Toxicity assessment of the biogenic amines cadaverine and putrescine in aquatic organisms

Cemeteries are among the most polluting sources of groundwater and surface water in the world. Necro-leachate, a liquid released during the putrefaction of corpses, is the main culprit of such pollution. Among the compounds in this liquid are the potentially toxic amines cadaverine and putrescine, which are still poorly understood in terms of the environmental health risks they pose. This study evaluated the acute toxicity of cadaverine and putrescine, using e�cient contamination bioindicators of aquatic environments. Danio rerio and Daphnia magna were exposed to both amines individually or in a mixture. Acute toxicity (LC 50 ) was then observed in both organisms after exposure to the amines individually. Cadaverine showed higher lethality for D. magna (LC 50 – 9.5 mg. L -1 ) and D. rerio (LC 50 - 335.5 mg. L -1 ) than did putrescine (LC 50 - 36.7 mg. L -1 and LC 50 - 452.6 mg. L -1 , respectively). In embryotoxicity tests with D. rerio (hatching rate and malformations), delayed hatching, vertebral column malformations, and pericardial edema were observed after exposure to an amine mixture. However, co-exposure to cadaverine and putrescine was not toxic to D. magna. The results of this study con�rmed the e�ciency of the bioindicators used to assess contaminants in cemeteries and raised awareness of the toxic potential of cadaveric decomposition by-products.


Introduction
One of the socio-environmental problems that most concern the world today is water pollution.The waste generated by a range of human actions contributes to this problem, waste that is currently considered the primary source of this contamination (Afangideh and Udokpoh 2022).
Within the last decade, the burial of bodies in cemeteries has aroused the interest of scientists, due to its polluting potential (Gonçalves et al. 2022).According to Batista et al. (2022), better planning is necessary regarding burial practices due to the risk of groundwater pollution from this type of activity.The risks pertaining to this type of pollution must be evaluated, as the degree of understanding with regard to the polluting loads of cemeteries remains insu cient (Franco et Rodrigues and Pacheco 2003).These studies point to the need for better monitoring of cemetery areas and to assess, in detail, the toxic effects of by-products coming from cadaveric decomposition (Batista et al. 2022).
Contamination from cemeteries has also been identi ed as a cause for concern by the World Health Organization (WHO) and the National Council for the Environment (CONAMA-Brazil), due to its impacts on the biological environment, soil, groundwater, and human health (Viegas et al. 2022).
Most necropolises were built without prior studies assessing the impacts on the environment and the local community (Neckel et al. 2021).According to Gonçalves et al. (2022), the increasing number of burials, especially during the SarsCov-2 pandemic, led to a worsening of the pollution scenario and to an alert about the potential ecotoxicological risks derived from the presence of necro-leachate in cemeteries (Gonçalves et al. 2022).
Cadaveric decomposition is the most relevant activity relating to the contamination of cemetery environments (Idehen and Ezenwa 2019;Zychowski and Bryndal 2015).During the decomposition of bodies, in addition to gases, a liquid called necro-leachate is generated (Batista et al. 2022).Necroleachate is composed of water, mineral salts and organic substances, including the biogenic amines (BAs) cadaverine (CAD) and putrescine (PUT); these substances are considered toxic and responsible for the fetid odors that can be found in bodily uids (Crisanto-Perrazo et al. 2022).Gómez et al. (2022) mention that the adult cadaver releases about 0.4-0.6L/kg in body weight of necro-leachate.The authors also warn that the physical-chemical conditions of the soil can facilitate the percolation of this liquid and the subsequent contamination of the water table.The necro-leachate may also carry within it microbiological agents and chemical compounds used in life by the body (Afangideh and Udokpoh 2022); in addition to this, substances used in the embalming process and in the construction of co ns are also released, both potentially raising the level of contamination in cemeteries (Silva et al. 2020).
CAD and PUT are by-products naturally synthesized in living organisms by the decarboxylation of amino acids.CAD (C 5 H 14 N 2 ) is a by-product of the amino acid lysine, and PUT (C 4 H 12 N 2 ), of ornithine (Franco et al. 2022;Gonçalves et al. 2022).In endogenous concentrations, these amines participate in important physiological processes, such as the synthesis of proteins, hormones, and nucleic acids.CAD and PUT are also related to cell proliferation and the maintenance of blood pressure and body temperature (Abuhlega and Ali 2022).In addition, BAs in uence membrane stability, in addition to acting as neurotransmitters.
However, when found in high concentrations, they can be toxic to different organisms, and pose a potential risk to the environment and public health (Dasa et al. 2022;Wójcik et al. 2020).The toxic level of BAs is di cult to determine, due to their speci c characteristics, as well as their concomitant presence with other amines.However, a maximum allowable level of total BAs has been suggested at 750-900 mg/kg body weight (Abuhlega and Ali 2022).
The zebra sh (Danio rerio) has been used in several ecotoxicological studies.The species emerged as a vertebrate model in the investigation of changes in embryonic development and the genotoxic, mutagenic and carcinogenic potential of aquatic contaminants (Anifowoshe et al. 2022).The main advantages of this model include its short life cycle, rapid reproductive maturity, extrauterine development, high egg production and transparent embryos, which permits easy phenotypic observation (Lee et al. 2022).Furthermore, according to Howe et al. (2013), the species' genetic makeup is 70% orthologous with that of human beings.
The planktonic water ea, Daphnia magna, is also an important bioindicator of freshwater toxicity, mainly due to its sensitivity to pollutants, genetic stability, and representativeness in the food chain.In addition, it allows the assessment of the acute and chronic effects of different chemical substances (Belaid and Sbartai 2021).D. magna are able to lter large amounts of water and suspended particles, thus allowing a greater potential to be affected by the ingestion of xenobiotics when compared to other aquatic organisms (Lovern and Hart 2022).
Considering that the environmental impacts caused by necro-leachate are still poorly understood, the objective of this study was to evaluate the effects of the acute toxicity of BAs CAD and PUT, isolated, or associated with each other.The ecotoxicological study was carried out with two important bioindicators of environmental quality (D. rerio and D. magna); the LC 50 in aquatic bioindicators and embryotoxicity in the sh was also evaluated.The results of this study have expanded our understanding with regard to BAs originating from cadaveric putrefaction, data that may serve to assist both in the elaboration of better norms for monitoring the effects of cemetery contaminants, as well as in the decision making of environmental agencies, regarding the regulation of installations and licensing of new cemeteries.

