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Sea urchin embryotoxicity test: proposal for a simplified bioassay
ARTICLE IN PRESS
Ecotoxicology and Environmental Safety 57 (2004) 123–128
Sea urchin embryotoxicity test: proposal for a simplified bioassay S. Manzo Environmental Department, ENEA C.R. Portici, Via Vecchio Macello, Portici (NA), I-80055, Italy Received 22 April 2002; received in revised form 18 September 2003; accepted 8 October 2003
Abstract Sea urchin embryotoxicity tests are widely used for evaluating the biological effects of contaminants in marine environments. The currently used traditional and standardized protocols are quite slow and laborious. The present work shows a modified bioassay (new embryotoxicity test; NET) in an attempt to speed up laboratory work using a limited number of fertilized eggs. Several experiments have been conducted both with a traditional bioassay and with the NET, using the same test conditions, in order to evaluate the reliability of the proposed simplified bioassay. Adult Paracentrotus lividus (Lamark) were collected from the Tyrrenian Sea (Bay of Naples) and embryos, reared in filtered seawater, were exposed to increasing potassium dichromate and copper sulfate concentrations. Then the EC50 was calculated. The analysis of the results evidenced good repeatability. The confidence limits in all tests overlapped; moreover, data correlation analysis between the results of both tests showed a high significant accordance (chromium, R2 ¼ 0:93; Po0:01; copper, R2 ¼ 0:86; Po0:05). In conclusion, the NET seems to be a good alternative to the traditional tests; it could be a first step toward a new routine ecotoxicological kit for seawater. r 2003 Elsevier Inc. All rights reserved. Keywords: Heavy metals; Embryotoxicity test; Simplified bioassay; Paracentrotus lividus
1. Introduction The use of sea urchin embryos and gametes in testing developmental, reproductive, and cytogenetic effects of chemicals and complex mixtures has been successfully developed by a number of laboratories worldwide (reviewed by Kobayashi, 1984; Pagano et al., 1986; Dinnel et al., 1988; Bay et al., 1993). It has been demonstrated that this kind of bioassay is rather sensitive to a number of pollutants (Kobayashi, 1984; Pagano et al., 1986; Trieff et al., 1995). In particular, available data indicate that early life stages of Paracentrotus lividus (Lamark) are very sensitive to metals (Brunetti et al., 1991; Warnau and Pagano, 1994; Warnau et al., 1996; Radenac et al., 2001). Moreover, water quality bioassays, by means of sea urchin embryos, have been used to monitor the biological effects of contamination in marine environments (Stebbing, 1985). Since the early findings of Wilson (1951), numerous reports have been published on biomonitoring of coastal waters (e.g., Bougis et al.,
Fax: +390-81-7723344. E-mail address: [email protected].
0147-6513/$ - see front matter r 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2003.10.007
1979; Chapman and Long, 1983; Pagano et al., 1989; Kobayashi, 1991; Torricelli et al., 2002). The available sea urchin embryotoxicity test protocols are generally based on the EPA standard method (US EPA, 1995). The differences among various protocols mainly depend on the sea urchin species used and on laboratory conditions (for P. lividus see Warnau et al., 1996; His et al., 1999; Radenac et al., 2001). Briefly, a sea urchin bioassay involves the exposure of a large number of eggs collected from a stock solution (about 300 eggs/mL) and in the analysis of 100 embryos in development for each replicate. This procedure is quite slow and laborious and can be affected by variability due to subsampling. The increasing interest in the biomonitoring of coastal ecosystems (estuaries, bays, harbors and other nearshore environments) gives rise to the need for developing innovative standardized procedures able to offer an acceptable compromise between reliability of scientific information and cost/time of the analyses. These procedures should at least meet the following criteria: be relatively ‘‘easy and cheap’’ to deploy, offer an acceptable evaluation of the ecosystem conditions, provide readily accessible and useful information and
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S. Manzo / Ecotoxicology and Environmental Safety 57 (2004) 123–128
be widely accepted by scientific and regulatory communities (Wells, 1999). Particularly, routine ecotoxicological monitoring requires simple, inexpensive and rapid procedures suitable as standard protocols by general laboratories (e.g., for the use aboard small research ships with limited space and equipment) (His et al., 1999). The present work introduces some modifications to the traditional procedures, aiming at simplifying and speeding up the laboratory work. In the modified bioassay, here called new embryotoxicity test (NET), a limited number of fertilized eggs (selected by means of a fertilization membrane) is used, in order to make the reading of embryos easier and faster, to evaluate mortality, and to avoid subsampling procedure. The aim of this work is to check the reliability of the proposed simplified sea urchin embryotoxicity test by evaluating its repeatability and comparing the results with those obtained using a traditional bioassay, performed under the same test conditions and with the same gamete pools. In this study embryos of P. lividus are utilized. This sea urchin is abundant in European coastal waters and has been frequently used in biological studies since the end of the past century (Monroy, 1986) and in ecotoxicological investigations (Dinnel et al., 1982; Klockner et al., 1985; Pagano et al., 1989).
