MATERIALS AND METHODS
Biologic material
Adult sea-urchins, Paracentrotus lividus, were gathered in the Praia da Aguda, located 8 km north of the Barrinha de Esmoriz / Lagoon of Paramos (BE/LP). After the adults were collected they were transferred into a tank filled with filtered sea water (FSA) and left for 2 days at 22 ºC to stabilise.
Compounds and chemical substances tested
For the different bioassays it was used high, medium and low concentrations of mixed polychlorinated biphenyls (PCBs) isomers and metal ions such as arsenite (As3+), arsenate (As5+) and manganese (Mn2+), shown in Table 1. The low concentrations correspond to the average concentration of this pollutants in the superficial and subterranean waters of BE/LP. With the use of the higher concentrations it was intended to study the effect of the potential accumulation and/or bioaccumulation of these pollutants.
The source of PCB’s was Aroclor 1254, from which a concentrated stock solution was made by solubilization of the commercial formulation in ethanol and further dilution in sterile deionised water. The stock solutions of the ions As3+, As5+ and Mn2+ were prepared by dilution of the salts NaAsO2 (sodium arsenite), Na2HAsO4.7H20 (heptahydrated sodium arsenate) and MnCl2.4H20 (tetrahydrated manganese chloride) in sterile deionised water. The final solutions, in the used concentrations, were obtained by dilutions of the stock solutions in sterile FSW (filtered sea water). In the PCBs solutions the ethanol concentration never exceeded 0.0001%, a value which was confirmed not to have any influence on the results.
All the reagents and chemicals used were purchased from Sigma-Aldrich Chemical, S.A. (Sintra, Portugal).
Gamete handling and fertilization conditions
Sea urchins were spawned using a 0.5 mL intra-celomatic injection of 0.2 M potassium chloride. The sperm was collected “dry”, directly from the gonopore, with a Pasteur pipette and packed in polypropylene microvials at 4ºC for up to a maximum period of 2 days. The oocytes were released directly to FSW. Later, they were collected, washed and conditioned at 4 ºC in fresh FSW. The subjects were then returned to the place of origin, after one week of recovery.
In the different bioassays it was always used a mixture of sperm and oocytes from 4 males and 4 females and each experiment was replicated at least twice. From the mixture of gametes, suspensions of sperm 1:100 000 and oocytes 1:100 were prepared in the different solutions to be tested. In the fertilization assay 10mL of sperm suspension were used to inseminate 20 mL of oocyte suspension (≈ 2 000 oocytes) and after fertilization the zygotes were incubated at 22ºC.
Toxicity tests induced in sperm and oocytes
In order to test the toxicity induced in gametes by Aroclor 1254 and the ions As3+, As5+ and Mn2+, both the sperm and the oocytes were incubated for 60 minutes at 22ºC in the solutions prepared with different concentrations of these pollutants. After the treatment period, the gametes were washed in sterile FSW and the sperm and oocytes from the control (FSW) were mixed with the sperm and oocytes from the different treatments according to the combinations shown in Table 2. After 30 minutes, 1, 3 and 72 hours, 2mL samples were withdrawn from each combination, and then fixed in 2mL of 8% formaldehyde for subsequent processing and analysis in accordance with the criteria set out in Table 3.
Toxicity assays induced in embryos
To test the induced toxicity by the various pollutants in the embryonic development, the used zygotes were previously fertilized in filtered sea water (FSW), with a well-developed fertilization membrane (30 minutes after fertilization). The zygotes were incubated, at 22ºC, in Petri dishes with 10mL of each of the solutions (Table 1) until they reached the pluteus stage. After 72 hours it was held a morphological analysis of live larvae, immobilized with a drop of 3:1 solution of ethanol-ether (v/v) before observation. For each treatment at least 100 larvae were observed using bright field microscopy and phase contrast microscopy with amplification of 400X and 600X. Changes in the larvae development were assessed with the defined criteria for larvae in the pluteus stage (Table 3).
Indicators of toxicity and genotoxicity
As toxicity indicators, are considered the viability of the gametes, the dynamics of the egg cleavage and the morphology of the larvae in the pluteus stage.
The sperm and oocytes viability was measured by the ability to fertilise 30 minutes after exposure to the various pollutants and was evaluated in 200 eggs. The eggs were considered to be fertilized if they showed an evident fertilization membrane (FM)
The dynamics of the egg cleavage was evaluated in 200 embryos 1 and 3 hours after fertilization, a period that corresponds to embryos in the stages of 2 to 32 cells. In the first and second stages of cleavage, are considered normally developed eggs all the fertilized eggs that are divided into 2 or 4 symmetrical fractions. All the eggs that were disproportionately divided or not divided were considered abnormal.
The assessment at the pluteus stage took place 72 hours after fertilization, by observing 100 larvae. The larvae considered to show a normal development had a visible gut and a well formed skeletal structure. Considered to show an abnormal development were the larvae with rudimentary skeleton, aberrant configurations and the ones that failed the transition from pre-pluteus.
The zygotes and embryos were observed by bright field and phase contrast microscopy, with magnification of 400X and 600X, with an Olympus BX 41 microscope. Images were collected with a Moticam 2300 video camera.
In assessing the genotoxicity induced by pollutants, the following cytogenetic parameters were considered: changes in mitotic activity and presence of cells with evidence of necrosis and apoptosis (Table 4).
Changes to the mitotic activity were evaluated by the cell division synchronism measured by the frequency of embryos in interphase (IE) and the average number of mitosis per embryo (MPE). The identification of embryos undergoing necrosis and apoptosis was made by the observing DNA, which has a characteristic pattern in these stages. The assessment of these parameters was performed in 100 embryos/treatment in stages from 2 to 32 cells, with an epifluorescence Olympus BX 41 microscope. The preparations were made with embryos fixed in formaldehyde 4% in PBS (v/v), in strips of 8 wells lined with Teflon. One drop of embryo suspension (20-30 embryos) was placed in each well and left to air dry. Then the DNA was stained with a DAPI (4,6-diamidino-2-phenylindole) solution in Citifluor®. The images were collected with a Moticam 2300 video camera.