Chemical substances evaluated
The BAs cadaverine (

Daphnia magna
Individuals of the species D. magna were cultivated in aquariums measuring 40 x 25 x 15 cm, containing water reconstituted with nutrients prepared according to USEPA (2002).The reconstituted water in the aquariums was changed twice a week.The organisms were kept in an acclimatized room under a controlled temperature (20 ± 2º C) and fed daily with algae of the species Raphidocelis subcapitata.

Danio rerio: LC 50 and embryos (FET test)
Preliminary tests were performed to determine the average lethal concentration for 50% of the exposed organisms (LC 50 -mg.L − 1 ).The concentrations used to obtain the LC 50 of CAD and PUT were 200, 400, 600, 800, and 1000 mg.L − 1 .The bioassays with a BAs mixture were performed using the concentrations in Table 1.
The FET test was performed with four different combinations of these CAD and PUT amines.In this assay, 3-hour post-fertilization (hpf) embryos were exposed to the mixtures for 96 hours, according to OECD protocol 236 (2013).A negative control was carried out with the embryo medium (35 mg of NaHCO 3 ; 60 mg of MgSO 4 ; 4 mg of KCl; 60 mg of CaSO 4 2H 2 O, in a nal volume of 1L of water), and a positive control was performed with dichloroaniline (3,5-Dichloroaniline ≥ 98%, Sigma Aldrich®, D55792).
Exposure was performed in 24-well polystyrene plates (n = 24 organisms per treatment).Embryos were individually maintained in 2 mL of test solution, under a 14/10h light/dark cycle at 26 ± 0.2 ºC in a B.O.D incubator.Embryos and larvae were evaluated and photographed every 24h using a stereomicroscope (Model SMZ 2 LED, software Optika View Version 7.1.1.5,Optika®), to determine the occurrence of lethality and malformations (spine, tail, pigmentation, edema, formation of somites, absorption of the yolk sac, and heartbeat).The hatching rate was calculated as the relation between the successful hatching of embryos and the total number of embryos in each replica.1.
Organisms were distributed on 12 polystyrene well plates in a total volume of 5 mL of test solution per well.For each treatment, the study used one plate, each containing two organisms, thus totaling 24 replicates per treatment.After a period of 24 and 48 h of exposure, the organisms were evaluated and compared to the control group with regard to their immobility.