2. Materials and methods Adult P. lividus (Lamark) were collected from the Tyrrenian Sea (Bay of Naples). Gametes were harvested and embryos were reared as described in detail by Pagano et al. (1986). Spawning was induced in sea urchins by injection of 1 mL 0.5 M KCl through the perioral membrane. Eggs were collected by placing spawning females separately in individual 250-mL beakers with filtered seawater (FSW; see below); ‘‘dry’’ sperm from each male was collected with an automatic pipette and stored in a sterile tube placed on ice. For each experiment, six individual females were selected for their appropriate egg quality (no immature forms, no debris, no fertilized eggs) and amount. Males were selected for sperm motility (checked under the microscope) and amount. The best three male and three female gametes were pooled and filtered through nylon cheesecloth (f ¼ 200 mm for eggs and 50 mm for sperm). The egg suspension (Stock solution) was diluted to a final concentration of 25–30 eggs/mL. Fertilization was carried out by adding 1 mL pooled sperm, diluted 1:1000 in FSW, to the egg suspension and by incubating at 1871 C for 15 min. Fertilization success in the stock solution was verified by the presence of the fertilization membrane in a random
sample of 100. Excess sperm was removed by decanting zygotes and resuspending them in FSW. The embryos were reared in filtered (0.45 mm) natural seawater (salinity 36%) at 1871 C, and then exposed to increasing concentrations of potassium dichromate and copper sulfate (nominal concentration range 2.9– 24 mg/L for potassium dichromate and 0.1–0.4 mg/L for copper sulfate). Experimental seawater and negative (blank) controls were provided by natural FSW collected from the same pristine site as the echinoids. Echinoids and seawater were always collected early in the morning of the starting day of a given experiment. Each experiment was run in at least three replicates, using six-well plates. Each toxicity test was specifically designed (following ‘‘range finding’’ tests) considering a toxicant concentration range allowing the calculation of EC50 (50% effect concentration) when the response data were combined with the measured toxicant data. Each embryo test response was adjusted for the control response using the Abbott formula (Finney, 1971). Between January and June, 13 tests for potassium dichromate and 5 tests for copper sulfate were conducted with the traditional method and the NET bioassay. 2.1. Conventional embryotoxicity test (CET) A volume corresponding to 250–300 fertilized eggs was transferred from the stock solution to test chambers (10 mL) containing toxicant solutions. For each treatment three replicates were prepared. The eggs were incubated at 1871 C for 48–50 h. After this period, 100 mL of 4% buffered formalin was added to each vessel and developmental abnormalities were observed by direct observation of 100 randomly chosen individuals. 2.2. NET bioassay Fertilized eggs were exposed to increasing toxicant concentrations in two steps. 1. Intermediate transfer of random fertilized eggs from stock solutions to rinse wells containing test toxicant concentrations. 2. Final transfer of 10 fertilized eggs (selected by means of a fertilization membrane) from rinse wells to test wells, each containing 5 mL of test solution (three replicates for each concentration). The intermediate transfer of fertilized eggs through rinsing wells minimizes the dilution in the test wells and allows for the simultaneous exposure of all the eggs to the toxicant solution. The selection of fertilized eggs was carried out under a stereomicroscope using a micropipette with a large opening (about 2 mm) to prevent
ARTICLE IN PRESS S. Manzo / Ecotoxicology and Environmental Safety 57 (2004) 123–128
injuries to the eggs. After a 48–50 h incubation period (1871 C), 100 mL of buffered formalin was added and normally developed plutei were counted. Test acceptability criteria for both methods were as follows:
3. Results Tables 1 and 2 show the EC50, NOEC, and %MSD for each test conducted with chromium and copper, respectively, for both NET and CET. The NET shows a %MSDo20 in 78% of cases, when chromium was used as a toxicant, and in all cases when copper was used. The %MSD varies between 45.6 and 10.3 with chromium and between 14.6 and 8.1 with copper. It is important to emphasize that NET and CET showed %MSD420 in exactly the same experiments. Generally, NET values are higher probably due to the lower number of embryos employed in the bioassay. The NOEC values for each NET experiment vary from 2.9 to 0.8 mg/L for chromium and from 60 to 40 mg/L for copper (depending on the steps in the different experiments) and are similar to the values found with CET. The EC50 (795% confidence limits) for chromium (Fig. 1A) and copper (Fig. 1B) indicates a good reproducibility for NET: the confidence limits overlap
percentage normally developed embryos in the control (blank) X80% and minimum significant difference (%MSD)p20 relative to the control.
*
*
125
2.3. Statistical analysis EC50 was calculated with probit analysis using response and toxicant concentration data for all the solutions; 95% confidence limits were also calculated. NOEC (no-observed-effect concentration) was calculated with Dunnett’s procedure and %MSD was calculated according to the US EPA (1995). The comparison between the two tests was carried out in terms of correlation analysis.