RESULTS
Detailed results of the toxicity bioassays with gametes of the sea-urchin P. lividus, are found in Tables 5 and 6. The toxicity was evaluated through the effect that the treatment of sperm or oocytes, with different concentrations of Aroclor 1254 and ions As3+, As5+ and Mn2+, had on the fertilization rate, the dynamics of the egg cleavage, and on the induction of damage transmitted to offspring .
Exposure of gametes to pollutants, in general, considerably reduces the fertilization rate (FR), noting that this reduction depends on the type of gametes, the pollutant and the intensity of treatment (Graph 1).
The results displayed by the toxicity test with sperm, show that the metals, particularly the ions As, are much more toxic to sperm than the mixture of PCBs congeners Aroclor 1254. The ion As3+, regardless of the concentration tested, induce the highest rate of failure in fertilization, exceeding the 36% rate of oocytes not fertilized when using sperm treated with the lowest concentration (1mM) and 50.6 % with the highest concentration (1mM).
The ion Mn2+ has an intermediate effect, and with the highest concentration (104mM) is considerably more toxic than the PCBs.
In contrast, the results of the toxicity test with the oocytes show that Aroclor 1254 is the most toxic pollutant to the female gametes and that the As ions are the least toxic. The reducing effect exerted by PCBs on the FR (15.3 % for the 10-2 mM treatment and 33% for the 10mM treatment) appears to result from changes that are induced in the structure of oocytes that, somehow, prevent their fertilization. Indeed, there is an almost total correlation between the frequency of oocytes with aberrant morphology (Figure 2e) and the rate of failure in fertilization. On the other hand, the slightly reducing effect exerted on the FR by the treatment of oocytes with ions As3+ and As5+, appears to result from changes occurring in oocytes at the cell membrane level. Almost all the non fertilized oocytes show an unchanged morphology but in the most drastic cases there is a breakage/dissolution of the plasma membrane (Figure 2d) indicating the beginning of the necrosis process and change of DNA organization (Figure 2g). This is also supported by the observation of polyspermy in several eggs (Figure 2c).
In the oocyte toxicity test, the ion Mn2+ also has an intermediate toxic effect. It is more toxic that the As ions and less toxic than Aroclor 1254. The type of changes induced in the oocytes is also mixed, showing morphology and/or characteristics similar to those induced by other pollutants.
Considering the array of results displayed it appears that the order of toxicity for the sperm toxicity test is As3+> As5+ Mn2+> Aroclor 1254 and for the oocyte toxicity test Aroclor 1254> Mn2+> As5+> As3+.
Effect on the egg cleavage dynamics
Observing the alterations of the egg cleavage dynamics shows that the treatment of gametes with different pollutants also produces changes in the sperm and oocytes that remain viable. These changes result in the drastic reduction of capacity and/or quality of the egg division (Graph 2). The effect, similar to that found in the reduction of the fertilization rate, also depends on the types of gametes tested, the pollutant and intensity of treatment. Overall, the changes are more pronounced in eggs derived from treated oocytes. However, all the tested pollutants induce a strong deleterious effect on the quality of sperm. These gametes are particularly sensitive to As3+, noting that the normal division of eggs, fertilized by sperm treated with this metal ion, is strongly committed to about half of the fertilized eggs.
Inhibition of the egg segmentation
In eggs fertilized by sperm treated with the pollutants, during 1h, there is a high frequency of eggs that have not yet started the first division 1h and 3 h after fertilization. This phenomenon was never observed with the control, where it was found that all the fertilized eggs were undergoing division, 1 and 3h after fertilization. The inhibitory effect of the egg cleavage is not very evident in the treatments with the As ions, but is observed in all tested concentrations of PCBs and Mn2+.
The inhibition of the egg cleavage is even higher in the fertilized eggs from oocytes previously exposed to various toxics. The phenomenon is induced by all pollutants, but is less evident in treatments with Mn2+. In the majority of eggs that did not divide that resulted from treatments with PCBs, the analysis of egg morphology (Figure 3b, c) and the chromatin organization (Figure 3f, g) suggests that a process of apoptosis started in most eggs (Figura 3f,g), 3h after fertilization; this was never observed in the control.
In the eggs originating from fertilized oocytes treated with As ions, the analysis of the egg morphology (Figure 3e) and the organisation of chromatin (Figure 3h) suggests the beginning of a necrosis process, 3 h after fertilization.
Anomalies in the egg segmentation
The treatment of gametes with the different pollutants also induces considerable disturbances in the segmentation of the egg.
In the eggs fertilized by sperm exposed to the pollutants, the failure rate of the normal egg segmentation is particularly high. The effect is more pronounced in treatments with As3+ and PCBs (Graph 2). The normal segmentation of the egg is also seriously compromised in eggs that resulted from treated oocytes. The treatment of oocytes with the higher concentrations induces inability of normal segmentation in more than 55 % of the eggs. The anomalies observed in the egg segmentation translate themselves into different levels of depth, depending on the treatment intensity at which the gametes were exposed. At the first and second segmentation, the lower concentrations usually induce the asymmetric division of the blastomeres (Figure 4d, f, g), associated with the reduction of the number of mitosis/embryo and improper chromosome segregation (Figure 4o, p). The higher concentrations, promote the incomplete segmentation of the egg (Figure 4b, c, g).
In eggs in more advanced segmentation stages (16 – 32 cells), the lower concentrations of pollutants also cause asymmetric segmentation (Figure 4j) and, in many embryos, the highest concentrations, trigger apoptosis (Figures 4k) and necrosis (Figure 4l).
The effect on the offspring quality
Table 6 shows the results concerning the quality of the embryos that originated from eggs fertilized by gametes exposed to the various pollutants and the toxicity bioassays in embryos – exposure to toxics post-fertilization. The assessment was made through morphological changes produced in the larvae development (Graph 3). The post-fertilization exposure to the different pollutants drastically reduces the quality of embryos in the pluteus stage. Treatment with the highest concentration of As3+, As5+ and Mn2+ adversely affects all the progeny, inducing the death of 40% of larvae (Graph 3) and deep skeletal deformities in the surviving larvae (Figure 5, b-e).
