Toxic effects of the antifouling agent irgarol 1051 on periphyton communities in coastal water microcosms

Pergamon

0025-326X(95)00223-5

Marine Pollution Bulletin, Vol. 32, No. 4, pp. 342-350, 1996 Copyright © 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0025-326X/96 $15.00 + 0.00

Toxic Effects of the Antifouling Agent Irgarol 1051 on Periphyton Communities in Coastal Water Microcosms BJORN DAHL and HANS BLANCK*

G6teborg University, Department of Plant Physiology, Botanical Institute, Carl Skottsbergs gata 22, S-413 19 Gfteborg, Sweden * A u t h o r to w h o m c o r r e s p o n d e n c e s h o u l d b e a d d r e s s e d .

In the late 1980s, a number of countries restricted the use of tri-n-butyltin (TBT) as an active ingredient in antifoufing paint for small boats, including pleasure craft. Irgarol 1051--2-(tert-butylamino)-4-(cyclopropylamino)-6-(methylthio)-l,3,5-triazine, belonging to the s-triazine group of herbicides--is now used in combination with copper in several antifouling products. Irgarol 1051 contamination of the marine environment was studied close to a marina on the west coast of Sweden. Highest concentrations (1.6 riM, 0.4 ltg 1-1) were observed during the peak of the boating season. To investigate the potential for toxic effects, marine periphyton communities were established on artificial substrata in 22-fitre flowthrough aquaria and exposed to Irgarol 1051 during a 3week period in April 1994. The algicide was continuously added at concentrations ranging from 0.06 to 260 riM. Irgarol 1051 was found to significantly (p <0.05) inhibit periphyton photosynthetic activity at 3.2 nM in short-term (hour) tests. Long-term (weeks) exposure produced effects at even lower concentrations. A significant change in community structure was found at 1 riM, which produced a shift towards tolerant species. Photosynthetic activity and algal biomass (chlorophyll a content) was significantly lowered at concentrations of 1 and 4 nM, respectively, indicating only minor functional redundancy in the communities. The most sensitive long-term effects were detected at 0.25-1 nM (0.063-0.25 lXg 1-1) of Irgarol 1051, which is within the concentration range detected in the contaminated areas around the marina. It can be concluded that the present use of Irgaroi 1051 is likely to damage microalgal communities in contaminated coastal waters. Copyright © 1996 Elsevier Science Ltd

Antifouling agents, which are used in paints to prevent biofouling of submerged surfaces in the sea, have been identified as a group of contaminants giving undesirable effects in the marine environment. In the 1980s, tri-nbutyltin (TBT) was found to severely damage nontarget populations of, for example, bivalves and 342

gastropods in the sea at extremely low concentrations (Bryan & Gibbs, 1991). The use of TBT in antifouling paint has since been restricted in a number of countries, and new formulations have been introduced on to the market. Copper compounds have largely replaced TBT as the active agent, often in combination with algicides such as Irgarol 1051 (2-(tert-butylamino)-4(cyclopropylamino)-6-(methylthio)-1,3,5-triazine). Irgarol 1051 belongs to the s-triazine group known as photosystem-II (PSII) inhibitors, with the inhibition of photosynthetic electron transport in chloroplasts as their biochemical mode of action (Moreland, 1980; Mets & Thiel, 1989; Holt, 1993). To the present authors' knowledge, there is no published information in the open literature about the toxicity of Irgarol 1051 to aquatic organisms. However, the producers have reported no-observed-effect concentrations (NOECs) for algal growth inhibition in the range of <0.28-2.6 nM with ECso values in the range of 1.8-19 nu (CibaGeigy, 1985; KemI, 1992). Irgarol 1051 is much less toxic to animals due to its specific mode of action. It is obvious that an algicide in antifouling paint should be toxic to marine algae on the treated surfaces. However, there will be a dissipation of Irgarol 1051 from the surface to the surrounding waters, with subsequent exposure of non-target algae. It is the environmental hazard to these organisms that is of concern. Irgarol 1051 contamination in the marine environment has recently been reported from the C6te d'Azur (Readman et al., 1993) and the southern coast of the UK (Gough et al., 1994). Concentrations of 0.0080.75 nM (0.002-0.19 lxg 1-i) were detected in coastal and estuarine areas, with even higher concentrations (up to 6.7 nM, 1.7 ~tg 1-1) in ports and marinas. Considering the available information, Irgarol 1051 has the potential of giving undesirable effects on algae in contaminated environments. The aim of the present investigation was (i) to determine the NOEC of Irgarol 1051 on marine periphyton communities after long-term exposure under ecologically realistic conditions and (ii) to

