acid (9cis/13cis-RA), 5,6epoxy-ATRA, 4keto-ATRA, 4-keto retinal, retinal and retinol. Nevertheless, retinol was not detected in any of the samples in concentrations above the limit of detection (LOD 60 ng g dw1) and therefore it is not reported in Table 1. The most potent retinoid, ATRA, was present in whole extracts at concentrations of 296 (Ma) and 848 (Ak) ng g dw1, but the most abundant retinoid in the tested samples was 4keto-ATRA at concentrations of 383 (Ma) and 2136 (Ak) ng g dw1, respectively. Regarding the other potent retinoids, 5,6-epoxy ATRA was detected at concentrations of 119 (Ma) and 222 (Ak) ng g dw1, and 9cis/13cis-RA were detected in the Ak extract at a concentration of 216 ng g dw1 (Table 1). Following fractionation, all analysed retinoid compounds eluted in the bioactive Fractions 4 and 5 for both samples with the exception of retinal and 4-keto retinal in the Ma sample which were also detected in the pooled non-bioactive fractions. However, retinal (similarly 4-keto retinal) has very low potency compared to

Fig. 2. Dose-response curves for zFET experiment at 120 hpf for extract Ma (A) and Ak (C) and comparison of malformation frequency in whole extracts and their respective fraction 5 at the same concentrations (B and D). Error bars represent standard deviation (N¼9, conducted as three independent experiments in triplicate with 20 embryos for each replicate and treatment group).

M. Pipal et al. / Chemosphere 241 (2020) 125061

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(mg g dw1) in the Ma sample (Table 1) and eluted mostly in Fraction 4 (MC YR and MC RR) and the “non-RA” fractions (MC YR and MC LR). No microcystins were detected in the Ak extract. 3.3. In vivo assays

Fig. 3. Dose-response curves for zFET experiment at 120 hpf for standard compound ATRA. Error bars represent standard deviation of three independent experiments performed in triplicate (N ¼ 9, 20 embryos for each replicate).

ATRA (ReP, Table 1), and even at relatively high concentrations detected in the Ma non-RA fraction (853 ng g dw1) it did not cause any detectable effect in the bioassays. The more polar retinoids such as 4keto-ATRA, as well as 4keto-retinal in the Ak extract, eluted in Fraction 4, while ATRA, 9cis/13cis-RA, 5,6epoxy-ATRA, and partially retinal and 4keto-retinal, eluted in Fraction 5. The explicability of retinoid-like activity by detected compounds was higher for the Ak extract, where we were able to explain 64% of the in vitro effect for the whole extract and 65% and 57% for Fraction 4 and 5, respectively. The explicability of the REQ in vitro by the detected compounds was lower for the Ma extract, with 18% explained for the whole extract and 17% for both Fractions 4 and 5 (Table 1). Microcystins were detected at mg per gram of dry weight levels

Embryos treated with ATRA as well as biomass extracts showed a range of typical malformations such as tail and craniofacial deformations or edemas. The frequency of spontaneous malformations in the controls was on average 3.4% ± 2.2% across all experiments. Complete dose-response curves for mortality and combined malformation frequency were determined (Figs. 2 and 3). The LC50 value was 5466 ng L1 (95% CI 5221e5722 ng L1; Table 2) and the EC50 for combined malformations was 1153 ng L1 (95% CI 1083e1228 ng L1; Table 2). The results of the in vivo experiments with the extracts and their fractions corresponded with the in vitro retinoid-like activity for both extracts. Malformations typical for retinoid signalling disruption were observed following exposure to both extracts and their Fractions 5. These were compared to the effects of the standard retinoid compound ATRA (Figs. 2e5). The effects showed high morphological similarity (Figs. 4 and 5) and the same doseresponse pattern in the severity of malformations, suggesting the presence of compounds affecting retinoid signalling in early vertebrate development. Among the observed malformations were uninflated gas bladder (ugb), tail tip (ttd), lower jaw (ljd), spine (sd), head (hd) and craniofacial deformations (cfd), heart edema (he) or complex malformations (Figs. 4 and 5). The severity of malformations increased with higher concentrations for all tested samples. At the lower concentrations (from 0.2 g dw L1 for whole extracts and 0.1 and 0.3 g dw L1 for Ma and Ak Fraction 5, respectively; from 0.8 mg L1 for ATRA, Figs. 2 and 3 and Table 3), uninflated gas bladder and tip tail deformations were most common (Table 3) as relatively mild deformations. More severe lower jaw and craniofacial deformations were detected along with yolk deformations and heart edemas (Figs. 4 and 5) in similar frequencies for the higher concentrations of the whole extracts and Fractions 5 (Table 3). The malformations caused by exposure to the Ma extract were detected at low concentrations of 0.2 g dw L1 corresponding to an REQ of 570 ng ATRA L1 (LOEC malformations, Table 2), and a dose-

Table 2 Individual lethal concentration (LC20), effective concentrations (EC20 and EC50) for malformations (with respective 95% confidence intervals) and LOEC concentrations for mortality and malformations for standard teratogen ATRA and extracts Ma and Ak and effective fractions determined in zFET (120 hpf). Fraction 4 of extract Ak did not reach EC20 up to the highest tested concentration (0.6 g dw L1).

LC20 LC50 EC20 malformation EC50 malformation LOEC mortality LOEC malformation

EC20 malformation EC50 malformation

ATRA [ng L1]

Ma [g dw L1]

Ma REQ [ng ATRA L1]

Ak [g dw L1]

Ak REQ [ng ATRA L1]

3837 (3553e4144) 5466 (5221e5722) 708 (628e798) 1153 (1083e1228) 2000 800

0.60 (0.59e0.62) 0.67 (0.58e0.77) 0.17 (0.16e0.19) 0.23 (0.22e0.24) 0.60 0.10

1715 (1671e1760) 1910 (1660e2204) 483 (441e528) 648 (616e681) 1710 285

0.34 (0.32e0.36) 0.40 (0.37e0.43) 0.11 (0.11 - 0e11) 0.13 (0.12e0.14) 0.60 0.20

981 (929e1037) 1148 (1075e1233) 311 (302e321) 372 (357e389) 1721 574

Ma F5 [g dw L1]