Table 1 EC50a (95% confidence limits), NOECb, and %MSDc in NET and CET (chromium) Test
Ad B Cd D E F G Hd I L M N O a
NET
CET
EC50 (mg/L as Cr)
NOEC (mg/L as Cr)
MSD %
EC50 (mg/L as Cr)
NOEC (mg/L as Cr)
MSD %
3.50 (2.53–5.04) 3.23 (3.11–3.47) 4.11 (2.89–4.26) 4.10 (3.66–4.94) 4.32 (4.24–4.39) 3.83 (3.36–4.13) 3.86 (3.78–4.00) 3.62 (3.08–3.99) 4.06 (3.49–4.31) 4.50 (4.15–4.77) 4.62 (4.39–4.77) 4.01 (3.49–4.36) 4.27 (3.97–4.47)
3.38
45
3.30
40
2.39
16
3.85
9
3.78
38
2.31
5
2.95
19
3.77
10
2.78
19
0.85
3
1.40
19
2.31
9
1.79
15
2.31
15
1.40
24
2.91
39
1.40
17
1.40
4
0.84
17
0.83
8
1.40
12
1.40
8
0.78
13
0.78
9
0.78
10
2.19 (2.11–4.74) 3.93 (3.85–3.96) 4.65 (4.58–4.73) 4.42 (4.17–4.61) 4.50 (4.44–4.54) 4.14 (4.07–4.21) 4.17 (3.88–4.35) 3.89 (3.47–5.18) 4.39 (4.14–4.52) 4.55 (4.31–4.79) 4.58 (4.02–5.14) 4.27 (4.15–4.39) 4.47 (4.40–4.55)
0.78
10
50% effect concentration. No effect concentration. c Minimun significant difference percentage. d Not used in calculation; see text. b
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Table 2 EC50a (95% confidence limits), NOEC,b and %MSDc in NET and CET (copper) Test
NET
A B C D E
CET
EC50 (mg/L as Cu)
NOEC (mg/L as Cu)
MSD %
EC50 (mg/L as Cu)
NOEC (mg/L as Cu)
MSD %
80 (79–86) 80 (76–84) 84 (80–88) 88 (79–96) 92 (88–92)
56
14
52
10
48
14
48
6
52
8
52
8
60
13
40
5
40
11
80 (76–156) 84 (84–88) 88 (84–92) 88 (84–88) 96 (92–96)
60
6
a
50% effect concentration. No effect concentration. c Minimun significant difference percentage. b
5.0
the corresponding CET EC50 mean values were 4.270.6 mg/L and 8776 mg/L. The correlation data analysis between CET and NET for potassium dichromate (only for the tests with %MSDo20) (Fig. 2A) shows a correlation of 93.2% with a P value much smaller than 0.01. The same statistical analysis using copper sulfate (Fig. 2B) as toxicant shows a correlation of about 86% and a P value of 0.019. Probably, the lower correlation value obtained with copper is due to the limited number of tests.
Cromium (mg/l)
4.5
4.0
3.5
3.0
2.5
2.0 O
N
M
L
I
G
F
E
D
B
(A)
4. Discussion
NET Test
100
The main objectives of this study can be stated as follows:
Copper (µg/l)
90
1. to assess the feasibility of a simpler and faster sea urchin embryotoxicity test and 2. to compare the sensitivity and repeatability of the new test with respect to the conventional method.
80
70
60
50 E
D
C
B
A
(B)
NET Test
Fig. 1. EC50 (error bars, 95% confidence limits) in NET for (A) chromium and (B) copper. Straight line=avg. EC50 dotted line=uncertainty range.
in all cases and all the values are within the uncertainty range (72 SD). NET EC50 mean values 7SD are 470.4 mg/L and 8575 mg/L for chromium and copper, respectively, and
The toxicity effects in both tests were studied under strictly identical experimental conditions to avoid the variability of responses due to different experimental parameters; this allows us to minimize the ‘‘high background noise’’ (Klo¨ckner et al., 1985). The MSD is a measure of the statistical sensitivity and it is dependent upon the test design and decreases with decreasing test variability (US EPA, 2000). The 20% limiting value is largely utilized as an ecotoxicological test acceptability criterion (US EPA, 2000); consequently it has been adopted for both NETs and CETs. The results obtained with NET show a good repeatability. NET MSD percentage is lower than 20% in the majority of tests and shows generally a decreasing trend in time due to improved operator skill.
ARTICLE IN PRESS
Chromium CET Test (mg/l)
S. Manzo / Ecotoxicology and Environmental Safety 57 (2004) 123–128
127
4.8
Table 3 Summary of some EC50a values for copper and chromium in P. lividus
4.6
Toxicant
EC50
Reference
4.4
Cu
72 mg/L 48–63 mg/L 50–100 mg/L 50–100 mg/L 80–92 mg/L 29–46 mg/L
Fernandez et al. (2001) Warnau et al. (1996) Kobayashi (1981) Radenac et al. (2001) Present work His et al. (1999)
Cr
0.52–5.20 mg/L 3.5–4.62 mg/L
Pagano et al. (1983) Present work
4.2
y= 0.65x + 1.7 4.0
2
R = 0.93 p < 0.0001
3.8
3.6 a
3.6
3.8
4.0
(A)
4.2
4.4
4.6
4.8
Copper CET Test (µg/l)
96
92
88
y = 1.05x - 0.02 84
2
R = 0.86 p < 0.02 80
80
(B)
50% effect concentration.
Chromium NET Test (mg/l)
84
88
92
96
Copper NET test (µg/l)
Fig. 2. Correlation analysis (dotted lines 95% confidence limits) between NET and traditional test for (A) chromium and (B) copper.
