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Sea-urchin Embryo Bioassay for in situ Evaluation of the Biological Quality of Coastal Seawater
Estuarine, Coastal and Shelf Science (2001) 52, 29–32 doi:10.1006/ecss.2000.0720, available online at http://www.idealibrary.com on
Sea-urchin Embryo Bioassay for in situ Evaluation of the Biological Quality of Coastal Seawater R. Beirasa, E. Va´zquez, J. Bellas, J. I. Lorenzo, N. Ferna´ndez, G. Macho, J. C. Marin˜o and L. Casas Departamento de Ecoloxı´a e Bioloxı´a Animal, University of Vigo, Galicia, Spain Received 6 July 2000, and accepted in revised form 17 September 2000 The Paracentrotus lividus sea-urchin embryo bioassay, consisting of incubation of fertilized eggs in test water and measurement of the percentage of four-armed plutei larvae developed after the incubation period (2–3 days), has been adapted for in situ evaluation of seawater quality in coastal areas. Mature sea-urchins are dissected in situ and fertilization is performed in the field; fertilized eggs are delivered into screw lid 50-ml cylinders with 20 m nylon mesh in both ends filled with sieved local seawater. The cylinders, tied to 60-cm ropes with weights on one end and buoys in the other one, are placed by scuba divers in the test sites at subtidal level and recovered after the incubation period. The contents of each cylinder are then transferred into a vial, fixed with formalin and observed directly under an inverted microscope to record the percentage (N=100) and size (length, N=25) of four-arm pluteus larvae. Our results show that the bioassay can discriminate between well known polluted and unpolluted sites, but further improvement is needed in order to: (1) take into account differences of temperature between sites; (2) minimize larval mortality due to reasons other than pollution. 2001 Academic Press
Keywords: bioassay; water quality; Paracentrotus lividus; embryo; larva
Introduction Estuaries are often highly productive ecosystems affected by urban development and industrial activities that strongly increase background levels of potentially harmful chemical and physical agents. The ever increasing number of xenobiotics and the effects of physicochemical parameters on their availability to marine organisms greatly complicates monitoring based on chemical analyses. Even if we had a priori knowledge of the kind of pollutants present, analytical chemistry allows determination of the degree and nature of pollution, but it does not provide evidence for biological consequences. Bioassays allow the detection of these effects by measuring biological responses on marine organisms, and particularly in their highly sensitive early life stages (His et al., 1999). Due to the ease of obtaining gametes and in vitro fertilization, sea-urchins have been used world-wide to provide biological material for embryo-larval bioassays to assess marine samples (e.g. Bougis et al., 1979; Kobayashi, 1981; Pagano et al., 1986; Dinnel & Stober, 1987; Vashchenko & Zhadan, 1993; Carr et al., 1996). Procedures for obtaining, conservation and manipulation of samples intended for bioassays a Corresponding author: Tel: +34-986812648; E-mail: rbeiras@ uvigo.es
0272–7714/01/010029+04 $35.00/0
are highly heterogeneous and can greatly affect the results of the test. Methodological standardization remains a bottleneck for the spread of routine biological assays in monitoring programmes. Performing the bioassay in place avoids the variability stemming from sample conservation and manipulation, and may thus help to obtain more consistent and comparable results. In situ bioassays should however follow simple and inexpensive methods in order to be suitable for routine use in biological monitoring. Materials and methods Keeping these aims in mind, in September 1998 and 1999 we conducted preliminary in situ bioassays at two selected sites in the Rı´a de Pontevedra (Galicia, NW Spain). One site in the inner part of the Rı´a (P3), was chosen to represent a polluted place on the basis of our own previous chemical and toxicological data, and another in the outer part of the Rı´a (P4), was chosen as a control. Location and general environmental characteristics of the sites are given in Table 1. Bioassay procedure Gametes were obtained by dissection from a single couple of adults. Eggs were transferred into a 2001 Academic Press
30 R. Beiras et al. T 1. Temperature (T), salinity (S), pH, dissolved oxygen, biological oxygen demand (BOD5, 95% confidence interval) and phosphate concentration (SD) in the seawater from the inner (Marı´n) and outer (Tulla) sites studied
Marı´n (P3) 4224 25 N 841 17 W Tulla (P4) 4220 20 N 848 55 W
21/7/98 16/9/98 7/9/99 14/9/99 21/7/98 16/9/98 7/9/99 14/9/99
T (C)
S
pH
O2 (mg l 1)
BOD5 (mg l 1)
PO4 (M)
17·0 16·5 20·1 18·0 17·4 19·4 19·8 17·0
32·9 32·7 33·6 33·7 34·4 33·1 34·1 34·1
7·6 7·7 8·0 8·1 7·6 7·8 8·0 8·1
8·5 5·3 8·0 8·7 13·5 7·5 8·6 9·3
7·030·627 6·960·566 n.m. n.m. 2·350·20 3·110·453 n.m. n.m.