Ma F5 REQ [ng ATRA L1]

Ak F5 [g dw L1]

Ak F5 REQ [ng ATRA L1]

0.07 (0.05e0.10) 0.15 (0.13e0.18)

122 (89e169) 246 (209e290)

0.25 (0.25e0.26) 0.30 (0.30e0.30)

406 (395e417) 478 (474e482)

8

M. Pipal et al. / Chemosphere 241 (2020) 125061

Fig. 4. Comparison of control embryo (A) with embryos from individual treatments with ATRA and extract Ma and its fractions showing typical phenotype at 120 hpf: individual typical malformations (BeI) and complex deformation at higher concentrations (J, K and L). Standard compound all-trans Retinoic acid (ATRA) at concentrations 1 200 ng L1 (D) and 2 000 ng L1 (G and J). Biomass extract Ma whole at concentrations 0.1 (B, REQ 285 ng ATRA L1), 0.2 (E, REQ 570 ng ATRA L1) and 0.6 g dw L1 (H and K, REQ 1710 ng ATRA L1). Biomass extract Ma fraction 5 at concentrations 0.1 (C, REQ 164 ng ATRA L1), 0.2 (F, REQ 328 ng ATRA L1) and 0.6 g dw L1 (I and L, REQ 984 ng ATRA L1). Used magnification: photos of whole embryos 30, head detail photos 90 (A, D, G and B, E, H) and 45 (C, F, I). ugb- uninflated gas bladder; ttd- tail tip deformation; he- heart edema; ljd- lower jaw deformation; sd- spine deformation, cfd- craniofacial deformation.

dependent increase in malformation severity was observed (Fig. 4 B, E, H and K). No effects on embryo development were detected after the exposure to Ma Fraction 4 up to the highest tested concentration of 0.6 g dw L1 (REQ 418 ng ATRA L1). Therefore, it was pooled together after the initial test with the other non-active fractions (F 1e3 and 6e12) at a concentration of 0.6 g dw L1. No effects were detected even in this mixture with other fractions at that concentration (Table 3). On the other hand, similar to the whole extract, a dose-dependent increase in severity of malformations was noted after the exposure to Ma Fraction 5. This is best illustrated in Fig. 4 along with the photos of the whole extract effects (Fig. 4C, F, I and L). At the lowest concentration (Ma Fraction 5, 0.1 g dw L1), only a small bend at the end of tails was visible, while tail deformation was more pronounced and deformation of the lower jaw became apparent at the concentration of 0.2 g dw L1 (Fig. 4 F). A large range of deformation severity was observed at the

concentration of 0.6 g dw L1, where for a smaller percent of embryos the listed malformations occurred in a less pronounced form (usually multiple malformations together), but most of the embryos suffered from complex deformations with severe edemas (Fig. 4 I and L; Table 3). The similarity of the phenotype detected in individual treatments (ATRA, Ma and its fractions) at comparable concentrations is also shown in Fig. 4. Comparable results were obtained after exposure to the Ak extract, where similar phenotype and malformations were also observed with a high resemblance to the effects caused by ATRA (Fig. 4 D, G and J) and with increasing severity at greater concentrations (Fig. 5). The observed teratogenicity corresponded to retinoid-like activity detected in vitro and was detected at the low concentration of 0.2 g dw L1 for the whole extract (LOEC malformations, REQ 574 ng ATRA L1) and at 0.3 g dw L1 (REQ 480 ng ATRA L1) for the Fraction 5. The Fraction 4 of the Ak extract caused

M. Pipal et al. / Chemosphere 241 (2020) 125061

9

Fig. 5. Comparison of control embryo (A) with embryos from individual treatments with extract Ak and its fractions showing typical phenotype at 120 hpf. Individual representative malformations for biomass extract Ak whole at concentrations 0.2 (C, REQ 574 ng ATRA L¡1), 0.3 (E, REQ 861ngATRA L¡1) and 0.6 g dw L¡1 (G, REQ 1721 ng ATRA L¡1) and Ak fraction 5 at concentrations 0.2 (B, REQ 320ngATRA L¡1), 0.3 (D, REQ 480 ng ATRA L¡1) and 0.6 g dw L¡1 (F and H, REQ 961 ng ATRA L¡1). Used magnification: photos of whole embryos 30£, head detail photos 90£. ugb- uninflated gas bladder; ttd- tail tip deformation; he- heart edema; ljd- lower jaw deformation, cfd- craniofacial deformation.

only mild malformations at the highest tested concentration of 0.6 g dw L1 (REQ 616 ng ATRA L1), such as uninflated gas bladder and tip tail deformations in relatively low frequency (Table 3). Occasional head and jaw deformations were also detected in the Ak Fraction 4 treatment, although less frequently and less pronounced than in the Ak Fraction 5 and whole extract at concentrations with comparable REQ values (Fig. 5 and Table 3). The results of the whole extract show a steep dose-response increase in malformation frequency (from 0 to almost 100% between concentrations of 0.1 and 0.2 g dw L1, Fig. 2). Therefore, the effects are better illustrated with

the increasing severity of malformations ranging from slightly malformed embryos at 0.2 g dw L1 to complex malformations at 0.3 g dw L1 (Fig. 5; Table 3), where it was apparent that, while there were 100% malformed embryos at both concentrations, the effects were more severe at the higher concentration. Calculated LC and EC values for the respective treatments are displayed in Table 2, both as concentration of biomass extract as well as the corresponding REQ value determined from detected bioactivity in vitro. The results show that both extracts of field cyanobacterial biomasses were more effective in zFET than

10 Table 3 Overview of frequencies of individual malformations in experiments including fractionated biomass extracts and ATRA. Results are shown as affected percent of surviving embryos with standard deviation (N ¼ 9, conducted as three independent experiments in triplicate with 20 embryos for each replicate). Statistically significant values are highlighted (Fisher exact test, p < 0.05). REQ in vitro levels in each exposure variant were derived from the REQs for individual samples in Table 1. Conc.