The extent of this effect depends on the treatment intensity, where a lower mortality rate and substantially lower larvae deformities are found in lower concentrations. In treatments with PCBs the same effects are seen although they are far less drastic.
A property of the metals tested seems to be the ability to inhibit the transition from larvae to the pluteus stage. In most tests, there was a high frequency of embryos on blastula and gastrula stages with pronounced malformations (Figure 5, f-h), which blocked the development before reaching the pluteus stage. This phenomenon is very pronounced with the As ions, less intense with Mn2+ and was not observed with PCBs. This suggests that the As ions have a more active action in the induction of malformations in the gastrulation process.
In a less intense way, the metals also have a negative effect on the quality of the offspring derived from eggs fertilized by gametes treated with these pollutants. The effect is considerably less evident in embryos from eggs fertilized by treated sperm.
For PCBs, no significant differences are observed between the type of changes produced and the frequency of individuals affected by embryo toxicity tests and in larvae originated from gametes treated with this pollutant.
This set of results shows that the embryo toxicity test is not as discriminative as the sperm toxicity test. However, the same trend of toxicity remains: As3+ ≥ As5+ ≥ Mn2+> Aroclor 1254.
Discussion
The echinoderm Paracentrotus lividus is recognized as an excellent bioindicator of contamination of marine ecosystems by heavy metals and organic compounds (Danis et al., 2005). In bioassays using the sea urchin, fertilization and toxicity tests induced in sperm and embryos, are widely widespread. In P. lividus, both are validated to assess the toxicity induced by polluted waters from both estuarine and coastal lagoon ecosystems (Arizzi Novelli et al., 2001; Volpi Ghirardini e Arizzi Novelli, 2001; Volpi Ghirardini et al., 2005), and in the assessment of the toxic effects of the heavy metals As3+, Cd2+, Cr3+, Ni2+, Pb2+, Cu2+, Zn2+ and Hg2+ (Warnau et al., 1996; Arizzi Novelli et al., 2003). However, very few studies on the sea urchin use oocyte toxicity tests (Au et al., 2001) and cytogenetic analysis (Saotome et al., 1999; Schweitzer et al., 1999; Dixon et al., 2002; Saotome e Hayashi, 2003). Thus, this is the first study to be developed in P. lividus that simultaneously involves, toxicity tests induced in sperm, oocytes and embryos and also cytological, embryological and cytogenetic evaluation parameters.
With this work, the toxic effect of Mn2+ is for the first time assessed in P. lividus. The results show that, in general, the P. lividus gametes are slightly sensitive to Mn. However, the sensitivity of oocytes is slightly larger than the sperm, which is demonstrated by the worst performance in the fertilization studies and the dynamics of the first egg divisions. Also the P. lividus larvae are more affected when they originate from oocytes exposed to Mn2+ for 1 h, than when resulting from oocytes fertilized by sperm undergoing the same treatment. The low toxicity of Mn in sperm could be a common feature between other species of se-urchins. In Arbacia punctulata, Young and Nelson (1974) found that the treatment of sperm with different concentrations of Mn2+ did not significantly reduce the sperm mobility, a function that in the sea urchin is directly related to the ability to fertilise (Berdishev et al., 1995; Au et al., 1999). The most obvious toxic manifestation is observed when the embryonic development takes place in the presence of Mn. In concentrations higher than 102mM it has a drastic effect on the quality of the larvae produced, causing a variety of different aberrations providing the potential to create a sensitive test for monitoring programmes. Similar results were seen in Anthocidaris crassipina by Kobayashi and Okamura (2004, 2005), suggesting that the greater toxicity of Mn in the sea urchin is induced at the embryonic development level.
The toxicity comparative study of the two ionic species of As show that the two ions induce similar effects in the sea urchin, but As3+ is considerably more toxic than As5+, confirming the toxicity relationship observed in other organisms (Koh et al., 2005). Regarding the fertilization capability, the P. lividus sperm is more sensitive to exposure of As ions than the oocytes, noting that the sperm treated with As drastically reduces the fertilization rate of non-treated oocytes. This sensitivity of the P. lividus male gametes to As was also observed by Arizzi et al. (2003) where in sperm toxicity assays a similar deleterious effect was observed. The greater sperm sensitivity, relatively to the oocytes, is also mentioned by Au et al. (1999), in which toxicity bioassays with Cd developed in Anthocidaris crassipina showed a worse performance in treated sperm relatively to the treated oocytes, both in fertilization and dynamics of the first egg cleavage tests.
However, our results show that the As is equally toxic to the female gametes when in concentrations above 102mM, inducing alterations in the dynamics of the egg cleavage and considerable damage in the quality of the offspring produced, measured in the pluteus stage. In the eggs fertilized with oocytes treated with As, there was a high percentage of eggs in which the mitosis was inhibited (27.6% and 36.1% at concentrations of 102 mM and 104mM, respectively). This indicator, when associated with the cells characteristic phenotype, which suggests the triggering of necrosis process (fragmentation/dissolution of the cell membrane and degradation of DNA), could be a good test for toxicity bioassays. Even the aberrations produced in the larvae pluteus stage and the high number of larvae with development blocked in stages prior to pre-pluteus, including blastula and gastrula stage with pronounced abnormalities, are a good indicator of toxicity.
The toxicity of As at the sperm level can be seen differently by changing the dynamics of the egg cleavage. Treatment of sperm with 1mM of As3+ is sufficient to induce asymmetrical division in more than 50% of eggs, giving rise to uneven blastomeres, improper chromosome segregation and reduction in the number of mitosis/embryo. This indicator is a very sensitive test with the potential to integrate programmes to monitor pollution in marine ecosystems. However, as noted with Mn, the toxicity of As at the sperm level does not have a significant effect on offspring, which seems to be a common pattern to various heavy metals. A similar effect was observed in another study, in toxicity bioassays of Cu, Ag, Cd and Hg conducted in P. lividus (Warnau et al., 1996).