7·01·02 1·40·44 0·560·110 0·660·180 0·240·030 0·150·007 0·210·025 0·200·030
n.m.=not measured.
Degree-days The biological responses recorded in this bioassay, embryogenesis and early larval growth, both depend on temperature. We chose 72 h as the incubation time in order to avoid interference on embryogenesis success, since this time is sufficient to achieve pluteus stage even at the lowest temperatures recorded at the sites studied. However the second biological response, early larval growth, must be expressed taking into account thermal differences among the sites studied. With this aim we calculated the larval growth rates per degree-day, following the most common degree-day model (Young & Young, 1998), where: DD=[(MaxMin)/2]To
where DD are the degree-days accumulated during 24 h, Max and Min are the daily maximum and minimum temperatures, and To is the temperature threshold for larval development, i.e. the temperature corresponding to 0 growth rate. In order to calculate To, an additional experiment was performed incubating P. lividus fertilized eggs at 14, 16, 18 and 20 C for 72 h following the methods described by Ferna´ ndez (1999). The results (Figure 1) showed a linear dependence of growth, measured
160 y = 11.283x – 76.333 R2 = 0.9647 120
Growth rate
measuring cylinder containing artificial seawater (see below). A few l of dry sperm were collected directly from the gonad with a Pasteur pipette, added to the egg suspension and carefully stirred to allow fertilization. Four samples of 20 l were taken and fixed with formalin for later checking of fertilization success (assessed by the percentage of eggs showing a fertilization membrane) and egg density. Fertilized eggs were delivered into 50 ml cages filled with 20 m-filtered fresh seawater from the study site. The cages (16 per site in 1998 and 10 per site in 1999) were tied to leaded ropes and placed under water at 2 m depth during low tide by divers. After 72 h the cages were recovered and the contents retained by the 20 m mesh bottom transferred into vials and fixed with formalin for later observation under an inverted microscope in order to record in each vial the percentage of 4-arm pluteus larvae developed (N=100) and the larval length (N=25), defined as the maximum dimension parallel to the postoral arm.
80
GR (µm/d) GR (µm/DD)
40 y = 0.0051x – 11.204
0 10
15 T (°C)
20
F 1. Growth rate (GR, recorded as length increase) obtained in laboratory after 72 h incubation of sea-urchin fertilized eggs at different temperatures. Notice that GR becomes temperature-independent when expressed on a degree-day (DD) basis. Diamonds: GK (m day 1); squares: GR (m DD 1).
In situ bioassays with sea-urchin embryos 31 T 2. Mean percentage of pluteus larvae and larval length (L) obtained after 72 h in situ incubation of fertilized sea-urchin eggs in cages placed at 2 m depth in two locations in the inner (Marı´n) and outer (Tulla) part of the Rı´a de Pontevedra (Galicia, NW Spain). One hundred individuals per cage were observed to record percentages, and 25 were used to record lengths; N=number of cages recovered. GR=growth rate, DD=degree days September 1998 % larvae
L (m)
Marı´n (P3) 95·33·38 15114·1*** (N=8) (N=8) Tulla (P4) 97·61·58 33041 (N=9) (N=9)
September 1999
GR (m DD
1
1·90·59 6·81·29
) % larvae
L (m)
0 — (N=10) 938·1 30071 (N=7) (N=7)