Volume 32/Number4/April 1996 measure concentrations of Irgarol 1051 over the boating season close to a small Swedish marina. In addition, concentrations of the herbicide diuron were measured due to its use as an antifouling algicide at this time. Marine periphyton communities were experimentally exposed to Irgarol 1051 in flow-through microcosms during April 1994. Biomass, community structure and periphyton photosynthetic activity were measured at the end of the experiments. M a t e r i a l s and M e t h o d s

Marine microcosms A marine microcosm system (Blanck & W/ingberg, 1988) at Kristineberg Marine Research Station by the Gullmar fiord on the west coast of Sweden (Fig. 1) was used (during April 1994) to investigate the influence of Irgarol 1051 exposure on periphyton colonization. Seawater was pumped from a depth of 3 m in the fjord into the laboratory by an air-driven teflonmembrane pump (Dominator Maskin, Jrnk6ping, Sweden). A nylon net with a 1 mm mesh size prevented larger organisms from reaching the experimental aquaria. An average continuous water flow of 214 ml min- ~ through each of the ten 22-1itre glass aquaria was achieved using a water-delivery system similar to the one described by Granmo & Kollberg (1972) modified by Molander et al. (1992). The mean residence time of water was 103 min. Stock solutions of Irgarol 1051--2-(tert-butylamino)-4-(cyclopropylamino)-6-(methylthio)- 1,3,5-triazine, > 97% purity (Ciba-Geigy, Basle, Switzerland)were prepared in acetone and stored at -20°C. Water solutions of Irgarol 1051 were prepared by diluting stock solutions 1000-fold with water purified by reversed osmosis. These Irgarol 1051 solutions were delivered to the aquaria at a flow rate of 2.0 ml min- l by means of a peristaltic pump (Ismatech IPN 26, Ziirich, Switzerland), giving a final concentration of acetone in the aquaria of 9 Ixl 1-1. The Irgarol 1051 solutions were prepared twice a week and kept in dim light to minimize the risk of photodegradation. Flow rates of seawater and Irgarol 1051 solutions were checked daily and adjusted when deviating from the desired values. Three of the 10 aquaria were designated

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Fig. 1 M a p of the investigated area on the west coast o f Sweden, with Kristineberg Marine Research Station indicated with an arrow (58"15'N, 11°27'E). Stations are referred to by numbers, with 1 at the site o f the m a r i n a a n d 6 in the outer archipelago.

controls with only co-solvent added. Irgarol 1051 was added to the seven remaining aquaria in a geometric concentration series (0.0625-256 nu) without replication. One-litre water samples were taken for quantitative analysis of Irgarol 1051 and diuron at the end of the experiment. Glass discs (1.5 cm 2, 170 per aquarium) for periphyton communities to colonize were mounted on polyethylene holders along the sides of each aquarium (Blanck & W/ingberg, 1988). The discs were cleaned in hot concentrated nitric acid, rinsed in purified water and finally rinsed in 70% ethanol before being submerged into the aquaria at the start of experiment. The aquaria were equipped with stirring devices to maintain thorough mixing and to minimize the boundary layer between bulk water and periphyton. Each aquarium was illuminated from above by two fluorescent tubes (Osram Lumilux Daylight L18W/12) that gave an average photon flux density of 124 gmol m -2 s -~ at the water surface and with a light/dark regime of 14/10 h following the sunrise and sunset at the time of the year.