Statistical analysis
To determine the average lethal concentration (LC 50 ) of CAD and PUT, as well as their con dence intervals (95%), the "Probit Analysis" method was used.Data were evaluated using Statgraphics Centurion XVII software, Version 1.17.04 (Stat Point Technologies 2014).The data obtained from the BA mixture were evaluated using the STATISTICA 7.0 program for the analysis of normality (Shapiro-Wilk) and homogeneity of variance (Levene).For the analysis of means comparison between treatments and control, this study used the GraphPad Prism 5.0 software.Data were submitted to the parametric ANOVA (Analysis of Variance) test, followed by the Dunnet post-test (p < 0.05).

Results
3.1 Acute toxicity bioassays with zebra sh embryos

Lethal concentration (LC 50 ) calculation
The lethality of embryos exposed to the BAs CAD or PUT was determined by the clotting of the embryo or by the absence of heartbeats.Data from negative and positive controls validated all the tests performed (OECD, 2013).The observed lethality after 24h of exposure was < 10% for the control group and 100% for the positive control.
The obtained results showed that the lethality rate was directly proportional to the increase in CAD concentration.All tested concentrations induced lethality in zebra sh embryos.After 96h of exposure, the lowest concentration tested (200 mg.L − 1 ) induced 21% lethality, while the highest (1000 mg.L − 1 ) induced 100% lethality.From these results, the average lethal concentration (LC 50 − 96h) of CAD was calculated (Fig. 1A), the value of which was 329.65 mg.L − 1 .
Embryos exposed to putrescine presented lethality for all tested concentrations, with dose-responsedependent characteristics.After 96h of exposure, the concentration of 200 mg.L − 1 caused 8% embryo lethality.The highest concentrations tested (C3-600 mg.L − 1 ; and C4-800 mg.L − 1 ), showed a lethality of 25% and 100%, respectively.The LC 50 of PUT (96h exposure) was of 449.56 mg.L − 1 (Fig. 1B).The increase in lethality was signi cant in all treatments with embryos exposed to CAD and PUT concomitantly (Fig. 2).Effects were observed within the rst 24h of exposure.Treatment M2 (LC 50 CAD + 1/10 LC 50 PUT) increased the lethality of individuals by 50% when compared to that of the control group.

Hatching delay and malformations
Table 3 displays data on the embryonic development stages (24-96 hpf) of the zebra sh.The control group organisms started hatching at 72 hpf.At the end of the 96-h exposure, the hatching rate of the control group was greater than 90%.
The different concentrations of CAD or PUT evaluated in this study did not induce changes in the hatching parameters of zebra sh eggs, when compared to those of the control group.However, every mixture of CAD and PUT caused morphological changes and a decrease in the egg hatching rate, except for M4, which had such a high lethality rate (> 90%) that it prevented the evaluation of these parameters (Fig. 3, 4, 5, 6).
All CAD + PUT mixtures evaluated in this study led to the delayed hatching of D. rerio eggs.The embryonic development stages (24-96 hpf) and zebra sh hatching delay images are shown in Fig. 3. Treatment M1, referring to the combination of lower concentrations (1/10 LC 50 CAD + 1/10 LC 50 PUT) had less in uence on this reduction in the hatching rate by 10%, when compared to the control group.On the other hand, the treatment that showed the greatest toxicity was a combination of the highest concentration of CAD with the lowest concentration of PUT (M2 -LC 50 CAD + 1/10 LC 50 PUT).This treatment reduced the hatching of embryos by 55% after 96h of exposure (Fig. 4).
The control group did not show signi cant morphological changes in any of the bioassays.The same occurred for the treatments carried out with isolated CAD and PUT, since none of the concentrations analyzed induced malformations in the embryonic zebra sh.The treatment performed with the M2 mixture (LC 50 CAD + 1/10 LC 50 PUT) showed the highest embryotoxicity.At this concentration, 21% of zebra sh embryos tested (FET test -96h of exposure) were carriers of morphological changes: pericardial edema and spinal de ciency malformations were the most observed.The M1 (1/10 LC 50 CAD + 1/10 LC 50 PUT) and M3 (LC 50 PUT + 1/10 LC 50 CAD) treatments induced malformations in 4%, including pericardial edema and spinal de ciency (Fig. 5).It was not possible to obtain malformation data for M4, as this concentration was toxic to D. rerio.The malformations found in the acute toxicity assessment for zebra sh exposed to the CAD and PUT mixture are displayed in Fig. 6.