[ng L1]

Control ATRA

0 400 800 1200 1600 2000 4000 6000 10000

Edema

Uninflated Gas Bladder

Deformations

Dead

Craniofacial

Spine

Tip tail

Yolk

Jaw

Complex

97 ± 1.0 95 ± 1.8 75 ± 3.3 46 ± 5.6 31 ± 0.6 21 ± 3.9 0.6 ± 1.0 e e

0 1.7 ± 1.7 1.1 ± 1.0 8.6 ± 4.6 17 ± 12 21 ± 15 0 e e

0 3.5 ± 0.1 10 ± 4.4 44 ± 17 52 ± 13 58 ± 5.8 1.3 ± 2.3 e e

1.1 ± 1.9 0.6 ± 1.0 1.1 ± 2.0 16 ± 19 20 ± 14 31 ± 16 1.3 ± 2.3 e e

0 0 5.6 ± 6.7 5.2 ± 3.0 10 ± 6.6 14 ± 18 1.3 ± 2.3 e e

0.6 ± 1.0 1.1 ± 2.0 10 ± 10 22 ± 13 36 ± 24 31 ± 24 1.3 ± 2.3 e e

0 2.3 ± 1.0 9.6 ± 3.4 24 ± 10 32 ± 16 23 ± 27 0±0 e e

0 1.1 ± 2.0 5.7 ± 1.0 16 ± 9.5 30 ± 22 40 ± 13 1.3 ± 2.3 e e

1.1 ± 1.0 2.3 ± 4.0 3.4 ± 4.5 9.1 ± 12 13 ± 14 27 ± 20 98 ± 3.5 100 e

1.7 ± 1.6 5.0 ± 0.0 2.7 ± 0.9 2.3 ± 2.6 6.0 ± 2.4 10 ± 1.4 18 ± 4.5 56 ±7.5 100

164 328 984 1710

98 ± 1.7 67 ± 3.1 40 ± 7.2 0 0 95 ± 6.8

0.6 ± 1.0 2.3 ± 1.0 13 ± 23 26 ± 12 4 ± 1.2 0

1.7 ± 2.9 6.5 ± 5.5 32 ± 38 40 ± 14 4.5 ± 1.2 1.8 ± 3.0

0 0 0 6.1 ± 4.6 3.0 ± 2.8 0

0.6 ± 1.0 13 ± 10 21 ± 14 21 ± 7.6 2.6 ± 2.8 0

0 20 ± 25 35 ± 18 42 ± 19 4.5 ± 1.2 0.6 ± 1.0

1.7 ± 2.9 14 ± 6.9 41 ± 22 39 ± 17 4.5 ± 1.2 0

1.1 ± 2.0 2.3 ± 2.6 5.9 ± 2.1 29 ± 11 4.5 ± 1.2 1.2 ± 2.0

0 0 0.6 ± 1.0 53 ± 20 96 ± 1.2 0

2.8 ± 1.9 5.0 ± 3.3 5.0 ± 5.0 8.9 ± 2.6 19 ± 12 4.2 ± 2.9

616 320 480 961 861 1721

100 ± 0.7 90 ± 4.2 97 ± 3.3 49 ± 3.0 0 0 e 97 ± 2.9

0 1.7 ± 1.8 0 3.4 ± 3.4 17 ± 2.6 20 ± 5.3 e 0

0 8.7 ± 7.0 1.1 ± 2.0 17 ± 16 36 ± 30 66 ± 26 e 3.4 ± 0.0

0.3 ± 0.6 0.6 ± 1.0 0 2.8 ± 4.8 21 ± 24 33 ± 18 e 0

0 0.6 ± 1.0 0 1.7 ± 1.7 5.4 ± 3.3 14 ± 9.5 e 1.1 ± 1.0

0 3.4 ± 3.0 2.3 ± 2.0 63 ± 14 34 ± 30 66 ± 26 e 0.6 ± 1.0

0.3 ± 0.6 2.3 ± 1.0 0.6 ± 1.0 6.8 ± 4.6 25 ± 20 47 ± 14 e 1.7 ± 1.7

0 1.1 ± 1.0 0 5.7 ± 1.0 32 ± 26 39 ± 20 e 0

0 1.2 ± 2.0 0 2.8 ± 2.0 63 ± 32 33 ± 26 100 0.6 ± 1.0

1.3 ± 1.3 3.3 ± 1.7 1.7 ± 1.7 1.7 ± 1.7 10 ± 4.4 7.2 ± 5.8 97 ± 7.8 2.2 ± 1.0

REQ in vitro [ng ATRA L1] Ma

[g dw L1]

Control Fraction 5

Whole non-RA

0 0.1 0.2 0.6 0.6 0.6

Ak

[g dw L1]