Periphyton photosynthetic activity The photosynthetic activity in periphyton was measured as the short-term incorporation of 14Ccarbon dioxide into acid-stable products (e.g. Blanck & W/ingberg, 1988; Molander & Blanck, 1992; Dahl & Blanck, 1996). The incubations of periphyton were performed in a thermostated waterbath set at the temperature in the aquaria. The light from fluorescent tubes (Osram Lumilux Daylight L18W/11) was filtered through 20 cm of water and regulated to give a photon flux density of 50 gmol m -2 s -1. Replicate glass discs (n = 4) from each of the aquaria were incubated in glass scintillation vials (20 ml) in 2 ml of filtered (Whatman GF/F) seawater. The incubation time was 30 min in light before the addition of 50 gl of a 14C-labelled bicarbonate solution. The radiolabelled bicarbonate solution was prepared by a 50-fold dilution of a stock solution (Amersham CFA2, 2 mCi m1-1, 55 mCi mmo1-1) with GF/F filtered seawater (approximately 1.9 mM total inorganic carbon) giving a final activity of 2 gCi (74 kBq) per 50-~tl aliquot. After 15 min, 100 gl of formaldehyde (37%) was added to terminate the carbon fixation activity. The water solutions were removed and the samples were acidified by adding 1 ml of concentrated acetic acid. The remaining inorganic carbon was driven off by a gentle stream of air, and at the same time the samples were dried at 60°C. The release of incorporated ~4C-carbon from the cells was enhanced by the addition of 1 ml of concentrated dimethylsulphoxide (Filbin & Hough, 1984) before adding 8 ml of a scintillation cocktail (Ready SafeTM, Beckman, Fullerton, CA, USA). The samples were thoroughly mixed and a liquid scintillation spectrometer (LS 3801, Beckman) was used to determine the amount of incorporated 14C-carbon. The radioactivities, as disintegrations per minute (dpm), were calculated from the counts per minute (cpm) data using an external standard technique and the appropriate correction factors for the sample quench characteristics and 343

Marine Pollution Bulletin machine efficiency. The activities from the photosynthesis measurements were corrected for abiotic 14C-carbon fixation, estimated as the amount of 14Ccarbon incorporated into samples killed by the addition of 100 txl of formaldehyde (37%) prior to the incubation.

Irgarol 1051 inhibition of photosynthesis The long-term inhibition of periphyton photosynthesis was estimated by comparing the photosynthetic activities of periphyton samples from the different aquaria. The measurements were made after 3 weeks of colonization. The short-term inhibition of photosynthesis was estimated using periphyton from each of the three control aquaria. The photosynthetic activity was measured, in this case, in a geometric concentration series (a factor of v / ~ between 1 and 316 riM) of Irgarol 1051 prepared in filtered (Whatman GF/F) seawater. Also, a control with only the co-solvent acetone added (10 Ixl 1-1) was employed in the test. Four replicates were used at each concentration, giving a total of 28 discs in each short-term test.

Algal biomass and relative abundances of algal species Algal biomass was estimated as the chlorophyll a content on the front surfaces of the glass discs. Pigments were extracted by DMSO in 30 min at 60°C (Hiscox & Israelstam, 1978) and analysed spectrophotometrically after the addition of an equal volume of 90% distilled acetone (Shoaf & Lium, 1976). The amount of chlorophyll a per disc was calculated using the equations of Jeffrey & Humphrey (1975). Three samples, each containing 6-8 glass discs, were analysed per aquarium. Taxonomic analysis of periphyton communities were made on three glass discs per aquarium, all sampled at the end of the experiment. The samples were preserved in 70% ethanol and stored at 4°C in darkness. One fresh sample per aquarium was analysed and video-recorded in order to facilitate later identification of algal species in the preserved samples. The presence of taxonomically different groups of algae, usually identified to the species level, was recorded in 50 randomly chosen fields (diameter of 252 lxm) per disc, using a phase contrast microscope at 1000× magnification. The increment of accumulated number of species vs number of analysed fields rapidly approaches zero after 35-40 fields (unpubl. results). The total area that was scanned on each disc was 0.05 mm 2, equivalent to 0.03% of the total disc area. Therefore, it is highly unlikely that the same area is analysed more than once. The relative abundance of each species was estimated as the number of fields where the species was observed, thus giving an abundance range of 0-50. Three different methods were used to evaluate the effects on community structure. The algal species richness and the Bray-Curtis dissimilarity index (Bray & Curtis, 1957), as described by Sheehan (1984) and Clarke (1993), were used to indicate relative deviations in community structure compared to the controls, whereas the non-metric multidimensional scaling 344

(MDS) made further use of the Bray-Curtis indices in order to reveal absolute differences between all community samples taken. The algal species richness was calculated as the number of algal taxa found on a sampling unit without considering their relative abundances. However, Bray-Curtis dissimilarity indices were calculated in order to include the information on relative abundances. Periphyton communities established under Irgarol 1051 exposure were compared to periphyton established at background levels. Communities with similar algal species richness or similar Bray-Curtis indices are not necessarily similar in their community structure. This is because the former measure disregards the identity of species and the latter only indicates the similarity relative to the control communities according to the procedure described above. However, using multivariate ordination techniques, such as the non-metric MDS, absolute measures of similarity can be obtained. Bray-Curtis dissimilarity indices for all possible interdisc comparisons were calculated and anlysed through non-metric MDS. The software used (Statistica, StatSoft, Tulsa, OK, USA, 1993) employed the rank-image and the monotone regression procedures to minimize the loss function, Kruskal's stress coefficient, in the iteration process (Schiffman et al., 1981). The resulting ordination projected the community structure observations in a two-dimensional display in which deviations from the ranked order of original similarity indices were minimized. The longer-term effect of Irgarol 1051 on community structure could then be visualized.