The EC50 values do not show significant variations: the confidence limits in all tests overlap and all the values fall within the uncertainty range (72 SD). Moreover, the NOEC values in experiments with the same concentration range are more or less the same. The EC50 values obtained with NET significantly correlate with the values obtained with CET, even if their 95% confidence limits are generally higher. Furthermore, EC50s determined in the present study are in good agreement with existing data on embryotoxicity of Cr and Cu in P. lividus obtained with the traditional bioassay (see Table 3). The conventional embryotoxicity method also shows a larger number of tests in which %MSD is lower than 20%, and the values are always lower than those found in the corresponding NETs. This is due to the use of a limited number of fertilized eggs which increases statistical variability. Preliminary experiments have been started in order to evaluate the optimal number of exposed embryos to be employed (S. Manzo, unpublished data) to minimize the test variability without losing the NET advantages. The experimental errors involved in NET and CET are, however, comparable,
due to the embryos’ heterogeneity and to the effective exposed embryo number. In fact, from one chamber to another there is a variability in the percentage of exposed embryos (due to the random sampling from the stock solution), and there is a variability in the counting of normal pluteus larvae due to the subsampling in each chamber test. A clear advantage of NET sea urchin embryotoxicity assay is that it simplifies and speeds up routine ecotoxicological monitoring. Fertilized eggs, selected at the beginning of the test, produce, after 48 h in the control chamber, healthy plutei at a satisfying rate. Further manipulation of fertilized eggs in NET does not produce any damage, but allows one to expose a limited number of fertilized eggs and makes the final counting of dead and living organisms and the evaluation of effects easier and faster.
5. Conclusion The classical sea urchin bioassay methodology is based on the measurements of a certain number of eggs whose fertilization ratio is not known in advance. NET uses fertilized eggs of P. lividus as a biological tool for assessing toxicity. This experimental procedure should make the analytical procedure easier, cheaper and less time consuming. Our results show that NET seems to be a good alternative to traditional methods considering the good test repeatability and the significant correlation of EC50 values between the two procedures. Moreover, NET could include mortality as a test endpoint because it involves a total count at the beginning and at the end of the test. The relatively lower sensitivity of NET, due to the limited number of exposed embryos, seems to be the only disadvantage, but it is largely within the test acceptability limit. Cryopreservation of fertilized eggs could be a useful tool to improve test speed and availability of test embryos all year around.
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Further investigation, using other toxicants and natural samples, is necessary to conclusively establish the feasibility of NET method.
Acknowledgments I gratefully thank Dr. C. Cremisini for critical review of the manuscript and fruitful suggestions and P. Sansone and co-workers at the Zoological Station of Naples for providing echinoids.
References Bay, S., Burgess, R., Nacci, D., 1993. Status and applications of Echinoid (Phylum Echinodermata) toxicity test methods. In: Landis, W.G., Hughes, J.S., Lewis, M.A. (Eds.), Environmental Toxicology Risk Assessment. ASTM STP 1179, Philadelphia, pp. 281–302. Bougis, P., Corre, M.C., Etienne, M., 1979. Sea urchin as a tool for assessment of the quality of sea water. Ann. Inst. Oceanogr. Paris 55, 21–26. Brunetti, R., Marin, M., Bressan, M., Zordan, M., Soggia, A., 1991. Effects of the chelating agent nitrilotriacetic acid (NTA) on the toxicity of metals (Cd, Cu, Zn and Pb) in the sea urchin Paracentrotus lividus Lmk. Vie Milieu 4, 39–43. Chapman, P.M., Long, E.R., 1983. The use of bioassays as part of a comprehensive approach to marine pollution assessment. Mar. Pollut. Bull. 14, 81–84. Dinnel, P.A., Pagano, G., Ossida, P.S., 1988. A sea urchin test system for marine environmental monitoring. In: Burke, R.D., Mladenov, P.V., Lambert, P., Parsley, R.L. (Eds.), Echinoderm Biology. Balkema, Rotterdam, pp. 611–619. Dinnel, P.A., Strober, Q.J., Crumley, C., Nakatani, R.E., 1982. Development of a sperm cell toxicity test for marine waters. In: Pearson, J.G., Foster, R.B., Bishop, W.E. (Eds.), Aquatic toxicology and hazard Assessment: Fifth Conference. ASTM STP 766, American Society of Testing and Materials, Philadelphia, pp. 82–98. Fernandez, N., Beiras, R., 2001. Combined Toxicity of dissolved Mercury with Copper, lead and Cadmium on Embryogenesis and Early Larval Growth of the Paracentrotus lividus Sea-Urchin. Ecotoxicology 10, 263–271. Finney, D.J., 1971. Probit Analysis. Cambridge University Press, London, 333pp. His, E., Heyvang, I., Geffard, O., De Mountadouin, X., 1999. A comparison between oyster (Crassostrea gigas) and sea urchin (Paracentrotus lividus) larval bioassay for toxicological studies. Water Res. 7, 1706–1718. Klockner, K., Roshental, H., Willfuhr, J., 1985. Invertebrate bioassay with North Sea water samples: structural effects on embryos and larvae of serpulids oyster and sea urchins. Helgol. Meeresunters. 39, 1–19. Kobayashi, N., 1981. Comparative toxicity of various chemicals, oil extracts and oil dispersant extracts to Canadian and Japanese sea urchin eggs. Publ. Seto Mar. Biol. Lab. 26, 123–133.