Control Fraction 4 Fraction 5

0.0 0.6 0.2 0.3 0.6 0.3 0.6 0.6

Whole non-RA

M. Pipal et al. / Chemosphere 241 (2020) 125061

ATRA

Normal

M. Pipal et al. / Chemosphere 241 (2020) 125061

expected solely from their ATRA concentrations equivalents (REQ) determined in vitro. Thus, in vivo teratogenicity of cyanobacterial extracts occurred at lower REQ levels compared to the in vivo effective concentrations of ATRA itself. The whole Ma and Ak extracts had 1.7-fold and 3-fold lower EC50 values for malformations, respectively, compared to the EC50 of pure ATRA (Table 2). The difference in observed developmental toxicity was similar when comparing the EC50 values for the Fractions 5 of the extracts and ATRA. 4. Discussion It is well described that RA plays a crucial role in vertebrate organogenesis and body patterning and that its synthesis and degradation is highly regulated to maintain specific concentrations in specific developing tissues (Hernandez et al., 2007; Shimozono et al., 2013). It is synthesized from vitamin A or retinol by the alcohol dehydrogenase (ADH), retinol dehydrogenase (RDH) and retinaldehyde dehydrogenase (RALDH) enzyme families (Ang et al., 1996; Duester, 2008) and constant levels are maintained by a complex of degrading enzymes such as CYP26 which plays a major role (Emoto et al., 2005; Oliveira et al., 2013). This complex regulation system has been shown to be vulnerable to disruption with extensive consequences (Duester, 2008; Oliveira et al., 2013). Abnormal RA concentration, either lower or higher, leads to severe developmental disorders, making it a potent teratogen and key regulator of developmental processes in vertebrates such as limb formation, body axis patterning, and skeletal, craniofacial, brain and heart development (Duester, 2008; Emoto et al., 2005; Spoorendonk et al., 2008; Stefanovic and Zaffran, 2017). We investigated samples prepared from two environmental cyanobacterial biomasses from reservoirs with developed water blooms. Despite the different taxonomic composition of the two samples, both elicited high retinoid-like activity in vitro. This bioactivity is in agreement with previous findings of retinoid-like activity detected in extracts and exudates from various species cultivated in the laboratory (Jonas et al., 2015, 2014; Priebojov
a
et al., 2017; Sychrov
et al., 2018; Smutna a et al., 2016) or from water bloom samples collected in the field (Jav urek et al., 2015; Sehnal et al., 2019; Wu et al., 2013). Recent studies document that retinoid-like activity is relatively common across water bodies with cyanobacterial bloom of various taxonomic composition (Sehnal et al., 2019). Our study provides a unique link between in vitro retinoid-like activity, teratogenic effects and retinoid concentrations in field samples. The detected retinoid-like activity corresponded with teratogenic effects observed in zFET for both extracts and their fractions. All bioactivity observed in vitro was caused by Fractions 4 and 5, where most of the detected retinoids were eluted. The bioactivity observed in vivo was also detected in Fraction 5, while Fraction 4 caused only minor effects for the Ak extract and no effects for the Ma extract at the highest tested concentration. In addition, a comparison of the teratogenic effects showed high morphological similarity to the effects caused by the standard retinoid ATRA. Currently available reports agree on the ability of cyanobacteria to produce retinoid-like compounds (Jaja-Chimedza et al., 2017;
et al., 2018; Sychrova
et al., 2016; Wu Jonas et al., 2014; Priebojova et al., 2013). While there are several reports on retinoid-like activity and retinoids produced by cyanobacterial blooms dominated by M. aeruginosa, this study is the first to report on the potential production of these compounds and the retinoid-like activity in a water bloom dominated by A. klebahnii. The detected level of retinoid-like activity is similar to previous findings for M. aeruginosa in a field sample from the same location (Jav urek et al., 2015) as well as a laboratory culture (Jonas et al., 2014).

11

Previous reports documented retinoid-like activity and/or production of retinoids in the biomass of laboratory cultured Aphanizomenon gracile and Aphanizomenon flos-aquae (Jonas et al., 2014;
et al., 2018; Wu et al., 2012). Retinoid-like activity was Priebojova also detected in our Ak field-collected extract dominated by A. klebahnii and its Fraction 4 and 5 where the REQ in vitro sum reached up to 92% of the whole extract activity on average. Similar results were obtained for the Ma extract obtained from biomass dominated by M. aeruginosa, where the sum of the retinoid-like activity of Fractions 4 and 5 reached on average up to 82% of the whole extract REQ in vitro. Thus, the data show relatively consistent results regarding the bioactivity and chemical composition before and after fractionation. The small difference of 18% in mean retinoid-like activity for the Ma extract and the sum of its fractions is generally within the biological variability of the used bioassay, but could be to some extent affected by minor losses during the fractionation or actually more than additive interactions among the individual fractions. The detected compounds document the ability of cyanobacteria to produce teratogenic retinoids like ATRA, 9cis-RA or 5,6epoxyATRA. These potent retinoids were eluted in the bioactive fractions, which confirms their contribution to the observed effects. The REQ in vitro could be explained to a relatively high degree, especially for the Ak sample and its fractions where the explicability by the detected compounds was 57e64%, while the compounds detected in the Ma extract and its fractions explained 17e18% of the effect detected in vitro. These findings suggest the presence of other unknown compounds, possibly other retinoids, contributing to bioactivity. Another possible explanation is that there may be a more-than-additive mixture effect of some compounds causing higher teratogenicity than expected based on the bioactivity of individual compounds (Table 1). The findings that extracts from both biomasses caused comparable retinoid-like activity and teratogenicity while only one of them contained a detectable amount of microcystins, and the fact that these microcystins in the Ma extract eluted in fractions with no adverse effects on development, support the negligible role of microcystins in the observed teratogenic effects. The teratogenic effects of the ATRA standard showed a typical phenotype similar to previous studies at comparable concentrations (EC50 100 - 1,100 ng L1 and LC50 5,190e85 000 ng L1 in Herrmann, 1995; Jonas et al., 2015; Selderslaghs et al., 2012, 2009; Wang et al., 2014) despite differences in the exposure method, such as light conditions, static or semi-static renewal of test solutions and age of embryos at test initiation. Among the other detected retinoids, only 9cis-RA and retinal were previously shown to act as teratogens in aquatic vertebrates, while no information is available on the teratogenicity of the other compounds in aquatic organisms. For example, 9cis-RA in Xenopus laevis had effective concentrations in the range of mg L1 (Kraft et al., 1994; Minucci et al., 1996; Zhu et al., 2014) and retinal in zebrafish had an EC50 for malformations of 512 ± 199 mg L1 (1.8 ± 0.7 mM) (Jaja-Chimedza et al., 2017). In previous studies, retinoids were detected in the environment but their effects on aquatic organisms were not investigated (Wu et al., 2013). In this study we were able to provide a link between the detection of certain compounds and their effects in vitro as well as in vivo, which provides further proof of cyanobacteria as important contributors of teratogenic retinoids to the aquatic environment (Jaja-Chimedza et al., 2017; Jonas et al., 2015). In accordance with previous reports (Jonas et al., 2015, 2014) we detected specific malformations caused by the cyanobacterial extracts and their fractions. These effects were typical for retinoid signalling disruption, including the aforementioned craniofacial and skeletal deformations (Figs. 4 and 5). Most notable was the resemblance and similar frequency of tip tail and jaw deformations between the

