Environmental Coastal Regions III, C.A. Brebbia, G.R. Rodriguez & E. Perez Martell (Editors) © 2000 WIT Press, www.witpress.com, ISBN 1-85312-827-9 Using bioassays for testing seawater quality in Greece A. Kungolos*, P. Samaras^, E. Koutseris* & G.P. SakellaropouW * Department of Planning and Regional Development and Department of Civil Engineering, University of Thessaly, Volos, Greece ^Chemical Process Engineering Research Institute, Thessaloniki, Greece Abstract The purpose of this study is to show the seawater quality in the Thermaikos Gulf, the Pagassitikos Gulf and Skiathos island in the Northern Aegean Sea. The assessment of coastal water quality presented here is based on two bioassays that use marine organisms as indicators of seawater quality, the invertebrate Artemia franciscana and the marine bioluminescent bacterium Vibrio fischeri. Bioassays are necessary in water pollution evaluations as physical and chemical tests alone are not sufficient to assess potential effects on aquatic organisms. According to our results, there was an improvement in coastal water quality in the Thermaikos Gulf between September 1997 and April- May 2000. In the Pagassitikos Gulf the coastal water quality was generally good in April- May 2000, while in October 1999 it was generally poor. Between the two bioassays that we used in this study, the Microtox test, which uses the marine bacterium Vibrio fischeri as a test organism, was more sensitive in detecting toxicity in seawater. 1 Introduction In Greece, the assessment of seawater quality contaminated with chemicals has been based on chemical analyses and the compliance or not of the measured concentrations with limit concentrations imposed by the legislation. This approach has quite some drawbacks which could be summarised as follows: The cost of a complete chemical analysis is very high, therefore only few chemical tests are performed, investigating the presence and concentration of a limited number of substances. On the other hand, with the number of new chemical substances entering the market increasing every year, one can never be sure that Environmental Coastal Regions III, C.A. Brebbia, G.R. Rodriguez & E. Perez Martell (Editors) © 2000 WIT Press, www.witpress.com, ISBN 1-85312-827-9 402 Environmental Coastal Regions HI the chemical analysis can cover all potentially hazardous chemicals. Finally, chemical tests alone do no give information concerning potential interactions of the chemical substances existing in the seawater. It has been shown, on more than one occasions, that mixtures of chemicals in water present at the level of current water quality criteria have caused severe toxic effects to aquatic organisms [1]. Because of all the above mentioned disadvantages associated with a monitoring strategy of water quality which depends exclusively on physical and chemical parameters, many countries have initiated including biological parameters in freshwater and seawater monitoring [2]. The European Union Commission has made a draft proposal in 1999, suggesting that the water quality criteria should not be only chemical but also ecological. In USA, EPA (Environmental Protection Agency) regulations require the use of an integrated strategy to achieve and maintain water quality standards. This strategy includes chemical specific analysis, biosurveys (organisms in receiving water body) and bioassays [2]. Bioassays do not measure the cause of the pollution, but they measure directly the polluted seawater effect on test organisms. In this study two bioassays have been used, the Anemia test, using the brine shrimp Artemia franciscana as a test organism, and the Microtox test, using the marine bacteria Vibrio fischeri as a test organism. Two coastal areas were investigated. Thermaikos gulf in Northern Greece, where the port city of Thessaloniki is located, and Pagassitikos gulf in Central Greece, where the port city of Volos is located. The Artemia [3,4] and Microtox [5] bioassays have been used extensively in the past for toxicity assessment of substances and mixtures of substances. The literature covering toxicity of environmental samples on these two organisms is more limited [6,7]. The authors of the present study had used Artemia franciscana in 1997 [6] for assessment of coastal water quality in Thessaloniki area. The purpose of this study includes the investigation of the use of marine organism bioassays for testing the ecological quality of coastal water, the comparison of coastal water quality at various time periods and the sensitivity comparison between Artemia and Microtox tests. 