Kobayashi, N., 1984. Marine ecotoxicological testing with echinoderms. In: Persoone, G., Jaspers, E., Claus, C. (Eds.), Ecotoxicological Testing for Marine Environment. State University of Ghent and Institute for Marine Scientific Research, Bredene, Belgium, pp. 341–405. Kobayashi, N., 1991. Marine pollution bioassay by using sea urchin eggs in the Tanabe Bay, Wakayama Prefecture, Japan, 1970–1987. Mar. Pollut. Bull. 23, 709–713. Monroy, A., 1986. A centennial debt of developmental biology to the sea urchin. Biol. Bull. Mar. Biol. Lab. 33, 330–337. Pagano, G., Cipollaro, M., Corsale, G., Esposito, A., Ragucci, E., Giordano, G.G., Trief, N.M., 1986. The sea urchin: bioassay for the assessment damage from environmental contaminants. In: Cairns Jr., J. (Ed.), Community Toxicity Testing. American Society for Testing and Materials, Philadelphia, pp. 67–92. Pagano, G., Corsale, G., Esposito, A., Dinnel, P.A., Romana, L.A., 1989. Use of sea urchin sperm and embryo bioassay in testing the sublethal toxicity of realistic pollutant level. Adv. Appl. Biotech. Ser. 5, 153–163. Pagano, G., Esposito, A., Bove, P., de Angelis, M., Rota, A., Giordano, G.G., 1983. The effects of hexavalent and trivalent chromium on fertilization and development in sea urchin. Environ. Res. 30, 442–452. Radenac, G., Fichet, D., Miramand, P., 2001. Bioaccumulation and toxicity of four dissolved metals in Paracentrotus lividus sea-urchin embryo. Mar. Environ. Res. 51, 151–166. Stebbing, A.R.D., 1985. Bioassay. In: Bayne, B.L., Brown, D.A., Burns, K., Dixon, D.R., Ivanovici, A.D., Livingstone, D.R., Lowe, D.M., Moore, M.N., Stebbing, A.R.S., Widdows, J. (Eds.), The Effects of Stress and Pollution on Marine Animals. Praeger, New York, pp. 133–137. Torricelli, L., Manzo, S., Accornero, A., Manfra, L., 2002. Biomonitoring of marine waters by use of microalgal tests: results from the Campania coastal zone (South Tyrrehenian Sea). Fresen. Environ. Bull. Lett. 11, 691–696. Trieff, N.M., Roman˜a, L.A., Esposito, A., Oral, R., Quiniou, F., Iaccarino, M., Alcock, N., Ramanujam, V.M.S., Pagano, G., 1995. Effluent from bauxite factory induces developmental and reproductive damage in sea urchins. Arch. Environ. Contam. Toxicol. 28, 173–177. US EPA, 1995. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to West Coast marine and estuarine organisms. EPA 600R95136, pp. 324–366. US EPA, 2000. Method guidance and recommendations for whole effluent toxicity (WET) testing (40 CFR Part 136). EPA 821B00004. Warnau, M., Pagano, G., 1994. Developmental toxicity of PbCl2 in the echinoid Paracentrotus lividus (Echinodermata). Bull. Environ. Contam. Toxicol. 53, 434–441. Warnau, M., Iaccarino, M., De Biase, A., Tempra, A., Jangoux, M., Dubois, P., Pagano, G., 1996. Spermiotoxicity and embryotoxicity of heavy metals in the echinoid Paracentrotus lividus. Environ. Toxicol. Chem. 15, 1931–1936. Wells, P.G., 1999. Monitoring the health of coastal marine ecosystems—the roles and challenges of microscale toxicity tests. Mar. Pollut. Bull. 39, 39–47. Wilson, D.P., 1951. A biological difference between natural waters. J. Mar. Biol. Assoc. UK 30, 1–21.
Ecotoxicology and Environmental Safety 57 (2004) 123–128
Sea urchin embryotoxicity test: proposal for a simplified bioassay S. Manzo Environmental Department, ENEA C.R. Portici, Via Vecchio Macello, Portici (NA), I-80055, Italy Received 22 April 2002; received in revised form 18 September 2003; accepted 8 October 2003
Abstract Sea urchin embryotoxicity tests are widely used for evaluating the biological effects of contaminants in marine environments. The currently used traditional and standardized protocols are quite slow and laborious. The present work shows a modified bioassay (new embryotoxicity test; NET) in an attempt to speed up laboratory work using a limited number of fertilized eggs. Several experiments have been conducted both with a traditional bioassay and with the NET, using the same test conditions, in order to evaluate the reliability of the proposed simplified bioassay. Adult Paracentrotus lividus (Lamark) were collected from the Tyrrenian Sea (Bay of Naples) and embryos, reared in filtered seawater, were exposed to increasing potassium dichromate and copper sulfate concentrations. Then the EC50 was calculated. The analysis of the results evidenced good repeatability. The confidence limits in all tests overlapped; moreover, data correlation analysis between the results of both tests showed a high significant accordance (chromium, R2 ¼ 0:93; Po0:01; copper, R2 ¼ 0:86; Po0:05). In conclusion, the NET seems to be a good alternative to the traditional tests; it could be a first step toward a new routine ecotoxicological kit for seawater. r 2003 Elsevier Inc. All rights reserved. Keywords: Heavy metals; Embryotoxicity test; Simplified bioassay; Paracentrotus lividus
1. Introduction The use of sea urchin embryos and gametes in testing developmental, reproductive, and cytogenetic effects of chemicals and complex mixtures has been successfully developed by a number of laboratories worldwide (reviewed by Kobayashi, 1984; Pagano et al., 1986; Dinnel et al., 1988; Bay et al., 1993). It has been demonstrated that this kind of bioassay is rather sensitive to a number of pollutants (Kobayashi, 1984; Pagano et al., 1986; Trieff et al., 1995). In particular, available data indicate that early life stages of Paracentrotus lividus (Lamark) are very sensitive to metals (Brunetti et al., 1991; Warnau and Pagano, 1994; Warnau et al., 1996; Radenac et al., 2001). Moreover, water quality bioassays, by means of sea urchin embryos, have been used to monitor the biological effects of contamination in marine environments (Stebbing, 1985). Since the early findings of Wilson (1951), numerous reports have been published on biomonitoring of coastal waters (e.g., Bougis et al.,
Fax: +390-81-7723344. E-mail address: [email protected].