12

M. Pipal et al. / Chemosphere 241 (2020) 125061

ATRA exposure and those of both extracts and their Fractions 5 (Figs. 4 and 5, Table 3). The individual malformations prominently associated with retinoid teratogenicity and the severity of their effects follow a similar dose-response pattern with a very steep dose-response increase across individual treatments (Figs. 2 and 3). These effects were observed already at 24 hpf (4 mg L1 ATRA, 0.6 g dw L1) suggesting that chorion does not significantly limit the bioavailability of retinoids since they are generally small molecules (ca 300 Da) and can therefore easily pass through chorion pores (ca 0.5 mm). In general, the comparison of the zFET and in vitro results showed very good agreement between the bioactivity and potency of the extracts and the distribution of the effects among the fractions. The in vitro assay clearly revealed the fractions with the greatest teratogenic effects, as well as those with the highest levels of potent retinoids. The comparison of results obtained for the ATRA standard and the field-collected extracts nevertheless suggest that both whole extracts and their respective Fractions 5 were more potent than would be expected solely from the REQ in vitro or chemical data (REQ chem). The Ma and Ak extracts had EC50 values for malformations at REQs of 648 ng ATRA L1 and 372 ng ATRA L1, respectively, while ATRA had an EC50 of 1153 ng L1. Similar results were observed for the LC50 values of the whole extracts compared to the LC50 of ATRA (Table 2). These results show that field-collected biomass extracts caused malformations at lower concentrations than would be expected from the REQs detected in vitro. Similarly, the Fractions 5 of the Ma and Ak extracts had EC50 values at REQs of 246 and 478 ng ATRA L1, respectively (Table 2), which indicate that the Fraction 5 of the Ma extract was more potent than the whole extract, despite a relatively similar retinoid content (Table 1). This may be caused by the influence of some other components of the complex mixture that could modify behaviour, metabolism or the effects of the active compounds in the whole extract compared to the Fraction 5 alone. There could also be antagonistic or additive/ synergistic effects that might affect the resulting toxicity in both the whole mixture and its fractions. This difference could also be affected by possible lower relative potency of some of the retinoids on mouse RARs compared to zebrafish, or even binding to other receptors like RXR. Therefore, despite high conservation of RAR receptors across species (Oliveira et al., 2013; Waxman and Yelon, 2011), these retinoids might elicit higher teratogenicity in vivo, while causing lower induction in vitro. The potency of the extracts might be also affected by differing efficiency of metabolisation of the retinoid precursors in zebrafish embryos compared to the in vitro system, which could increase the internal concentration of bioactive forms, mainly ATRA. Nevertheless, the employed in vitro model also possesses metabolic capacity and has a functional pathway that can convert less potent retinoids to transcriptionally active metabolites (Chen and Reeses, 2011). Production of RA precursors like carotenoids has been reported for another cyanobacteria species Cylindrospermopsis raciborskii (Jaja-Chimedza et al., 2017; Wu et al., 2013). Carotenoids were shown to contribute to teratogenicity and might influence the differences in the potency of extracts in vitro compared to in vivo since metabolic capacity and metabolisation of carotenoids to bioactive retinoids can differ between the in vitro assay and zFET models (Jaja-Chimedza et al., 2017). However, the described carotenoids were effective only at a much higher mM concentration range (hundreds of mg to mg L1; Jaja-Chimedza et al., 2017), while the effective concentrations of the retinoids detected in this study are in the nM range (ng to mg L1). There might also be further compounds with higher potency that were not analysed, especially other RA derivatives such as 13cis or 9cis 4-keto-RA previously detected in the environment (Wu et al., 2012, 2010), and possibly others. Detection of a broader spectrum of compounds (and availability of their analytical standards) would

enable more precise mixture characterization and improve the interpretation of the observed effects and allow for better prediction of the suspected mixture effects. There might also be other compounds contributing to the teratogenicity that are eluted in the same fraction as the majority of the detected potent retinoids, but which do not trigger RAR directly. Ultimately there is little information about the teratogenicity of most of the detected retinoids, which makes full characterization of the relation between the in vivo and in vitro results difficult and thus further investigation is necessary to clarify the observed differences. The effective concentrations observed in our study are of considerable environmental relevance since the EC20 REQ values (Table 2) of the whole extracts for malformations correspond or are close to previously reported retinoid-like activity detected in surface waters. Recent report on retinoid-like activity associated with cyanobacterial blooms in the environment detected the greatest REQ in water 263 ng L1 at the same location, where sample Ak was collected at a different time (Sehnal et al., 2019). Previously reported retinoid-like activity in water surrounding the cyanobacterial bloom from which sample Ma was collected reached REQ 19 ng L1 (Jav urek et al., 2015). Moreover, the REQ in water surrounding the cyanobacterial bloom where sample Ak was collected reached up to 1202 ng ATRA L1 (Priebojova et al., in preparation), which is well above the REQ levels effective in vivo. Similarly, the retinoids detected in this and other studies were observed also in waters surrounding water blooms (Jav urek et al., 2015; Sehnal et al., 2019; Wu et al., 2012). Despite the variable contents of retinoids and levels of retinoid-like activity in waters with water bloom development the so far available reports document environmental relevance of our results and common occurrence of compounds with retinoid-like activity. The relevance is further supported by the reports of retinoid-like activity in exudates of M. aeruginosa laboratory cultures where REQs reached 474e1081 ng ATRA L1 (Jav urek et al., 2015) and 383e1078 ng ATRA L 1 or in exudates of other cyanobacteria which reached up to 6243 ng ATRA L1
et al., 2018). Those results document the ability of (Priebojova cyanobacteria to release compounds with retinoid-like activity into their surroundings in concentrations that could cause severe teratogenicity in vertebrates. Nevertheless, the actual concentrations in water bodies probably fluctuate significantly in time and are affected by the composition and state of the cyanobacterial bloom as well as by environmental conditions. Furthermore, the teratogenicity EC50 REQs for both extracts are comparable to REQs detected in cyanobacterial biomasses in both the environment (Jav urek et al., 2015; Sehnal et al., 2019) and laboratory cultures
et al., 2018). Based on the dry weight yield of the (Priebojova sampled biomass and the cell densities of the biomass prior to lyophilization (Table S1 in Supplementary Information), we can estimate the corresponding cell densities that would result in extract concentrations after releasing the entire cell content (Equation S(1)). In other words, we can determine how many cells were extracted for the calculated effective concentrations. The EC50 concentrations of the whole extracts used in this study correspond to compound content from cyanobacterial bloom biomass at cell densities of 9.55  106 cells mL1 for Ma and 1.08  106 cells mL1 for Ak. These are 10-fold lower relative cell densities than those observed in the environment during sample collection at both locations (Table S1; Ma, 1.23  107 cells mL1; Ak, 1.08  107 cells mL1) and also lower than routinely observed cell densities in the
zkova
et al., 2017). environment (Jav urek et al., 2015; Procha Therefore, it is expected that the occurrence of compounds with retinoid-like activity detected within the biomasses of this study may be very common in surface waters (Sehnal et al., 2019) and may also potentially adversely affect aquatic vertebrates populations by increasing the incidence of developmental