GR (m DD 1) — 4·82·04
***Significantly smaller than P4 (P<0·001).
as length increase (GR, m day 1), on temperature (T, C), following the equation: GR=11·3T76·3 (R2 =0·97). From this equation To was calculated as 6·8 C. Results and discussion After 72 h incubation of the fertilized eggs in the clean waters from the control site (Tulla) >90% of the embryos developed into pluteus larvae approximately 300 m long. In both the 1998 and 1999 experiments the bioassay detected impaired embryo development at the inner site (Marı´n). The first year percentage of larvae is not significantly different from the control site but larvae are markedly smaller (P<0·001). Average water temperature during the three days was higher in Tulla (17·9 C) than in Marı´n (16·3 C). When these differences are taken into account and growth (length increase) rates (GR) are expressed on a degree-day (DD) basis the values at the control site are again much higher than the inner site. Mean GR in Tulla was 6·81·28 m DD 1 and in Marı´n 1·90·55, (P<0·001). In the next year the toxicity at Marı´n was higher and no larvae were obtained, with embryogenesis arrested at blastula stage or occasionally even at morula stage. The bioassay was therefore suitable for qualitative assessment of the seawater and it also provided a measure of the degree of pollution, making a distinction between the moderate toxicity found in 1998 and the high toxicity found in 1999. The main advantages of this in situ bioassay are: (1) it avoids the interference caused by sampling, storage and manipulation of the samples; and (2) it allows evaluation of the seawater quality with no need of obtaining an elutriate from the sediments to
be used as a surrogate to assess the impact on the organisms living in the water column. The main limitation, in turn, is that it requires field operations that can not be delayed, such as recovery and fixation of the cage contents after 72 h and, particularly, fertilization (and thus mature biological material) just prior to the placement of the cages. In summary, this short-term in situ bioassay requires a minimum of field material and technical skills and provides enough sensitivity for direct assessment of seawater quality, avoiding the false positives and false negatives associated with sample collection, handling and storage of environmental samples intended for laboratory bioassays. These advances, unfortunately, do not affect the problem of obtaining suitable and homogeneous biological material all year round, which still limits the incorporation of embryo-larval bioassays as a routine technique in monitoring programmes. Acknowledgements We thank L. Pombar and Z. Romero for their helpful technical assistance. This study was partially funded by Xunta de Galicia (XUGA 30116A96) and CICYT (AMB99-0946). References Bougis, P., Corre, M. C. & E u tienne, M. 1979 Sea-urchin larvae as a tool for assessment of the quality of sea water. Annales de l’Institut Oceanographique Paris 55, 21–26. Carr, R. S. 1996 Sediment quality assessment studies of Tampa Bay, Florida. Environmental Toxicology and Chemistry 15, 1218– 1231. Dinnel, P. A. & Stober, Q. J. 1987 Application of the sea urchin sperm bioassay to sewage treatment efficiency and toxicity in marine waters. Marine Environmental Research 21, 121–133. Ferna´ ndez, N. 1999 Toxicidad del mercurio, cobre, cadmio y plomo sobre la embrioge´nesis del erizo de mar (Paracentrotus lividus). MSc Thesis. University of Vigo.
32 R. Beiras et al. His, E., Beiras, R. & Seaman, M. N. L. 1999 The assessment of marine pollution- Bioassays with bivalve embryos and larvae. In Advances in Marine Biology (Southward, A. I., Tyler, P. A. & Young, C. M., eds). Academic Press, London, vol. 37, pp. 1– 178. Kobayashi, N. 1981 Comparative toxicity of various chemicals, oil extracts and oil dispersant extracts to canadian and japanese sea urchin eggs. Publications of the Seto. Marine Biological Laboratory XXVI, 123–133. Pagano, M., Cipollaro, M., Corsale, G., Esposito, A., Ragucci, E., Giordano, G. G. & Trieff, N. M. 1986 The sea urchin bioassay for
the assessment of damage from environmental contaminants. Community Toxicity Testing, ASTM STP 920 (John Cairns, Jr., ed.). American Society for Testing and Materials, Philadelphia. Vashchenko, M. A. & Zhadan, P. M. 1993 Bioassays of bottom sediments of Peter Velikiy Gulf (Sea of Japan) with sexual cells, embryos and larvae of the sea urchin. Oceanology 33, 102–104. Young, L. J. & Young, J. H. 1998 Statistical Ecology. Kluwer Academic Publishers, Boston, 565 pp.