0147-6513/$ - see front matter r 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2003.10.007
1979; Chapman and Long, 1983; Pagano et al., 1989; Kobayashi, 1991; Torricelli et al., 2002). The available sea urchin embryotoxicity test protocols are generally based on the EPA standard method (US EPA, 1995). The differences among various protocols mainly depend on the sea urchin species used and on laboratory conditions (for P. lividus see Warnau et al., 1996; His et al., 1999; Radenac et al., 2001). Briefly, a sea urchin bioassay involves the exposure of a large number of eggs collected from a stock solution (about 300 eggs/mL) and in the analysis of 100 embryos in development for each replicate. This procedure is quite slow and laborious and can be affected by variability due to subsampling. The increasing interest in the biomonitoring of coastal ecosystems (estuaries, bays, harbors and other nearshore environments) gives rise to the need for developing innovative standardized procedures able to offer an acceptable compromise between reliability of scientific information and cost/time of the analyses. These procedures should at least meet the following criteria: be relatively ‘‘easy and cheap’’ to deploy, offer an acceptable evaluation of the ecosystem conditions, provide readily accessible and useful information and
ARTICLE IN PRESS 124
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be widely accepted by scientific and regulatory communities (Wells, 1999). Particularly, routine ecotoxicological monitoring requires simple, inexpensive and rapid procedures suitable as standard protocols by general laboratories (e.g., for the use aboard small research ships with limited space and equipment) (His et al., 1999). The present work introduces some modifications to the traditional procedures, aiming at simplifying and speeding up the laboratory work. In the modified bioassay, here called new embryotoxicity test (NET), a limited number of fertilized eggs (selected by means of a fertilization membrane) is used, in order to make the reading of embryos easier and faster, to evaluate mortality, and to avoid subsampling procedure. The aim of this work is to check the reliability of the proposed simplified sea urchin embryotoxicity test by evaluating its repeatability and comparing the results with those obtained using a traditional bioassay, performed under the same test conditions and with the same gamete pools. In this study embryos of P. lividus are utilized. This sea urchin is abundant in European coastal waters and has been frequently used in biological studies since the end of the past century (Monroy, 1986) and in ecotoxicological investigations (Dinnel et al., 1982; Klockner et al., 1985; Pagano et al., 1989).
2. Materials and methods Adult P. lividus (Lamark) were collected from the Tyrrenian Sea (Bay of Naples). Gametes were harvested and embryos were reared as described in detail by Pagano et al. (1986). Spawning was induced in sea urchins by injection of 1 mL 0.5 M KCl through the perioral membrane. Eggs were collected by placing spawning females separately in individual 250-mL beakers with filtered seawater (FSW; see below); ‘‘dry’’ sperm from each male was collected with an automatic pipette and stored in a sterile tube placed on ice. For each experiment, six individual females were selected for their appropriate egg quality (no immature forms, no debris, no fertilized eggs) and amount. Males were selected for sperm motility (checked under the microscope) and amount. The best three male and three female gametes were pooled and filtered through nylon cheesecloth (f ¼ 200 mm for eggs and 50 mm for sperm). The egg suspension (Stock solution) was diluted to a final concentration of 25–30 eggs/mL. Fertilization was carried out by adding 1 mL pooled sperm, diluted 1:1000 in FSW, to the egg suspension and by incubating at 1871 C for 15 min. Fertilization success in the stock solution was verified by the presence of the fertilization membrane in a random
sample of 100. Excess sperm was removed by decanting zygotes and resuspending them in FSW. The embryos were reared in filtered (0.45 mm) natural seawater (salinity 36%) at 1871 C, and then exposed to increasing concentrations of potassium dichromate and copper sulfate (nominal concentration range 2.9– 24 mg/L for potassium dichromate and 0.1–0.4 mg/L for copper sulfate). Experimental seawater and negative (blank) controls were provided by natural FSW collected from the same pristine site as the echinoids. Echinoids and seawater were always collected early in the morning of the starting day of a given experiment. Each experiment was run in at least three replicates, using six-well plates. Each toxicity test was specifically designed (following ‘‘range finding’’ tests) considering a toxicant concentration range allowing the calculation of EC50 (50% effect concentration) when the response data were combined with the measured toxicant data. Each embryo test response was adjusted for the control response using the Abbott formula (Finney, 1971). Between January and June, 13 tests for potassium dichromate and 5 tests for copper sulfate were conducted with the traditional method and the NET bioassay. 