The embryo toxicity test, considered highly sensitive in the evaluation of toxicity induced by heavy metals (Warnau et al., 1996; Kobayashi e Okamura, 2004, Arizzi Novelli et al., 2001; Volpi Ghirardini et al., 2005), revealed itself hardly effective in the lowest concentration of As3+ (1mM), which confirms the results obtained by Arizzi et al. (2003). However, it is quite effective for higher concentrations of As3+ superior to 102 mM.
For polychlorinated biphenyls (PCBs), it is not known any toxicity study in P. lividus, but recently Danis et al. (2005) showed that this specie is an efficient bioaccumulator of PCBs because it is not sensitive to these compounds. The results of this work show that the mixture of PCBs congeners, Arocolor 1254, is not very toxic to the P. lividus larvae, suggesting that the embryonic development is not a very sensitive indicator to integrate toxicity tests on embryos. Similar results were found in similar studies in Lytechinus pictus and Stongylocentrotus purpuratus (Weisberg et al., 1996, 1997; Schweitzer et al., 1999, 2000) and in Arbacia punctulata (Adams, 1983). Our results show that this indicator only becomes effective in the presence of high concentrations of PCBs (10 mM Aroclor 1254), resulting in a considerable percentage of larvae with skeletal deformities (19%) and death of some larvae (4.8%). However, tests for toxicity developed with gametes treated for 1h with Arocolor 1254 proved to be effective in detecting low concentrations of PCBs (10-5 mM Aroclor 1254), particularly those developed with oocytes. In these assays, with any of the tested concentrations, there were degenerative changes of oocytes, reduction in the fertilization efficiency, formation of aberrant pluteus and death of larvae. These effects, which demonstrate the great sensitivity of these gametes to Aroclor 1254, are identical to those referred by Adams (1988) in an experiment conducted with treated oocytes of A. punctulata, but where the fertilization and the whole embryonic development took place in the presence of Aricolor 1254.
Similarly to what it has been observed in toxicity assays with As, also with PCBs the indicator that proved most effective was the dynamics of the first egg cleavage. In all the tested concentrations, there was asymmetrical division of the egg, improper chromosome segregation, reduction of the number of mitosis/embryo and complete inhibition of mitosis, with consequent lack of segmentation, in a considerable percentage of eggs. These cytological and cytogenetic indicators, associated with the characteristic phenotype of most cells that show signs of apoptosis, like the formation of bubbles in the cell membrane and excessive condensation and/or fragmentation of DNA, may constitute a robust and sensitive test, appropriate for monitoring of these persistent organic pollutants. The introduction of complementary diagnostic tools to detect in a more sensitive way the triggering of apoptosis, as the Comet assay – Single Cell Gel” or the “ Tunel – Terminal dTUP nick end labelling assay, may provide grater strength to the test.
The comparative results of toxicity induced by polychlorinated biphenyls (PCBs) and by the ions As3+, As5+ and Mn2+, arising from the implementation of this model, confirms the effectiveness of some tests in the detection of these pollutants and show that some are quite specific. The relevance of these results can be the possibility to discriminate the bioavailability of some pollutants in projects for biomonitorization of polluted coastal marine ecosystems.
This study shows that for some assays there are evaluation parameters that show great specificity:
Sperm induced toxicity tests
i. Fertilization rate. Suitable for the detection of As, in particular ion As3+. It may allow to discriminate the presence of As relatively to the presence of Mn and the tested PCBs congeners (total 7), which have a considerably more modest performance in this test. Order of observed effectiveness - As3+> As5+> Mn2+> Aroclor 1254.
ii. Alteration in the egg cleavage dynamics. Very sensitive test in detecting As3+ and PCBs. Established order of effectivity - As3+> Aroclor 1254> Mn2+> As5+.
There are parameters that seem to allow to discriminate the presence of PCBs relatively to As3+. The toxicity induced by PCBs in sperm is reflected in a strong inhibition in the egg cleavage. This effect is residual in As. Moreover, the effect produced by As3+ is reflected in a high frequency of eggs with aberrant division, effect substantially lower in treatments with PCBs.
iii. Transmission of damage to the offspring. Test with little sensitivity, but effective for high concentrations of PCBs and Mn2+. Order of effectiveness - Aroclor 1254 > Mn2+ > As5+ > As3+.
Toxicity tests induced in oocytes
i. fertilization rate. Test with little sensitivity, but effective for high concentrations of PCBs. The inhibition of fertilization, due to the aberrant morphology of the oocytes, is a special feature that can discriminate the presence of this compound relatively to metals, in particular the As ions. Order of effectiveness - Aroclor 1254 > Mn2+ > As5+ > As3+.
ii. Alteration of the cleavage dynamics of the egg. Sensitive test in the detection of all toxics. Order of observed effectiveness - Aroclor 1254 ≥ As3+ ≥ As5+ ≥ Mn2+.
The inhibition of the egg segmentation allows to discriminate the presence of PCBs and As ions, relatively to Mn. The presence of cells with a phenotype characteristic of apoptosis (bubbles formation in the cell membrane and condensation / fragmentation of DNA) can discriminate the presence of PCBs relatively to the As ions. The presence of cells with a phenotype suggesting necrosis (fragmentation/dissolution of the cell membrane and degradation of DNA) allows discriminating the presence of As ions relatively to PCBs.
iii. Transmission of damage to the offspring. Test that detects the studied pollutants when in medium and high concentrations. Established order of effectiveness - As3+ ≥ Mn2+ ≥ As5+> Aroclor 1254.
The presence of larvae with development blocked in stages prior to pre-pluteus (blastula and gastrula with pronounced malformations) allows to discriminate the presence of As ions relatively to Mn and PCBs.
Toxicity test in embryos
i. Quality of larvae in the pluteus stage. Sensitive test in the detection of As and Mn. Order of effectiveness - As3+ ≥ As5+ ≥ Mn2+ > Aroclor 1254.
Discriminates the presence of As ions relatively to Mn and PCBs, when considering larvae with blocked development previous to pluteus.