2 Materials and methods Coastal water samples were collected in glass containers. Sampling was done in shallow waters from piers or docks. The coastal water samples were transferred to the laboratory within 6 hours from the sampling time. The bioassays were performed either the same day or after 24 hours. In the latter case coastal water samples were maintained at 4 °C. The bioassays Artoxkit M test [3], and Microtox were used in this study. Hatching of Artemia franciscana was done with the help of a NO VITAL 504 incubator. Larvae of the brine shrimp Artemia franciscana were hatched from cysts. Cyst hatching was initiated 48 hours prior to the start of toxicity tests. Three replicates were done for each sample and 10 organisms were transferred into each well. The multiwell plate was put in the incubator at 25±1 °C in Environmental Coastal Regions III, C.A. Brebbia, G.R. Rodriguez & E. Perez Martell (Editors) © 2000 WIT Press, www.witpress.com, ISBN 1-85312-827-9 Environmental Coastal Regions HI 403 darkness for 24 hours for the actual test. The inhibitions caused by the coastal water on the bioluminescence of Vibrio fischeri were measured with a Microtox 500 analyser (Azur Environmental, USA). 3 Results and Discussion A map of Thessaloniki area is shown in Figure 1. Coastal waters samples were taken from 7 different sites, which are also shown in Figure 1. Sampling site A was in Potamos, Epanomi, which is after Epanomi cape, where Thermaikos Gulf Figure 1. Map of Thessaloniki area indicating the sampling sites for coastal water samples. Environmental Coastal Regions III, C.A. Brebbia, G.R. Rodriguez & E. Perez Martell (Editors) © 2000 WIT Press, www.witpress.com, ISBN 1-85312-827-9 404 Environmental Coastal Regions III becomes very wide and opens to the Aegean Sea. Potamos beach is very popular for swimming within Thessaloniki residents. Sampling site two was in the fishing village of Mihaniona, between the capes of Megalo Emvolo and Epanomi. The other 5 sampling sites were in the inner Thermaikos Gulf (Gulf of Thessaloniki). Sampling site C was in Perea and sampling site D in Aretsu, Kalamaria, between the capes of Megalo Emvolo and Micro Emvolo. The other three sampling sites were in the inner Gulf of Thessaloniki (Thessaloniki Bay), in the area which is supposed to be most polluted from anthropogenic activities. Sampling site E was in Analipsi, Thessaloniki in the heart of the east part of the city. Sampling site F was in the port of Thessaloniki. Sampling site G was in Kalohori, close to the industrial area of Thessaloniki. The effect of coastal water from the 7 different sampling sites on the survival of Artemia franciscana is given in Table 1. As it is presentedin this table, toxicity of natural seawater on Artemia franciscana was generally low, with the exception of sampling site F, the port of the city. The authors of the present study had tested the toxicity of coastal water on Artemia franciscana, with samples from the same sampling sites during September 1997 [6]. Table 1. Toxicity assessment of Thermaikos Gulf with Artemia franciscana as test organism. Symbol Sampling site Tests Toxicity range performed (%) A Potamos, Epanomi 2 0 0-10 B Mihaniona 3 C Perea 3 0-20 D Aretsu, Thessaloniki 3 0-5 E Analipsi, Thessaloniki 3 0 F Port, Thessaloniki 3 10-30 G Kalohori 3 0-10 Comparison between the two tests can be seen in Figure 2. Figure 2 shows a clear improvement in coastal water quality between September 1997 and April- May 2000. One possible reason for this improvement is the extension of the treating capacity of Thessaloniki city municipal wastewater treatment plant between 1997 and 2000. The improvement in coastal water quality is very prominent in Analipsi area (sampling site E). This result can be attributed to the fact that domestic effluents are no more discharged untreated to Thessaloniki Bay in that area [6]. Another possible explanation for the improvement of coastal water quality between September 1997 and May 2000 is the fact that rainfall height in Greece is generally high in the period from January to May, but very low from July to October. Therefore, dilution of toxicants is very low in September and potential toxicity of seawater higher. Environmental Coastal Regions III, C.A. Brebbia, G.R. Rodriguez & E. Perez Martell (Editors) © 2000 WIT Press, www.witpress.com, ISBN 1-85312-827-9 Environmental Coastal Regions III 405 100-i Septemberl997 80- April-May 2000 60- 40- H 20- D E F Sampling sites Figure 2: Comparison of coastal water toxicity between September 1997 and April - May 2000 in Thessaloniki area, using Artemia franciscana as test organism (D: Aretsu, E: Analipsi, F: Port, Thessaloniki, G: Kalohori). The effect of coastal water from the 7 different sampling sites on the bioluminescence of Vibrio fischeri is shown in Table 2. These results were deduced by the Microtox 500 analyser. The Microtox test uses a sublethal effect, the inhibition on bioluminescence as an endpoint and it is, therefore, more sensitive than Artemia test. Figure 3 shows the comparison of Artemia franciscana and Vibrio fischeri sensitivity in detecting coastal water toxicity in Thessaloniki area during April - May 2000. It is clear that Microtox test is more Table 2. Toxicity assessment of Thermaikos Gulf with Vibrio fischeri as test organism. Symbol Sampling site Tests Toxicity range performed (%) A Potamos 3 0 B Mihaniona 3 0 C Perea 3 0-10 D Aretsu, Thessaloniki 3 0-10 E Analipsi, Thessaloniki 3 5-10 F Port, Thessaloniki 3 0-40 G Kalohori 3 0 Environmental Coastal Regions III, C.A.