2.1. Conventional embryotoxicity test (CET) A volume corresponding to 250–300 fertilized eggs was transferred from the stock solution to test chambers (10 mL) containing toxicant solutions. For each treatment three replicates were prepared. The eggs were incubated at 1871 C for 48–50 h. After this period, 100 mL of 4% buffered formalin was added to each vessel and developmental abnormalities were observed by direct observation of 100 randomly chosen individuals. 2.2. NET bioassay Fertilized eggs were exposed to increasing toxicant concentrations in two steps. 1. Intermediate transfer of random fertilized eggs from stock solutions to rinse wells containing test toxicant concentrations. 2. Final transfer of 10 fertilized eggs (selected by means of a fertilization membrane) from rinse wells to test wells, each containing 5 mL of test solution (three replicates for each concentration). The intermediate transfer of fertilized eggs through rinsing wells minimizes the dilution in the test wells and allows for the simultaneous exposure of all the eggs to the toxicant solution. The selection of fertilized eggs was carried out under a stereomicroscope using a micropipette with a large opening (about 2 mm) to prevent
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injuries to the eggs. After a 48–50 h incubation period (1871 C), 100 mL of buffered formalin was added and normally developed plutei were counted. Test acceptability criteria for both methods were as follows:
3. Results Tables 1 and 2 show the EC50, NOEC, and %MSD for each test conducted with chromium and copper, respectively, for both NET and CET. The NET shows a %MSDo20 in 78% of cases, when chromium was used as a toxicant, and in all cases when copper was used. The %MSD varies between 45.6 and 10.3 with chromium and between 14.6 and 8.1 with copper. It is important to emphasize that NET and CET showed %MSD420 in exactly the same experiments. Generally, NET values are higher probably due to the lower number of embryos employed in the bioassay. The NOEC values for each NET experiment vary from 2.9 to 0.8 mg/L for chromium and from 60 to 40 mg/L for copper (depending on the steps in the different experiments) and are similar to the values found with CET. The EC50 (795% confidence limits) for chromium (Fig. 1A) and copper (Fig. 1B) indicates a good reproducibility for NET: the confidence limits overlap
percentage normally developed embryos in the control (blank) X80% and minimum significant difference (%MSD)p20 relative to the control.
*
*
125
2.3. Statistical analysis EC50 was calculated with probit analysis using response and toxicant concentration data for all the solutions; 95% confidence limits were also calculated. NOEC (no-observed-effect concentration) was calculated with Dunnett’s procedure and %MSD was calculated according to the US EPA (1995). The comparison between the two tests was carried out in terms of correlation analysis.
Table 1 EC50a (95% confidence limits), NOECb, and %MSDc in NET and CET (chromium) Test
Ad B Cd D E F G Hd I L M N O a
NET
CET
EC50 (mg/L as Cr)
NOEC (mg/L as Cr)
MSD %
EC50 (mg/L as Cr)
NOEC (mg/L as Cr)
MSD %
3.50 (2.53–5.04) 3.23 (3.11–3.47) 4.11 (2.89–4.26) 4.10 (3.66–4.94) 4.32 (4.24–4.39) 3.83 (3.36–4.13) 3.86 (3.78–4.00) 3.62 (3.08–3.99) 4.06 (3.49–4.31) 4.50 (4.15–4.77) 4.62 (4.39–4.77) 4.01 (3.49–4.36) 4.27 (3.97–4.47)
3.38
45
3.30
40
2.39
16
3.85
9
3.78
38
2.31
5
2.95
19
3.77
10
2.78
19
0.85
3
1.40
19
2.31
9
1.79
15
2.31
15
1.40
24
2.91
39
1.40
17
1.40
4
0.84
17
0.83
8
1.40
12
1.40
8
0.78
13
0.78
9
0.78
10
2.19 (2.11–4.74) 3.93 (3.85–3.96) 4.65 (4.58–4.73) 4.42 (4.17–4.61) 4.50 (4.44–4.54) 4.14 (4.07–4.21) 4.17 (3.88–4.35) 3.89 (3.47–5.18) 4.39 (4.14–4.52) 4.55 (4.31–4.79) 4.58 (4.02–5.14) 4.27 (4.15–4.39) 4.47 (4.40–4.55)
0.78
10
50% effect concentration. No effect concentration. c Minimun significant difference percentage. d Not used in calculation; see text. b
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126
Table 2 EC50a (95% confidence limits), NOEC,b and %MSDc in NET and CET (copper) Test
NET
A B C D E
CET
EC50 (mg/L as Cu)
NOEC (mg/L as Cu)
MSD %
EC50 (mg/L as Cu)
NOEC (mg/L as Cu)
MSD %
80 (79–86) 80 (76–84) 84 (80–88) 88 (79–96) 92 (88–92)
56
14
52
10
48
14
48
6
52
8
52
8
60
13
40
5
40
11
80 (76–156) 84 (84–88) 88 (84–92) 88 (84–88) 96 (92–96)
60
6
a
50% effect concentration. No effect concentration. c Minimun significant difference percentage. b
5.0
the corresponding CET EC50 mean values were 4.270.6 mg/L and 8776 mg/L. The correlation data analysis between CET and NET for potassium dichromate (only for the tests with %MSDo20) (Fig. 2A) shows a correlation of 93.2% with a P value much smaller than 0.01. The same statistical analysis using copper sulfate (Fig. 2B) as toxicant shows a correlation of about 86% and a P value of 0.019. Probably, the lower correlation value obtained with copper is due to the limited number of tests.