M. Pipal et al. / Chemosphere 241 (2020) 125061

abnormalities (Stuart et al., 2004; Wu et al., 2013). Blooms often grow in warm shallow reservoirs or are brought by wind to reservoir coasts which many organisms use as habitats for reproduction and offspring development. The effects of blooms can be especially relevant at the end of the cyanobacterial vegetation period when the bloom is dying off and higher amounts of cell contents are released. It is especially concerning that the cyanobacterial species dominating blooms investigated in this study are widespread and very often form dense water blooms (Huisman et al., 2018). Our findings strongly support the hypothesis that compounds with retinoid-like activity produced by cyanobacterial blooms are the main driver of teratogenicity in exposed organisms and might play a role in some localized problems in aquatic vertebrate populations (Wu et al., 2012). 5. Conclusions This study provides new evidence for the production of retinoids by naturally occurring cyanobacterial water blooms, their retinoid-like activity and teratogenic effects. We were able to characterize the teratogenicity of compounds from two natural water bloom biomasses dominated by different cyanobacterial species. Fractionation of the biomass extracts allowed us to link observed teratogenicity to fractions with detected retinoid-like activity and potent retinoid compounds. The detected retinoid compounds play a major role in the observed effects, although there are still unknown factors affecting the observed toxicity, especially in vivo. Moreover, our results suggest that detected microcystins play little to no role in the observed teratogenic effects. Previous studies along with our results show that aquatic organisms are exposed to mixtures of retinoid compounds in the environment. Fractionation has been proven as an effective way to study the potential association of specific compounds produced by cyanobacteria with observed effects, and allowed us to strengthen the link between the compounds and their bioactivity. Additionally, the presented concentrations of individual retinoids, in combination with their contribution to the total in vitro effect, indicate the relevance of single detected retinoids towards the detected bioactivity. The high conservation of retinoid signalling pathways among vertebrates also indicates potential risks to other vertebrates. Teratogenic effects detected after exposure to extracts of the fieldcollected biomasses at comparable REQ concentrations as detected in cyanobacterial water blooms emphasize the need to consider retinoid compounds and their effects on aquatic organisms. Further studies should identify additional compounds within the effective fraction contributing to in vitro retinoid-like activity as well as teratogenicity. It is especially important to focus on the in vivo effects of the compounds detected in Fraction 5, which showed the greatest teratogenic effects as well as the greatest in vitro retinoidlike activity. It is also crucial to consider that the adverse effects of these compounds in aquatic environments may be further amplified in combination with other stressors including decreased water quality and oxygen levels or changes in pH, which are also caused by cyanobacterial blooms. Their impacts in water bodies might be further exacerbated by increased occurrence of cyanobacterial blooms and wider distribution of invasive species expected as a consequence of global change. Acknowledgement This study was supported by Czech Science Foundation, Czechia, Project No. 18-15199S and RECETOX Research Infrastructure (Projects LM2015051 and CZ.02.1.01/0.0/0.0/16_013/0001761 from Ministry of Education, Youth and Sports of the Czech Republic).