Biologic material
Adult sea-urchins, Paracentrotus lividus, were gathered in the Praia da Aguda, located 8 km north of the Barrinha de Esmoriz / Lagoon of Paramos (BE/LP). After the adults were collected they were transferred into a tank filled with filtered sea water (FSA) and left for 2 days at 22 ºC to stabilise.
Compounds and chemical substances tested
For the different bioassays it was used high, medium and low concentrations of mixed polychlorinated biphenyls (PCBs) isomers and metal ions such as arsenite (As3+), arsenate (As5+) and manganese (Mn2+), shown in Table 1. The low concentrations correspond to the average concentration of this pollutants in the superficial and subterranean waters of BE/LP. With the use of the higher concentrations it was intended to study the effect of the potential accumulation and/or bioaccumulation of these pollutants.
The source of PCB’s was Aroclor 1254, from which a concentrated stock solution was made by solubilization of the commercial formulation in ethanol and further dilution in sterile deionised water. The stock solutions of the ions As3+, As5+ and Mn2+ were prepared by dilution of the salts NaAsO2 (sodium arsenite), Na2HAsO4.7H20 (heptahydrated sodium arsenate) and MnCl2.4H20 (tetrahydrated manganese chloride) in sterile deionised water. The final solutions, in the used concentrations, were obtained by dilutions of the stock solutions in sterile FSW (filtered sea water). In the PCBs solutions the ethanol concentration never exceeded 0.0001%, a value which was confirmed not to have any influence on the results.
All the reagents and chemicals used were purchased from Sigma-Aldrich Chemical, S.A. (Sintra, Portugal).
Gamete handling and fertilization conditions
Sea urchins were spawned using a 0.5 mL intra-celomatic injection of 0.2 M potassium chloride. The sperm was collected “dry”, directly from the gonopore, with a Pasteur pipette and packed in polypropylene microvials at 4ºC for up to a maximum period of 2 days. The oocytes were released directly to FSW. Later, they were collected, washed and conditioned at 4 ºC in fresh FSW. The subjects were then returned to the place of origin, after one week of recovery.
In the different bioassays it was always used a mixture of sperm and oocytes from 4 males and 4 females and each experiment was replicated at least twice. From the mixture of gametes, suspensions of sperm 1:100 000 and oocytes 1:100 were prepared in the different solutions to be tested. In the fertilization assay 10mL of sperm suspension were used to inseminate 20 mL of oocyte suspension (≈ 2 000 oocytes) and after fertilization the zygotes were incubated at 22ºC.
Toxicity tests induced in sperm and oocytes
In order to test the toxicity induced in gametes by Aroclor 1254 and the ions As3+, As5+ and Mn2+, both the sperm and the oocytes were incubated for 60 minutes at 22ºC in the solutions prepared with different concentrations of these pollutants. After the treatment period, the gametes were washed in sterile FSW and the sperm and oocytes from the control (FSW) were mixed with the sperm and oocytes from the different treatments according to the combinations shown in Table 2. After 30 minutes, 1, 3 and 72 hours, 2mL samples were withdrawn from each combination, and then fixed in 2mL of 8% formaldehyde for subsequent processing and analysis in accordance with the criteria set out in Table 3.
Toxicity assays induced in embryos
To test the induced toxicity by the various pollutants in the embryonic development, the used zygotes were previously fertilized in filtered sea water (FSW), with a well-developed fertilization membrane (30 minutes after fertilization). The zygotes were incubated, at 22ºC, in Petri dishes with 10mL of each of the solutions (Table 1) until they reached the pluteus stage. After 72 hours it was held a morphological analysis of live larvae, immobilized with a drop of 3:1 solution of ethanol-ether (v/v) before observation. For each treatment at least 100 larvae were observed using bright field microscopy and phase contrast microscopy with amplification of 400X and 600X. Changes in the larvae development were assessed with the defined criteria for larvae in the pluteus stage (Table 3).
Indicators of toxicity and genotoxicity
As toxicity indicators, are considered the viability of the gametes, the dynamics of the egg cleavage and the morphology of the larvae in the pluteus stage.
The sperm and oocytes viability was measured by the ability to fertilise 30 minutes after exposure to the various pollutants and was evaluated in 200 eggs. The eggs were considered to be fertilized if they showed an evident fertilization membrane (FM)
The dynamics of the egg cleavage was evaluated in 200 embryos 1 and 3 hours after fertilization, a period that corresponds to embryos in the stages of 2 to 32 cells. In the first and second stages of cleavage, are considered normally developed eggs all the fertilized eggs that are divided into 2 or 4 symmetrical fractions. All the eggs that were disproportionately divided or not divided were considered abnormal.
The assessment at the pluteus stage took place 72 hours after fertilization, by observing 100 larvae. The larvae considered to show a normal development had a visible gut and a well formed skeletal structure. Considered to show an abnormal development were the larvae with rudimentary skeleton, aberrant configurations and the ones that failed the transition from pre-pluteus.
The zygotes and embryos were observed by bright field and phase contrast microscopy, with magnification of 400X and 600X, with an Olympus BX 41 microscope. Images were collected with a Moticam 2300 video camera.
In assessing the genotoxicity induced by pollutants, the following cytogenetic parameters were considered: changes in mitotic activity and presence of cells with evidence of necrosis and apoptosis (Table 4).
Changes to the mitotic activity were evaluated by the cell division synchronism measured by the frequency of embryos in interphase (IE) and the average number of mitosis per embryo (MPE). The identification of embryos undergoing necrosis and apoptosis was made by the observing DNA, which has a characteristic pattern in these stages. The assessment of these parameters was performed in 100 embryos/treatment in stages from 2 to 32 cells, with an epifluorescence Olympus BX 41 microscope. The preparations were made with embryos fixed in formaldehyde 4% in PBS (v/v), in strips of 8 wells lined with Teflon. One drop of embryo suspension (20-30 embryos) was placed in each well and left to air dry. Then the DNA was stained with a DAPI (4,6-diamidino-2-phenylindole) solution in Citifluor®. The images were collected with a Moticam 2300 video camera.