Sea-urchin Embryo Bioassay for in situ Evaluation of the Biological Quality of Coastal Seawater R. Beirasa, E. Va´zquez, J. Bellas, J. I. Lorenzo, N. Ferna´ndez, G. Macho, J. C. Marin˜o and L. Casas Departamento de Ecoloxı´a e Bioloxı´a Animal, University of Vigo, Galicia, Spain Received 6 July 2000, and accepted in revised form 17 September 2000 The Paracentrotus lividus sea-urchin embryo bioassay, consisting of incubation of fertilized eggs in test water and measurement of the percentage of four-armed plutei larvae developed after the incubation period (2–3 days), has been adapted for in situ evaluation of seawater quality in coastal areas. Mature sea-urchins are dissected in situ and fertilization is performed in the field; fertilized eggs are delivered into screw lid 50-ml cylinders with 20 m nylon mesh in both ends filled with sieved local seawater. The cylinders, tied to 60-cm ropes with weights on one end and buoys in the other one, are placed by scuba divers in the test sites at subtidal level and recovered after the incubation period. The contents of each cylinder are then transferred into a vial, fixed with formalin and observed directly under an inverted microscope to record the percentage (N=100) and size (length, N=25) of four-arm pluteus larvae. Our results show that the bioassay can discriminate between well known polluted and unpolluted sites, but further improvement is needed in order to: (1) take into account differences of temperature between sites; (2) minimize larval mortality due to reasons other than pollution. 2001 Academic Press
Keywords: bioassay; water quality; Paracentrotus lividus; embryo; larva
Introduction Estuaries are often highly productive ecosystems affected by urban development and industrial activities that strongly increase background levels of potentially harmful chemical and physical agents. The ever increasing number of xenobiotics and the effects of physicochemical parameters on their availability to marine organisms greatly complicates monitoring based on chemical analyses. Even if we had a priori knowledge of the kind of pollutants present, analytical chemistry allows determination of the degree and nature of pollution, but it does not provide evidence for biological consequences. Bioassays allow the detection of these effects by measuring biological responses on marine organisms, and particularly in their highly sensitive early life stages (His et al., 1999). Due to the ease of obtaining gametes and in vitro fertilization, sea-urchins have been used world-wide to provide biological material for embryo-larval bioassays to assess marine samples (e.g. Bougis et al., 1979; Kobayashi, 1981; Pagano et al., 1986; Dinnel & Stober, 1987; Vashchenko & Zhadan, 1993; Carr et al., 1996). Procedures for obtaining, conservation and manipulation of samples intended for bioassays a Corresponding author: Tel: +34-986812648; E-mail: rbeiras@ uvigo.es
0272–7714/01/010029+04 $35.00/0
are highly heterogeneous and can greatly affect the results of the test. Methodological standardization remains a bottleneck for the spread of routine biological assays in monitoring programmes. Performing the bioassay in place avoids the variability stemming from sample conservation and manipulation, and may thus help to obtain more consistent and comparable results. In situ bioassays should however follow simple and inexpensive methods in order to be suitable for routine use in biological monitoring. Materials and methods Keeping these aims in mind, in September 1998 and 1999 we conducted preliminary in situ bioassays at two selected sites in the Rı´a de Pontevedra (Galicia, NW Spain). One site in the inner part of the Rı´a (P3), was chosen to represent a polluted place on the basis of our own previous chemical and toxicological data, and another in the outer part of the Rı´a (P4), was chosen as a control. Location and general environmental characteristics of the sites are given in Table 1. Bioassay procedure Gametes were obtained by dissection from a single couple of adults. Eggs were transferred into a 2001 Academic Press
30 R. Beiras et al. T 1. Temperature (T), salinity (S), pH, dissolved oxygen, biological oxygen demand (BOD5, 95% confidence interval) and phosphate concentration (SD) in the seawater from the inner (Marı´n) and outer (Tulla) sites studied
Marı´n (P3) 4224 25 N 841 17 W Tulla (P4) 4220 20 N 848 55 W
21/7/98 16/9/98 7/9/99 14/9/99 21/7/98 16/9/98 7/9/99 14/9/99
T (C)
S
pH
O2 (mg l 1)
BOD5 (mg l 1)
PO4 (M)
17·0 16·5 20·1 18·0 17·4 19·4 19·8 17·0
32·9 32·7 33·6 33·7 34·4 33·1 34·1 34·1
7·6 7·7 8·0 8·1 7·6 7·8 8·0 8·1
8·5 5·3 8·0 8·7 13·5 7·5 8·6 9·3
7·030·627 6·960·566 n.m. n.m. 2·350·20 3·110·453 n.m. n.m.
