Using Bioassays for Testing Seawater Quality in Greece A. Kungolos, P. Samaras^, E. Koutseris & G.P. Sakellaropouw * Depar

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

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 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.

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 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

406 Environmental Coastal Regions HI sensitive than Anemia test. Artemia franciscana has been found to be an organism tolerant to certain toxicants [8]. In addition, Artemia test is a very useful test for seawater toxicity assessment, because it is a simple and it can be performed with the naked eye, without need for expensive analysers. Microtox test is a reliable test for detecting seawater toxicity [5]. Still, when detecting seawater toxicity with Vibrio fischeri, one should be careful to use standard of same salinity with the sample to be tested. The salinity in our coastal water samples was generally high, between 38 and 40 ppt. Figure 4 shows the 13 sampling sites in Magnesia prefecture in Central Greece. Two sampling sites A and B were in Skiathos island in the open Aegean Sea. Nine sampling sites were in Pagassitikos Gulf. Sampling sites L and M are in a small bay in the opening of Pagassitikos Gulf. All these beaches are used for swimming, with the exception of sampling site F which is the port of Volos. Among these 13 sampling sites, only sites E (Agria) and F (Volos Port) are close to industrial areas. The other sampling sites are affected only by municipal sewage and effluents from foodstuff processing small units, mainly olive oil mills. There are more than 40 operating olive oil mills in Magnesia prefecture. Their season for operation is usually between October and January.

100-.

80- Artemia

Vibrio fischeri * 60- £

* 40-1 H

D E

Sampling sites

Figure 3: Comparison of Artemia franciscana and Vibrio fischeri sensitivity in detecting coastal water toxicity in Thessaloniki area during April - May 2000 (C: Perea, D: Aretsu, E: Analipsi, F: Port, Thessaloniki).

Environmental Coastal Regions III 407

. j*i \®»X!' "* " " - ' "* ^^V**5SS? ^ ^^//^KA^ '-^!|i?^^ * f^^ *\ * ww, &*«•**,A:» ''***" H?*^" *?\"- ^^%St^'**'^ «« •• ^ I

, *" MKu^tiW^''' "WW*^"""™ SAW ^*->J -' V " " "J'*V*'^ ^^ )?^*O ^ tSO\, -**1"»t -•»•*''"- " < **-

Figure 4. Map of Volos area indicating the sampling sites for coastal water samples.

It can be seen in Table 3 that in October 1999 coastal water toxicity was generally higher than one would expect in areas which are not much affected by heavy industries. On the other hand in May 2000, the coastal water toxicity was generally very low, in most cases it was zero. Figure 5 shows the comparison of coastal water toxicity between October 1999 and May 2000 in Magnesia prefecture coastal areas. It is clear that coastal water toxicity was generally low in May 2000, but relatively high in October 1999. It was also observed in Thessaloniki area (see Figure 2) that toxicity was lower in spring than in early autumn. One possible explanation could be the fact that October comes after a long dry season, therefore toxic pollutants are not so much diluted. On the other hand, the operation start-up of many olive mills in October could have affected the coastal water toxicity. Another possible

408 Environmental Coastal Regions HI

Table 3. Toxicity assessment of Pagassitikos Gulf with Artemia franciscana as test organism.

Symbol Sampling site May 1999 October 1999 May 2000 A Skiathos -_ 10-50 0-20 B Koukounaries 10-50 0 C Kala Nera - 20 30 _ D Kato Gatzea 0 0 E Agria - 20-40 0 F Port, Volos 0-5 20 0 G Alykes beach 0-5 10 0-10

H Aghialos - 40 0 - I Almyros beach _ 0-20 0-30 J Amaliapoli 0-10 0-10 - K Sourpi beach 0 0 L Rodia beach - 0 0 M Lihoura beach - 10-40 0

explanation is the fact that a lot of tourists use to visit these beaches in the season from June to September, increasing the amount of untreated sewage discharging to the sea, since there is no municipal wastewater treatment plant in the tourist beaches of Magnesia prefecture.

lOO-i

October 1999

May 2000

B E F G Sampling sites

Figure 5: Comparison of coastal water toxicity between October 1999 and May 2000 in Volos area, using Artemia franciscana as test organism (A: Skiathos, B: Koukounaries, E: Agria, F: Port, Volos, G: Alykes, H: Aghialos, J: Amaliapoli).

Environmental Coastal Regions III 409

4 Conclusions

Bioassays can be a very useful tool in assessing the coastal water quality. In this study, two bioassays, Artemia test and Microtox test were used for assessing the coastal water quality from 7 different sites in Thessaloniki area and 13 different sites in Magnesia prefecture. It was found that there was a clear improvement in coastal water quality in Thessaloniki between September 1997 and April- May 2000. Microtox test was found to be more sensitive than Artemia test in detecting seawater toxicity. In addition, due to the fact that no single marine organism is sensitive to all potential toxicants, it is essential that a battery of bioassays should be used for a proper assessment of seawater quality. Coastal water quality was generally very good in Magnesia prefecture in May 2000. On the other hand, it was not so good in October 1999. It is suggested that there should be more tests in future with bioassays covering the whole year and that bioassays should be combined with chemical tests for a proper assessment of seawater quality.

Acknowledgement

The authors of this study express their gratitude to Sewerage Authority of

Thessaloniki S.A. and Prefecture of Magnesia Development Company S.A. for financial support on this study.

References

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Using Bioassays for Testing Seawater Quality in Greece A. Kungolos*, P. Samaras^, E. Koutseris* & G.P. Sakellaropouw * Depar

Using Bioassays for Testing Seawater Quality in Greece A. Kungolos, P. Samaras^, E. Koutseris & G.P. Sakellaropouw * Depar