RESULTS
Detailed results of the toxicity bioassays with gametes of the sea-urchin P. lividus, are found in Tables 5 and 6. The toxicity was evaluated through the effect that the treatment of sperm or oocytes, with different concentrations of Aroclor 1254 and ions As3+, As5+ and Mn2+, had on the fertilization rate, the dynamics of the egg cleavage, and on the induction of damage transmitted to offspring .
Exposure of gametes to pollutants, in general, considerably reduces the fertilization rate (FR), noting that this reduction depends on the type of gametes, the pollutant and the intensity of treatment (Graph 1).
The results displayed by the toxicity test with sperm, show that the metals, particularly the ions As, are much more toxic to sperm than the mixture of PCBs congeners Aroclor 1254. The ion As3+, regardless of the concentration tested, induce the highest rate of failure in fertilization, exceeding the 36% rate of oocytes not fertilized when using sperm treated with the lowest concentration (1mM) and 50.6 % with the highest concentration (1mM).
The ion Mn2+ has an intermediate effect, and with the highest concentration (104mM) is considerably more toxic than the PCBs.
In contrast, the results of the toxicity test with the oocytes show that Aroclor 1254 is the most toxic pollutant to the female gametes and that the As ions are the least toxic. The reducing effect exerted by PCBs on the FR (15.3 % for the 10-2 mM treatment and 33% for the 10mM treatment) appears to result from changes that are induced in the structure of oocytes that, somehow, prevent their fertilization. Indeed, there is an almost total correlation between the frequency of oocytes with aberrant morphology (Figure 2e) and the rate of failure in fertilization. On the other hand, the slightly reducing effect exerted on the FR by the treatment of oocytes with ions As3+ and As5+, appears to result from changes occurring in oocytes at the cell membrane level. Almost all the non fertilized oocytes show an unchanged morphology but in the most drastic cases there is a breakage/dissolution of the plasma membrane (Figure 2d) indicating the beginning of the necrosis process and change of DNA organization (Figure 2g). This is also supported by the observation of polyspermy in several eggs (Figure 2c).
In the oocyte toxicity test, the ion Mn2+ also has an intermediate toxic effect. It is more toxic that the As ions and less toxic than Aroclor 1254. The type of changes induced in the oocytes is also mixed, showing morphology and/or characteristics similar to those induced by other pollutants.
Considering the array of results displayed it appears that the order of toxicity for the sperm toxicity test is As3+> As5+ Mn2+> Aroclor 1254 and for the oocyte toxicity test Aroclor 1254> Mn2+> As5+> As3+.
Effect on the egg cleavage dynamics
Observing the alterations of the egg cleavage dynamics shows that the treatment of gametes with different pollutants also produces changes in the sperm and oocytes that remain viable. These changes result in the drastic reduction of capacity and/or quality of the egg division (Graph 2). The effect, similar to that found in the reduction of the fertilization rate, also depends on the types of gametes tested, the pollutant and intensity of treatment. Overall, the changes are more pronounced in eggs derived from treated oocytes. However, all the tested pollutants induce a strong deleterious effect on the quality of sperm. These gametes are particularly sensitive to As3+, noting that the normal division of eggs, fertilized by sperm treated with this metal ion, is strongly committed to about half of the fertilized eggs.
Inhibition of the egg segmentation
In eggs fertilized by sperm treated with the pollutants, during 1h, there is a high frequency of eggs that have not yet started the first division 1h and 3 h after fertilization. This phenomenon was never observed with the control, where it was found that all the fertilized eggs were undergoing division, 1 and 3h after fertilization. The inhibitory effect of the egg cleavage is not very evident in the treatments with the As ions, but is observed in all tested concentrations of PCBs and Mn2+.
The inhibition of the egg cleavage is even higher in the fertilized eggs from oocytes previously exposed to various toxics. The phenomenon is induced by all pollutants, but is less evident in treatments with Mn2+. In the majority of eggs that did not divide that resulted from treatments with PCBs, the analysis of egg morphology (Figure 3b, c) and the chromatin organization (Figure 3f, g) suggests that a process of apoptosis started in most eggs (Figura 3f,g), 3h after fertilization; this was never observed in the control.
In the eggs originating from fertilized oocytes treated with As ions, the analysis of the egg morphology (Figure 3e) and the organisation of chromatin (Figure 3h) suggests the beginning of a necrosis process, 3 h after fertilization.
Anomalies in the egg segmentation
The treatment of gametes with the different pollutants also induces considerable disturbances in the segmentation of the egg.
In the eggs fertilized by sperm exposed to the pollutants, the failure rate of the normal egg segmentation is particularly high. The effect is more pronounced in treatments with As3+ and PCBs (Graph 2). The normal segmentation of the egg is also seriously compromised in eggs that resulted from treated oocytes. The treatment of oocytes with the higher concentrations induces inability of normal segmentation in more than 55 % of the eggs. The anomalies observed in the egg segmentation translate themselves into different levels of depth, depending on the treatment intensity at which the gametes were exposed. At the first and second segmentation, the lower concentrations usually induce the asymmetric division of the blastomeres (Figure 4d, f, g), associated with the reduction of the number of mitosis/embryo and improper chromosome segregation (Figure 4o, p). The higher concentrations, promote the incomplete segmentation of the egg (Figure 4b, c, g).
In eggs in more advanced segmentation stages (16 – 32 cells), the lower concentrations of pollutants also cause asymmetric segmentation (Figure 4j) and, in many embryos, the highest concentrations, trigger apoptosis (Figures 4k) and necrosis (Figure 4l).
The effect on the offspring quality
Table 6 shows the results concerning the quality of the embryos that originated from eggs fertilized by gametes exposed to the various pollutants and the toxicity bioassays in embryos – exposure to toxics post-fertilization. The assessment was made through morphological changes produced in the larvae development (Graph 3). The post-fertilization exposure to the different pollutants drastically reduces the quality of embryos in the pluteus stage. Treatment with the highest concentration of As3+, As5+ and Mn2+ adversely affects all the progeny, inducing the death of 40% of larvae (Graph 3) and deep skeletal deformities in the surviving larvae (Figure 5, b-e).