7·01·02 1·40·44 0·560·110 0·660·180 0·240·030 0·150·007 0·210·025 0·200·030
n.m.=not measured.
Degree-days The biological responses recorded in this bioassay, embryogenesis and early larval growth, both depend on temperature. We chose 72 h as the incubation time in order to avoid interference on embryogenesis success, since this time is sufficient to achieve pluteus stage even at the lowest temperatures recorded at the sites studied. However the second biological response, early larval growth, must be expressed taking into account thermal differences among the sites studied. With this aim we calculated the larval growth rates per degree-day, following the most common degree-day model (Young & Young, 1998), where: DD=[(MaxMin)/2]To
where DD are the degree-days accumulated during 24 h, Max and Min are the daily maximum and minimum temperatures, and To is the temperature threshold for larval development, i.e. the temperature corresponding to 0 growth rate. In order to calculate To, an additional experiment was performed incubating P. lividus fertilized eggs at 14, 16, 18 and 20 C for 72 h following the methods described by Ferna´ ndez (1999). The results (Figure 1) showed a linear dependence of growth, measured
160 y = 11.283x – 76.333 R2 = 0.9647 120
Growth rate
measuring cylinder containing artificial seawater (see below). A few l of dry sperm were collected directly from the gonad with a Pasteur pipette, added to the egg suspension and carefully stirred to allow fertilization. Four samples of 20 l were taken and fixed with formalin for later checking of fertilization success (assessed by the percentage of eggs showing a fertilization membrane) and egg density. Fertilized eggs were delivered into 50 ml cages filled with 20 m-filtered fresh seawater from the study site. The cages (16 per site in 1998 and 10 per site in 1999) were tied to leaded ropes and placed under water at 2 m depth during low tide by divers. After 72 h the cages were recovered and the contents retained by the 20 m mesh bottom transferred into vials and fixed with formalin for later observation under an inverted microscope in order to record in each vial the percentage of 4-arm pluteus larvae developed (N=100) and the larval length (N=25), defined as the maximum dimension parallel to the postoral arm.
80
GR (µm/d) GR (µm/DD)
40 y = 0.0051x – 11.204
0 10
15 T (°C)
20
F 1. Growth rate (GR, recorded as length increase) obtained in laboratory after 72 h incubation of sea-urchin fertilized eggs at different temperatures. Notice that GR becomes temperature-independent when expressed on a degree-day (DD) basis. Diamonds: GK (m day 1); squares: GR (m DD 1).
In situ bioassays with sea-urchin embryos 31 T 2. Mean percentage of pluteus larvae and larval length (L) obtained after 72 h in situ incubation of fertilized sea-urchin eggs in cages placed at 2 m depth in two locations in the inner (Marı´n) and outer (Tulla) part of the Rı´a de Pontevedra (Galicia, NW Spain). One hundred individuals per cage were observed to record percentages, and 25 were used to record lengths; N=number of cages recovered. GR=growth rate, DD=degree days September 1998 % larvae
L (m)
Marı´n (P3) 95·33·38 15114·1*** (N=8) (N=8) Tulla (P4) 97·61·58 33041 (N=9) (N=9)
September 1999
GR (m DD
1
1·90·59 6·81·29
) % larvae
L (m)
0 — (N=10) 938·1 30071 (N=7) (N=7)