13

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.125061. References €ro }, N., Kiss, G., Gyo } ri, J., Vehovszky, A., Acs, A., Kov
acs, A.W., Csepregi, J.Z., To
ts, N., Farkas, A., 2013. The ecotoxicological evaluation of CylinKova drospermopsis raciborskii from Lake Balaton (Hungary) employing a battery of bioassays and chemical screening. Toxicon 70C, 98e106. https://doi.org/ 10.1016/j.toxicon.2013.04.019. 
Ang, H.L., Deltour, L., Hayamizu, T.F., Zgombi c-Knight, M., Duester, G., 1996. Retinoic acid synthesis in mouse embryos during gastrulation and craniofacial development linked to class IV alcohol dehydrogenase gene expression. J. Biol. Chem. 271, 9526e9534. https://doi.org/10.1074/jbc.271.16.9526. Berry, J.P., Gibbs, P.D.L., Schmale, M.C., Saker, M.L., 2009. Toxicity of cylindrospermopsin, and other apparent metabolites from Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum, to the zebrafish (Danio rerio) embryo. Toxicon 53, 289e299. https://doi.org/10.1016/j.toxicon.2008.11.016.
ha, L., Babica, P., Marsa
lek, B., 2009. Toxins produced in cyanobacterial water Bla blooms - toxicity and risks. Interdiscip. Toxicol. 2, 36e41. https://doi.org/ 10.2478/v10102-009-0006-2.
, K., Babica, P., Vrskova
, D., Marsa
lek, B., Bla
ha, L., 2006. Burýskov
a, B., Hilscherova Toxicity of complex cyanobacterial samples and their fractions in Xenopus laevis embryos and the role of microcystins. Aquat. Toxicol. 80, 346e354. https://doi.org/10.1016/j.aquatox.2006.10.001. Chen, Y., Reeses, D.H., 2011. The retinol signaling pathway in mouse pluripotent P19 cells. J. Cell. Biochem. 112, 2865e2872. https://doi.org/10.1002/jcb.23200. Duester, G., 2008. Retinoic acid synthesis and signaling during early organogenesis. Cell 134, 921e931. https://doi.org/10.1016/j.cell.2008.09.002. Emoto, Y., Wada, H., Okamoto, H., Kudo, A., Imai, Y., 2005. Retinoic acidmetabolizing enzyme Cyp26a1 is essential for determining territories of hindbrain and spinal cord in zebrafish. Dev. Biol. 278, 415e427. https://doi.org/ 10.1016/j.ydbio.2004.11.023. Gardiner, D., Ndayibagira, a., Grün, F., Blumberg, B., 2003. Deformed frogs and environmental retinoids. Pure Appl. Chem. 75, 2263e2273. https://doi.org/ 10.1351/pac200375112263. Gardiner, D.M., Hoppe, D.M., 1999. Environmentally induced limb malformations in mink frogs (Rana septentrionalis). J. Exp. Zool. 284, 207e216. https://doi.org/ 10.1002/(SICI)1097-010X(19990701)284:2<207::AID-JEZ10>3.0.CO;2-B. Ghazali, I. El, Saqrane, S., Carvalho, A.P., Ouahid, Y., Oudra, B., Del Campo, F.F., Vasconcelos, V., 2009. Compensatory growth induced in zebrafish larvae after pre-exposure to a Microcystis aeruginosa natural bloom extract containing microcystins. Int. J. Mol. Sci. 10, 133e146. https://doi.org/10.3390/ijms10010133. Halliday, T., 1998. A declining amphibian conundrum. Nature 394, 418e419. Heatwole, H., Wilkinson, J.W., 2013. Amphibian Biology, Volume 11, Part 3: Status of Conservation and Decline of Amphibians: Eastern Hemisphere: Western Europe, Amphibian Biology. Pelagic Publishing. Hernandez, R.E., Putzke, A.P., Myers, J.P., Margaretha, L., Moens, C.B., 2007. Cyp 26 enzymes generate the retinoic acid response pattern necessary for hindbrain development. Development 134, 177e187. https://doi.org/10.1242/dev.02706. Herrmann, K., 1995. Teratogenic effects of retinoic acid and related substances on the early development of the zebrafish (Brachydanio rerio) as assessed by a novel scoring system. Toxicol. In Vitro 9, 267e283. Huisman, J., Codd, G.A., Paerl, H.W., Ibelings, B.W., Verspagen, J.M.H., Visser, P.M., 2018. Cyanobacterial blooms. Nat. Rev. Microbiol. 16, 471e483. https://doi.org/ 10.1038/s41579-018-0040-1. ISO 7346, 1996. ISO 7346-1:1996 - Water Quality – Determination of the Acute Lethal Toxicity of Substances to a Freshwater Fish [Brachydanio Rerio HamiltonBuchanan (Teleostei, Cyprinidae)] – Part 1: Static Method. Jaja-Chimedza, A., Saez, C., Sanchez, K., Gantar, M., Berry, J.P., 2015. Identification of teratogenic polymethoxy-1-alkenes from Cylindrospermopsis raciborskii, and taxonomically diverse freshwater cyanobacteria and green algae. Harmful Algae 49, 156e161. https://doi.org/10.1016/j.hal.2015.09.010. Jaja-Chimedza, A., Sanchez, K., Gantar, M., Gibbs, P., Schmale, M., Berry, J.P., 2017. Carotenoid glycosides from cyanobacteria are teratogenic in the zebrafish (Danio rerio) embryo model. Chemosphere 174, 478e489. https://doi.org/ 10.1016/j.chemosphere.2017.01.145. Jav urek, J., Sychrov
a, E., Smutn
a, M., Bittner, M., Kohoutek, J., Adamovský, O.,
kov

, K., 2015. Retinoid compounds assoNova a, K., Smetanov
a, S., Hilscherova ciated with water blooms dominated by Microcystis species. Harmful Algae 47, 116e125. https://doi.org/10.1016/j.hal.2015.06.006. Jonas, A., Buranova, V., Scholz, S., Fetter, E., Novakova, K., Kohoutek, J., Hilscherova, K., 2014. Retinoid-like activity and teratogenic effects of cyanobacterial exudates. Aquat. Toxicol. 155, 283e290. https://doi.org/10.1016/ j.aquatox.2014.06.022. Jonas, A., Scholz, S., Fetter, E., Sychrova, E., Novakova, K., Ortmann, J., Benisek, M., Adamovsky, O., Giesy, J.P., Hilscherova, K., 2015. Endocrine, teratogenic and neurotoxic effects of cyanobacteria detected by cellular in vitro and zebrafish embryos assays. Chemosphere 120, 321e327. https://doi.org/10.1016/ j.chemosphere.2014.07.074. Kaya, K., Shiraishi, F., Uchida, H., Sano, T., 2011. A novel retinoic acid analogue, 7-

14

M. Pipal et al. / Chemosphere 241 (2020) 125061

hydroxy retinoic acid, isolated from cyanobacteria. Biochim. Biophys. Acta Gen. Subj. 1810, 414e419. https://doi.org/10.1016/j.bbagen.2010.11.009. Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., Schilling, T.F., 1995. Stages of embryonic development of the zebrafish. Am. J. Anat. 203, 253e310. Kraft, J.C., Schuht, T.I.M., Juchau, M., Kimelmant, D., 1994. The retinoid X receptor ligand, 9-cis-retinoic acid, is a potential regulator of early Xenopus development. Proc. Natl. Acad. Sci. U. S. A. 91, 3067e3071. Lawrence, C., 2007. The husbandry of zebrafish (Danio rerio): a review. Aquaculture 269, 1e20. Minucci, S., Saintjeannet, J.-P., Toyama, R., Scita, G., Deluca, L., Taira, M., Levin, A.A., Ozato, K., Dawid, I.B., 1996. Retinoid X receptor-selective ligands produce malformations in Xenopus embryos. Proc. Natl. Acad. Sci. U.S.A. 93, 1803e1807. 
, T., Kiviranta, H., Verta, M., Nov
ak, J., Benísek, M., Pacherník, J., Janosek, J., Sidlov a
ha, L., Hilscherov
Giesy, J.P., Bla a, K., 2007. Interference of contaminated sediment extracts and environmental pollutants with retinoid signaling. Environ. Times 26, 1591e1599. https://doi.org/10.1897/06-513R.1. Oberemm, A., Becker, J., Codd, G.A., Steinberg, C., 1999. Effects of cyanobacterial toxins and aqueous crude extracts of cyanobacteria on the development of fish and amphibians. Environ. Toxicol. 14, 77e88. https://doi.org/10.1002/(SICI) 1522-7278(199902)14:1<77::AID-TOX11>3.0.CO;2-F. OECD, 2013. Test No. 236: Fish Embryo Acute Toxicity (FET) Test, OECD Guidelines for the Testing of Chemicals, Section 2, pp. 1e22. ~ a, B., 2013. Retinoic acid Oliveira, E., Casado, M., Raldúa, D., Soares, A., Barata, C., Pin receptors’ expression and function during zebrafish early development. J. Steroid Biochem. Mol. Biol. 138, 143e151. https://doi.org/10.1016/ j.jsbmb.2013.03.011.
, J., Hilscherov