Assessment of acute toxicity in D. magna
The results of the toxicity for D. magna exposed to the BAs CAD and PUT showed that CAD is highly toxic to the microcrustacean.All evaluated concentrations of these BAs were lethal for D. magna, when compared to that of the control group.The observed lethality for the control group was < 20%.
The lowest concentration tested (C1-10 mg.L − 1 ) caused a lethality of 65% of the individuals.
The results obtained with PUT also showed lethality for D. magna.However, the acute toxicity of PUT was lower than that of CAD.Treatment C1 (10 mg.L − 1 ) resulted in 20% lethality, while C2 and C3 caused 10% lethality when compared to the control group.The treatments with the highest concentrations (C4, C5 and C6) caused 100, 65 and 70% lethality, respectively.The LC 50 value (36.74 mg.L − 1 ) obtained in the 48h of exposure assays appears in Fig. 7B.
The lethality results obtained with D. magna for the treatments with the mixture of CAD and PUT amines were not statistically different from those of the control.Concentrations M1 and M3 induced 25% lethality; M2, 8%; M4, 29%; and the control, 17%.The results are shown in Fig. 8.

Discussion
Cemeteries are among the most silent and continuous sources of soil and groundwater contamination.The decomposition of human as well as of animal carcasses should be considered a natural phenomenon.However, during the decomposition process, toxic metabolites are produced that can pose a risk both to the physical environment (water and soil) and to the biology of the contaminated site, in addition to threatening human health (Zhou et al. 2021).The necro-leachate generated from this process can in ltrate the soil and contaminate groundwater, in addition to contributing to the increase of microbiological contamination from heavy metals and toxic organic pollutants (Franco et al. 2022).
From the results of the tests carried out with D. rerio and D. magna, exposed separately to BAs, it was possible to infer that CAD was more lethal than PUT, although the hatching delay values, and morphological changes, were not statistically different from those of the control group.
The assays performed with D. rerio exposed to the CAD and PUT mixtures show that these amines, when associated with each other, can increase the lethality rate, and induce delays in hatching and malformations in zebra sh larvae.Some authors also cite that CAD and PUT mixtures are more toxic than isolated amines, due to synergistic effects occurring between them, which potentiates the toxicity of other BAs such as histamine and tyramine (Donato et al. 2022;Saad et al. 2022).Among the morphological changes in zebra sh co-exposed to the BAs evaluated in this study, most notable are the pericardial edema and spinal de ciencies.
Pericardial edema is identi ed by increased uid in the pericardium.This malformation is a cardiovascular abnormality resulting from both an increased permeability of blood vessels and the accumulation of contaminants in tissues (Vallim et  The cytotoxic effect of CAD and PUT during zebra sh embryogenesis may be explained by its nucleophilic and polycationic nature at physiological pH.According to Pelka et al. (2017), the lipophilic characteristics, the electrical charge, and the structural conformation of a molecule, can all play an important role in the absorption of chemicals through the chorion of the zebra sh embryo.Furthermore, they may interact with negatively charged chemical species of DNA, RNA, and proteins (Kumar et al. 2021).This effect was reported by Del Rio et al. ( 2019) for HT29 intestinal cell cultures exposed to these BAs.The authors correlated the cytotoxicity of CAD and PUT with the possibility of amine interaction with phospholipids (of an anionic nature) and by the destabilization of cell membranes.This information may explain the appearance of malformations in the present study, but further studies are necessary to con rm this hypothesis.
The D. magna organisms showed greater sensitivity to an isolated BA when compared to that of the acute toxicity tests using zebra sh.CAD also showed a higher level of toxicity for this organism when compared to PUT (CAD LC 50 of 9.51 mg.L − 1 and PUT LC 50 of 36.74 mg.L − 1 ).However, the BA mixture did not signi cantly increase the lethality of exposed individuals at any of the evaluated treatments.
Although there are no scienti c references regarding an assessment of the acute toxicity of these BAs in D. magna, studies by Spadotim (2015) carried out using water samples near a cemetery in the city of Limeira, São Paulo State, Brazil, con rmed the acute toxicity of these samples in relation to the microcrustaceans D. magna and Ceriodaphnia dubia.The authors also related this toxic effect on the test organisms to the high zinc concentration recorded in the evaluated waters.According to Wieczorek et