The extent of this effect depends on the treatment intensity, where a lower mortality rate and substantially lower larvae deformities are found in lower concentrations. In treatments with PCBs the same effects are seen although they are far less drastic.
A property of the metals tested seems to be the ability to inhibit the transition from larvae to the pluteus stage. In most tests, there was a high frequency of embryos on blastula and gastrula stages with pronounced malformations (Figure 5, f-h), which blocked the development before reaching the pluteus stage. This phenomenon is very pronounced with the As ions, less intense with Mn2+ and was not observed with PCBs. This suggests that the As ions have a more active action in the induction of malformations in the gastrulation process.
In a less intense way, the metals also have a negative effect on the quality of the offspring derived from eggs fertilized by gametes treated with these pollutants. The effect is considerably less evident in embryos from eggs fertilized by treated sperm.
For PCBs, no significant differences are observed between the type of changes produced and the frequency of individuals affected by embryo toxicity tests and in larvae originated from gametes treated with this pollutant.
This set of results shows that the embryo toxicity test is not as discriminative as the sperm toxicity test. However, the same trend of toxicity remains: As3+ ≥ As5+ ≥ Mn2+> Aroclor 1254.
Discussion
The echinoderm Paracentrotus lividus is recognized as an excellent bioindicator of contamination of marine ecosystems by heavy metals and organic compounds (Danis et al., 2005). In bioassays using the sea urchin, fertilization and toxicity tests induced in sperm and embryos, are widely widespread. In P. lividus, both are validated to assess the toxicity induced by polluted waters from both estuarine and coastal lagoon ecosystems (Arizzi Novelli et al., 2001; Volpi Ghirardini e Arizzi Novelli, 2001; Volpi Ghirardini et al., 2005), and in the assessment of the toxic effects of the heavy metals As3+, Cd2+, Cr3+, Ni2+, Pb2+, Cu2+, Zn2+ and Hg2+ (Warnau et al., 1996; Arizzi Novelli et al., 2003). However, very few studies on the sea urchin use oocyte toxicity tests (Au et al., 2001) and cytogenetic analysis (Saotome et al., 1999; Schweitzer et al., 1999; Dixon et al., 2002; Saotome e Hayashi, 2003). Thus, this is the first study to be developed in P. lividus that simultaneously involves, toxicity tests induced in sperm, oocytes and embryos and also cytological, embryological and cytogenetic evaluation parameters.
With this work, the toxic effect of Mn2+ is for the first time assessed in P. lividus. The results show that, in general, the P. lividus gametes are slightly sensitive to Mn. However, the sensitivity of oocytes is slightly larger than the sperm, which is demonstrated by the worst performance in the fertilization studies and the dynamics of the first egg divisions. Also the P. lividus larvae are more affected when they originate from oocytes exposed to Mn2+ for 1 h, than when resulting from oocytes fertilized by sperm undergoing the same treatment. The low toxicity of Mn in sperm could be a common feature between other species of se-urchins. In Arbacia punctulata, Young and Nelson (1974) found that the treatment of sperm with different concentrations of Mn2+ did not significantly reduce the sperm mobility, a function that in the sea urchin is directly related to the ability to fertilise (Berdishev et al., 1995; Au et al., 1999). The most obvious toxic manifestation is observed when the embryonic development takes place in the presence of Mn. In concentrations higher than 102mM it has a drastic effect on the quality of the larvae produced, causing a variety of different aberrations providing the potential to create a sensitive test for monitoring programmes. Similar results were seen in Anthocidaris crassipina by Kobayashi and Okamura (2004, 2005), suggesting that the greater toxicity of Mn in the sea urchin is induced at the embryonic development level.
The toxicity comparative study of the two ionic species of As show that the two ions induce similar effects in the sea urchin, but As3+ is considerably more toxic than As5+, confirming the toxicity relationship observed in other organisms (Koh et al., 2005). Regarding the fertilization capability, the P. lividus sperm is more sensitive to exposure of As ions than the oocytes, noting that the sperm treated with As drastically reduces the fertilization rate of non-treated oocytes. This sensitivity of the P. lividus male gametes to As was also observed by Arizzi et al. (2003) where in sperm toxicity assays a similar deleterious effect was observed. The greater sperm sensitivity, relatively to the oocytes, is also mentioned by Au et al. (1999), in which toxicity bioassays with Cd developed in Anthocidaris crassipina showed a worse performance in treated sperm relatively to the treated oocytes, both in fertilization and dynamics of the first egg cleavage tests.
However, our results show that the As is equally toxic to the female gametes when in concentrations above 102mM, inducing alterations in the dynamics of the egg cleavage and considerable damage in the quality of the offspring produced, measured in the pluteus stage. In the eggs fertilized with oocytes treated with As, there was a high percentage of eggs in which the mitosis was inhibited (27.6% and 36.1% at concentrations of 102 mM and 104mM, respectively). This indicator, when associated with the cells characteristic phenotype, which suggests the triggering of necrosis process (fragmentation/dissolution of the cell membrane and degradation of DNA), could be a good test for toxicity bioassays. Even the aberrations produced in the larvae pluteus stage and the high number of larvae with development blocked in stages prior to pre-pluteus, including blastula and gastrula stage with pronounced abnormalities, are a good indicator of toxicity.
The toxicity of As at the sperm level can be seen differently by changing the dynamics of the egg cleavage. Treatment of sperm with 1mM of As3+ is sufficient to induce asymmetrical division in more than 50% of eggs, giving rise to uneven blastomeres, improper chromosome segregation and reduction in the number of mitosis/embryo. This indicator is a very sensitive test with the potential to integrate programmes to monitor pollution in marine ecosystems. However, as noted with Mn, the toxicity of As at the sperm level does not have a significant effect on offspring, which seems to be a common pattern to various heavy metals. A similar effect was observed in another study, in toxicity bioassays of Cu, Ag, Cd and Hg conducted in P. lividus (Warnau et al., 1996).