GR (m DD 1) — 4·82·04
***Significantly smaller than P4 (P<0·001).
as length increase (GR, m day 1), on temperature (T, C), following the equation: GR=11·3T76·3 (R2 =0·97). From this equation To was calculated as 6·8 C. Results and discussion After 72 h incubation of the fertilized eggs in the clean waters from the control site (Tulla) >90% of the embryos developed into pluteus larvae approximately 300 m long. In both the 1998 and 1999 experiments the bioassay detected impaired embryo development at the inner site (Marı´n). The first year percentage of larvae is not significantly different from the control site but larvae are markedly smaller (P<0·001). Average water temperature during the three days was higher in Tulla (17·9 C) than in Marı´n (16·3 C). When these differences are taken into account and growth (length increase) rates (GR) are expressed on a degree-day (DD) basis the values at the control site are again much higher than the inner site. Mean GR in Tulla was 6·81·28 m DD 1 and in Marı´n 1·90·55, (P<0·001). In the next year the toxicity at Marı´n was higher and no larvae were obtained, with embryogenesis arrested at blastula stage or occasionally even at morula stage. The bioassay was therefore suitable for qualitative assessment of the seawater and it also provided a measure of the degree of pollution, making a distinction between the moderate toxicity found in 1998 and the high toxicity found in 1999. The main advantages of this in situ bioassay are: (1) it avoids the interference caused by sampling, storage and manipulation of the samples; and (2) it allows evaluation of the seawater quality with no need of obtaining an elutriate from the sediments to
be used as a surrogate to assess the impact on the organisms living in the water column. The main limitation, in turn, is that it requires field operations that can not be delayed, such as recovery and fixation of the cage contents after 72 h and, particularly, fertilization (and thus mature biological material) just prior to the placement of the cages. In summary, this short-term in situ bioassay requires a minimum of field material and technical skills and provides enough sensitivity for direct assessment of seawater quality, avoiding the false positives and false negatives associated with sample collection, handling and storage of environmental samples intended for laboratory bioassays. These advances, unfortunately, do not affect the problem of obtaining suitable and homogeneous biological material all year round, which still limits the incorporation of embryo-larval bioassays as a routine technique in monitoring programmes. Acknowledgements We thank L. Pombar and Z. Romero for their helpful technical assistance. This study was partially funded by Xunta de Galicia (XUGA 30116A96) and CICYT (AMB99-0946). References Bougis, P., Corre, M. C. & E u tienne, M. 1979 Sea-urchin larvae as a tool for assessment of the quality of sea water. Annales de l’Institut Oceanographique Paris 55, 21–26. Carr, R. S. 1996 Sediment quality assessment studies of Tampa Bay, Florida. Environmental Toxicology and Chemistry 15, 1218– 1231. Dinnel, P. A. & Stober, Q. J. 1987 Application of the sea urchin sperm bioassay to sewage treatment efficiency and toxicity in marine waters. Marine Environmental Research 21, 121–133. Ferna´ ndez, N. 1999 Toxicidad del mercurio, cobre, cadmio y plomo sobre la embrioge´nesis del erizo de mar (Paracentrotus lividus). MSc Thesis. University of Vigo.
32 R. Beiras et al. His, E., Beiras, R. & Seaman, M. N. L. 1999 The assessment of marine pollution- Bioassays with bivalve embryos and larvae. In Advances in Marine Biology (Southward, A. I., Tyler, P. A. & Young, C. M., eds). Academic Press, London, vol. 37, pp. 1– 178. Kobayashi, N. 1981 Comparative toxicity of various chemicals, oil extracts and oil dispersant extracts to canadian and japanese sea urchin eggs. Publications of the Seto. Marine Biological Laboratory XXVI, 123–133. Pagano, M., Cipollaro, M., Corsale, G., Esposito, A., Ragucci, E., Giordano, G. G. & Trieff, N. M. 1986 The sea urchin bioassay for
the assessment of damage from environmental contaminants. Community Toxicity Testing, ASTM STP 920 (John Cairns, Jr., ed.). American Society for Testing and Materials, Philadelphia. Vashchenko, M. A. & Zhadan, P. M. 1993 Bioassays of bottom sediments of Peter Velikiy Gulf (Sea of Japan) with sexual cells, embryos and larvae of the sea urchin. Oceanology 33, 102–104. Young, L. J. & Young, J. H. 1998 Statistical Ecology. Kluwer Academic Publishers, Boston, 565 pp.