, T., Sychrova
, E., Smutna
, M., 2018. Priebojova a, K., Proch
azkova Intracellular and extracellular retinoid-like activity of widespread cyanobacterial species. Ecotoxicol. Environ. Saf. 150, 312e319. https://doi.org/10.1016/ j.ecoenv.2017.12.048.
zkova
, T., Sychrov

, B., Ve
, J., Kohoutek, J., Lepsova
Procha a, E., Jav urkova cerkova
celova
, O., Bla
ha, L., Hilscherova
, K., 2017. Phytoestrogens and sterols in Ska waters with cyanobacterial blooms - analytical methods and estrogenic potencies. Chemosphere 170, 104e112. https://doi.org/10.1016/ j.chemosphere.2016.12.006.
zkova
, T., Smutna
, M., Kohoutek, J., Lepsov

, O., Sehnal, L., Procha a-Sk
acelova
, K., 2019. Widespread occurrence of retinoids in water bodies Hilscherova associated with cyanobacterial blooms dominated by diverse species. Water Res. 156, 136e147. https://doi.org/10.1016/j.watres.2019.03.009. Selderslaghs, I.W.T., Blust, R., Witters, H.E., 2012. Feasibility study of the zebrafish assay as an alternative method to screen for developmental toxicity and embryotoxicity using a training set of 27 compounds. Reprod. Toxicol. 33, 142e154. https://doi.org/10.1016/j.reprotox.2011.08.003. Selderslaghs, I.W.T., Van Rompay, A.R., De Coen, W., Witters, H.E., 2009. Development of a screening assay to identify teratogenic and embryotoxic chemicals using the zebrafish embryo. Reprod. Toxicol. 28, 308e320. https://doi.org/

10.1016/j.reprotox.2009.05.004. Shimozono, S., Iimura, T., Kitaguchi, T., Higashijima, S.-I., Miyawaki, A., 2013. Visualization of an endogenous retinoic acid gradient across embryonic development. Nature 496, 363e366. https://doi.org/10.1038/nature12037.
, M., Priebojov

, J., Hilscherov
Smutna a, J., Ve cerkova a, K., 2017. Retinoid-like compounds produced by phytoplankton affect embryonic development of Xenopus laevis. Ecotoxicol. Environ. Saf. 138, 32e38. https://doi.org/10.1016/ j.ecoenv.2016.12.018. Spoorendonk, K.M., Peterson-Maduro, J., Renn, J., Trowe, T., Kranenbarg, S., Winkler, C., Schulte-Merker, S., 2008. Retinoic acid and Cyp26b1 are critical regulators of osteogenesis in the axial skeleton. Development 135, 3765e3774. https://doi.org/10.1242/dev.024034. Stefanovic, S., Zaffran, S., 2017. Mechanisms of retinoic acid signaling during cardiogenesis. Mech. Dev. 143, 9e19. https://doi.org/10.1016/j.mod.2016.12.002. Stuart, S.N., Chanson, J.S., Cox, N.A., Young, B.E., Rodrigues, A.S.L., Fischman, D.L., Waller, R.W., 2004. Status and trends of Amphibian declines and extinctions worldwide. Science 306, 1783e1786. https://doi.org/10.1126/science.1103538, 80.
, M., Nova
kov
Sychrov
a, E., Priebojov
a, J., Smutna a, K., Kohoutek, J., Hilscherov
a, K., 2016. Characterization of total retinoid-like activity of compounds produced by three common phytoplankton species. Harmful Algae 60, 157e166. https:// doi.org/10.1016/j.hal.2016.11.002. Wang, Y., Chen, J., Du, C., Li, C., Huang, C., Dong, Q., 2014. Characterization of retinoic acid-induced neurobehavioral effects in developing zebrafish. Environ. Toxicol. Chem. 33, 431e437. https://doi.org/10.1002/etc.2453. Waxman, J.S., Yelon, D., 2011. Zebrafish retinoic acid receptors function as contextdependent transcriptional activators. Dev. Biol. https://doi.org/10.1016/ j.ydbio.2011.01.022. Wu, X., Hu, J., Jia, A., Peng, H., Wu, S., Dong, Z., 2010. Determination and occurrence of retinoic acids and their 4-oxo metabolites in Liaodong Bay, China, and its adjacent rivers. Environ. Toxicol. Chem. 29, 2491e2497. https://doi.org/10.1002/ etc.322. Wu, X., Jiang, J., Hu, J., 2013. Determination and occurrence of retinoids in a eutrophic lake (Taihu Lake, China): cyanobacteria blooms produce teratogenic retinal. Environ. Sci. Technol. 47, 807e814. https://doi.org/10.1021/es303582u. Wu, X., Jiang, J., Wan, Y., Giesy, J.P., Hu, J., 2012. Cyanobacteria blooms produce teratogenic retinoic acids. Proc. Natl. Acad. Sci. U. S. A. 109, 9477e9482. https:// doi.org/10.1073/pnas.1200062109. Zhu, J., Yu, L., Wu, L., Hu, L., Shi, H., 2014. Unexpected phenotypes of malformations induced in Xenopus tropicalis embryos by combined exposure to triphenyltin and 9-cis-retinoic acid. J. Environ. Sci. (China) 26, 643e649. https://doi.org/ 10.1016/S1001-0742(13)60474-X. Zi, J., Pan, X., MacIsaac, H.J., Yang, J., Xu, R., Chen, S., Chang, X., 2018. Cyanobacteria blooms induce embryonic heart failure in an endangered fish species. Aquat. Toxicol. 194, 78e85. https://doi.org/10.1016/j.aquatox.2017.11.007.