The embryo toxicity test, considered highly sensitive in the evaluation of toxicity induced by heavy metals (Warnau et al., 1996; Kobayashi e Okamura, 2004, Arizzi Novelli et al., 2001; Volpi Ghirardini et al., 2005), revealed itself hardly effective in the lowest concentration of As3+ (1mM), which confirms the results obtained by Arizzi et al. (2003). However, it is quite effective for higher concentrations of As3+ superior to 102 mM.
For polychlorinated biphenyls (PCBs), it is not known any toxicity study in P. lividus, but recently Danis et al. (2005) showed that this specie is an efficient bioaccumulator of PCBs because it is not sensitive to these compounds. The results of this work show that the mixture of PCBs congeners, Arocolor 1254, is not very toxic to the P. lividus larvae, suggesting that the embryonic development is not a very sensitive indicator to integrate toxicity tests on embryos. Similar results were found in similar studies in Lytechinus pictus and Stongylocentrotus purpuratus (Weisberg et al., 1996, 1997; Schweitzer et al., 1999, 2000) and in Arbacia punctulata (Adams, 1983). Our results show that this indicator only becomes effective in the presence of high concentrations of PCBs (10 mM Aroclor 1254), resulting in a considerable percentage of larvae with skeletal deformities (19%) and death of some larvae (4.8%). However, tests for toxicity developed with gametes treated for 1h with Arocolor 1254 proved to be effective in detecting low concentrations of PCBs (10-5 mM Aroclor 1254), particularly those developed with oocytes. In these assays, with any of the tested concentrations, there were degenerative changes of oocytes, reduction in the fertilization efficiency, formation of aberrant pluteus and death of larvae. These effects, which demonstrate the great sensitivity of these gametes to Aroclor 1254, are identical to those referred by Adams (1988) in an experiment conducted with treated oocytes of A. punctulata, but where the fertilization and the whole embryonic development took place in the presence of Aricolor 1254.
Similarly to what it has been observed in toxicity assays with As, also with PCBs the indicator that proved most effective was the dynamics of the first egg cleavage. In all the tested concentrations, there was asymmetrical division of the egg, improper chromosome segregation, reduction of the number of mitosis/embryo and complete inhibition of mitosis, with consequent lack of segmentation, in a considerable percentage of eggs. These cytological and cytogenetic indicators, associated with the characteristic phenotype of most cells that show signs of apoptosis, like the formation of bubbles in the cell membrane and excessive condensation and/or fragmentation of DNA, may constitute a robust and sensitive test, appropriate for monitoring of these persistent organic pollutants. The introduction of complementary diagnostic tools to detect in a more sensitive way the triggering of apoptosis, as the Comet assay – Single Cell Gel” or the “ Tunel – Terminal dTUP nick end labelling assay, may provide grater strength to the test.
The comparative results of toxicity induced by polychlorinated biphenyls (PCBs) and by the ions As3+, As5+ and Mn2+, arising from the implementation of this model, confirms the effectiveness of some tests in the detection of these pollutants and show that some are quite specific. The relevance of these results can be the possibility to discriminate the bioavailability of some pollutants in projects for biomonitorization of polluted coastal marine ecosystems.
This study shows that for some assays there are evaluation parameters that show great specificity:
Sperm induced toxicity tests
i. Fertilization rate. Suitable for the detection of As, in particular ion As3+. It may allow to discriminate the presence of As relatively to the presence of Mn and the tested PCBs congeners (total 7), which have a considerably more modest performance in this test. Order of observed effectiveness - As3+> As5+> Mn2+> Aroclor 1254.
ii. Alteration in the egg cleavage dynamics. Very sensitive test in detecting As3+ and PCBs. Established order of effectivity - As3+> Aroclor 1254> Mn2+> As5+.
There are parameters that seem to allow to discriminate the presence of PCBs relatively to As3+. The toxicity induced by PCBs in sperm is reflected in a strong inhibition in the egg cleavage. This effect is residual in As. Moreover, the effect produced by As3+ is reflected in a high frequency of eggs with aberrant division, effect substantially lower in treatments with PCBs.
iii. Transmission of damage to the offspring. Test with little sensitivity, but effective for high concentrations of PCBs and Mn2+. Order of effectiveness - Aroclor 1254 > Mn2+ > As5+ > As3+.
Toxicity tests induced in oocytes
i. fertilization rate. Test with little sensitivity, but effective for high concentrations of PCBs. The inhibition of fertilization, due to the aberrant morphology of the oocytes, is a special feature that can discriminate the presence of this compound relatively to metals, in particular the As ions. Order of effectiveness - Aroclor 1254 > Mn2+ > As5+ > As3+.
ii. Alteration of the cleavage dynamics of the egg. Sensitive test in the detection of all toxics. Order of observed effectiveness - Aroclor 1254 ≥ As3+ ≥ As5+ ≥ Mn2+.
The inhibition of the egg segmentation allows to discriminate the presence of PCBs and As ions, relatively to Mn. The presence of cells with a phenotype characteristic of apoptosis (bubbles formation in the cell membrane and condensation / fragmentation of DNA) can discriminate the presence of PCBs relatively to the As ions. The presence of cells with a phenotype suggesting necrosis (fragmentation/dissolution of the cell membrane and degradation of DNA) allows discriminating the presence of As ions relatively to PCBs.
iii. Transmission of damage to the offspring. Test that detects the studied pollutants when in medium and high concentrations. Established order of effectiveness - As3+ ≥ Mn2+ ≥ As5+> Aroclor 1254.
The presence of larvae with development blocked in stages prior to pre-pluteus (blastula and gastrula with pronounced malformations) allows to discriminate the presence of As ions relatively to Mn and PCBs.
Toxicity test in embryos
i. Quality of larvae in the pluteus stage. Sensitive test in the detection of As and Mn. Order of effectiveness - As3+ ≥ As5+ ≥ Mn2+ > Aroclor 1254.
Discriminates the presence of As ions relatively to Mn and PCBs, when considering larvae with blocked development previous to pluteus.