Conclusion
The data from the present study con rm the toxic potential of the BAs CAD and PUT on the aquatic organisms D. rerio and D. magna.Delays in hatching and malformations during the embryonic development of the sh, as well as an increased lethality for the water ea D. magna were observed.Although isolated BAs did not provide signi cant toxic effects, compared to the control group, CAD showed higher toxicity values than PUT.Mixtures of these substances have already triggered more worrying toxic effects.These data on the toxicity of BA mixtures reinforce the need for studies that assess impacts on the soil and groundwater around cemeteries, as these amines are released concomitantly during the cadaveric putrefaction process.
Given these observations, it is possible to conclude that the test organisms used in the study were e cient in assessing the toxicity of the BAs CAD and PUT.The proven toxicity of these amines highlights the requirement for more e cient environmental management in cemeteries, which should help to control necropolis pollution.

Declarations Funding
We would like to thank CAPES (Coordenação de Aperfeiçoamento de Pessoa de Nível Superior) for supplying a scholarship to the rst author and Embrapa Environment for providing support for the development of this research.

Figure 1 A
Figure 1

Table 1
Cadaverine (CAD) and putrescine (PUT) concentrations (LC 50 mg.L − 1 ) used in the toxicity evaluations of mixtures of these biogenic amines for Danio rerio and Daphnia magna.LC 50 -mg.L − 1 ).After the preliminary tests, the following concentrations were those de nitive to obtain the LC 50 : 10; 16; 25.6; 40.96; 65.54; 104.86 mg.L − 1 , respectively C1 up to C6.The concentrations for the BAs mixtures are shown in Table 2.3.2Daphnia magna acute toxicity test D. magna neonates, which were younger than 24h old, were exposed for 48h to the following treatments: control (only reconstituted water), CAD, PUT and a mixture of both substances (CAD + PUT).Preliminary tests (0; 0.1; 1; 10 and 100 mg.L − 1 ) were performed to determine the mean lethal concentration for 50% of the individuals (
al. 2022; Wu et al. 2019).According to Cooman et al. (2022), another common phenotype observed in embryotoxicity assays is spinal deformities after exposure to different xenobiotics.Studies showed that tail curvature may be associated with neuroin ammation caused by environmental pollutants (Iqubal et al. 2020; Massei et al. 2019).
(Neckel et al. 2017 PUT increased the lethality of the aphid Cinara cupressi, as well as signi cantly reducing the fecundity of these organisms in up to ve generations.According to Netto et al. (2021) and Oliveira et al. (2022), ecotoxicological tests performed with nontarget organisms is an attractive strategy to estimate tolerable concentrations of toxic compounds present in the environment.Thus, the tests carried out here with the bioindicators D. rerio and D. magna, to evaluate the toxicity of BAs in cemetery environments, present important data that may assist in providing consistent information about the risks and environmental tolerances of this type of contamination.A study presenting results from with the BAs CAD and PUT is unprecedented because although the toxicity of these amines in food is well known, little is known about the environmental impacts and effects on human health of BAs derived from putrefactive processes in cemeteries.Considering the high and continuous production of these amines in necropolises, the increase in burials in the last two years, resulting from the SARS-CoV-2 Pandemic Gonçalves et al. (2022), as well as the absence of management and treatment of e uents emitted by decomposing bodies, and the possibility of contamination of groundwater near cemeteries(Neckel et al. 2017), these amines should be further investigated, regarding their risks to the environment and human health.The originality of this study also lies in the type of evaluation carried out, with bioindicators and biomarkers of recognized e ciency for monitoring quality and/or environmental impacts(Belaid andSbartai 2021; Anifowoshe et al. 2022; Lee et al. 2022; Howe et al. 2013) indicated by important environmental agencies around the world (e.g.OECD, USPEA, CONAMA-BR).