Cromium (mg/l)
4.5
4.0
3.5
3.0
2.5
2.0 O
N
M
L
I
G
F
E
D
B
(A)
4. Discussion
NET Test
100
The main objectives of this study can be stated as follows:
Copper (µg/l)
90
1. to assess the feasibility of a simpler and faster sea urchin embryotoxicity test and 2. to compare the sensitivity and repeatability of the new test with respect to the conventional method.
80
70
60
50 E
D
C
B
A
(B)
NET Test
Fig. 1. EC50 (error bars, 95% confidence limits) in NET for (A) chromium and (B) copper. Straight line=avg. EC50 dotted line=uncertainty range.
in all cases and all the values are within the uncertainty range (72 SD). NET EC50 mean values 7SD are 470.4 mg/L and 8575 mg/L for chromium and copper, respectively, and
The toxicity effects in both tests were studied under strictly identical experimental conditions to avoid the variability of responses due to different experimental parameters; this allows us to minimize the ‘‘high background noise’’ (Klo¨ckner et al., 1985). The MSD is a measure of the statistical sensitivity and it is dependent upon the test design and decreases with decreasing test variability (US EPA, 2000). The 20% limiting value is largely utilized as an ecotoxicological test acceptability criterion (US EPA, 2000); consequently it has been adopted for both NETs and CETs. The results obtained with NET show a good repeatability. NET MSD percentage is lower than 20% in the majority of tests and shows generally a decreasing trend in time due to improved operator skill.
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Chromium CET Test (mg/l)
S. Manzo / Ecotoxicology and Environmental Safety 57 (2004) 123–128
127
4.8
Table 3 Summary of some EC50a values for copper and chromium in P. lividus
4.6
Toxicant
EC50
Reference
4.4
Cu
72 mg/L 48–63 mg/L 50–100 mg/L 50–100 mg/L 80–92 mg/L 29–46 mg/L
Fernandez et al. (2001) Warnau et al. (1996) Kobayashi (1981) Radenac et al. (2001) Present work His et al. (1999)
Cr
0.52–5.20 mg/L 3.5–4.62 mg/L
Pagano et al. (1983) Present work
4.2
y= 0.65x + 1.7 4.0
2
R = 0.93 p < 0.0001
3.8
3.6 a
3.6
3.8
4.0
(A)
4.2
4.4
4.6
4.8
Copper CET Test (µg/l)
96
92
88
y = 1.05x - 0.02 84
2
R = 0.86 p < 0.02 80
80
(B)
50% effect concentration.
Chromium NET Test (mg/l)
84
88
92
96
Copper NET test (µg/l)
Fig. 2. Correlation analysis (dotted lines 95% confidence limits) between NET and traditional test for (A) chromium and (B) copper.
The EC50 values do not show significant variations: the confidence limits in all tests overlap and all the values fall within the uncertainty range (72 SD). Moreover, the NOEC values in experiments with the same concentration range are more or less the same. The EC50 values obtained with NET significantly correlate with the values obtained with CET, even if their 95% confidence limits are generally higher. Furthermore, EC50s determined in the present study are in good agreement with existing data on embryotoxicity of Cr and Cu in P. lividus obtained with the traditional bioassay (see Table 3). The conventional embryotoxicity method also shows a larger number of tests in which %MSD is lower than 20%, and the values are always lower than those found in the corresponding NETs. This is due to the use of a limited number of fertilized eggs which increases statistical variability. Preliminary experiments have been started in order to evaluate the optimal number of exposed embryos to be employed (S. Manzo, unpublished data) to minimize the test variability without losing the NET advantages. The experimental errors involved in NET and CET are, however, comparable,
due to the embryos’ heterogeneity and to the effective exposed embryo number. In fact, from one chamber to another there is a variability in the percentage of exposed embryos (due to the random sampling from the stock solution), and there is a variability in the counting of normal pluteus larvae due to the subsampling in each chamber test. A clear advantage of NET sea urchin embryotoxicity assay is that it simplifies and speeds up routine ecotoxicological monitoring. Fertilized eggs, selected at the beginning of the test, produce, after 48 h in the control chamber, healthy plutei at a satisfying rate. Further manipulation of fertilized eggs in NET does not produce any damage, but allows one to expose a limited number of fertilized eggs and makes the final counting of dead and living organisms and the evaluation of effects easier and faster.
5. Conclusion The classical sea urchin bioassay methodology is based on the measurements of a certain number of eggs whose fertilization ratio is not known in advance. NET uses fertilized eggs of P. lividus as a biological tool for assessing toxicity. This experimental procedure should make the analytical procedure easier, cheaper and less time consuming. Our results show that NET seems to be a good alternative to traditional methods considering the good test repeatability and the significant correlation of EC50 values between the two procedures. Moreover, NET could include mortality as a test endpoint because it involves a total count at the beginning and at the end of the test. The relatively lower sensitivity of NET, due to the limited number of exposed embryos, seems to be the only disadvantage, but it is largely within the test acceptability limit. Cryopreservation of fertilized eggs could be a useful tool to improve test speed and availability of test embryos all year around.
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Further investigation, using other toxicants and natural samples, is necessary to conclusively establish the feasibility of NET method.
Acknowledgments I gratefully thank Dr. C. Cremisini for critical review of the manuscript and fruitful suggestions and P. Sansone and co-workers at the Zoological Station of Naples for providing echinoids.
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