Inferential statistics In the analysis of the acute effect of Irgarol 1051 on periphyton photosynthetic activity, an ANOVA was used and when significant (p< 0.05) it was followed by Dunnett's test. Data were log10 transformed and tested for variance heterogeneity (Cochran's test) and normal distribution (Z2 test) in order to meet assumptions for parametric tests before ANOVA. The short-term NOEC was calculated as the highest tested concentration not significantly (p>0.05) differing from the activity of the controls. Three controls and seven unreplicated Irgarol 1051 levels were employed to investigate the long-term effects of Irgarol 1051. The long-term NOEC was estimated as the highest tested concentration at which the response of periphyton did not deviate from the 95% confidence interval of the response in the controls. The long-term lowest-observed-effect concentration (LOEC) was accordingly estimated as the concentration following the NOEC. EC50 values were quantified from the generated concentration-effect curves by interpolation.

Analysis of lrgarol 1051 and diuron In addition to the analysis of experimental waters, subsurface (20-30 cm depth) coastal water samples were also collected from the field. Samples were taken each month at the marina of Fiskeb/ickskil (Fig. 1, station 1) from May to September 1994. Samples were also collected at a reference station in the outer archipelago (station 5) on the same occasions. At the end of June,

Volume 32/Number 4/April 1996

samples were taken along an axis from the marina to the outer archipelago (stations 1 to 6) in order to investigate any gradients in contamination. Samples were collected in 1-1itre glass bottles, which had been pre-washed and rinsed in ethanol and which were equipped with teflon lids. Thirty millilitres of dichloromethane was used for preservation of the samples. In addition, during 1993 three samples were taken at stations 1 and 5 in a pilot survey with a similar sampling technique. Irgarol 1051 and diuron in seawater were analysed according to the SLU (Swedish Agricultural University, Uppsala) procedure for nonpolar and semipolar pesticides in drinking water (Akerblom et al., 1990), optimized to monitor the two compounds. Pesticides were extracted on arrival at the laboratory (on the same day) after the addition of surrogate standards, using liquid-liquid extraction with dichloromethane. Pesticides were quantified by GC-MS, using selective ion monitoring (SIM) in the EI-mode. Three selective ions, 182, 238 and 253 amu, were used to confirm the presence of Irgarol. Two surrogate standards were used: 1. the triazine terbutryn to correct for recovery of Irgarol; and 2. one normal surrogate standard (ethion) to monitor and compensate method performance for other semipolar pesticides, including diuron.

the 3-week long experiment. Analysed Irgarol 1051 concentrations in the water were 58+ 13%, n = 6 (SD), of the nominal values (Table 1). In the following, the longer-term Irgarol 1051 concentrations will be denoted by their nominal values. Algal biomass, measured as the chlorophyll a content on the discs, was lowered in communities exposed to Irgarol 1051 (Fig. 3). At a nominal exposure level of 4 nM, biomass was 28% of the controls and thus significantly lower. Also, the photosynthetic activity was lower in periphyton affected by Irgarol 1051 (Fig. 4). At 1 nt~ periphyton showed less than 50% of the photosynthetic activity of the controls. The ECs0 values for reduction of biomass and photosynthetic activity were calculated to be 2.7 and 0.82 nM, respectively. The effect on photosynthetic activity per unit of chlorophyll a was even stronger (Fig. 5). There was a decreasing O 150

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exposure. The chlorophylla content is expressed as relative to the average content in periphyton from the three control aquaria (1.23 lxg disc-l). Error bars show standard deviations (n=3). Dotted lines indicate the 95% confidencelimits of the controls. Observations significantlydifferent from the pooled controls (p<0.05) are indicated by stars. Nominal Irgarol 1051 concentrations are given. 345

Marine Pollution Bulletin

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Long-term concentration of Irgarol 1051 [nM) Fig. 4 Long-term effects of Irgarol 1051 on periphyton photosynthetic activity. Periphyton samples from each aquarium were transferred to filtered 0 V h a t m a n G F / F ) Irgarol-1051-free seawater and photosynthesis were measured as the incorporation of 14C-carbon dioxide at the end of the 3-week experiment. Activities are expressed as relative to the average activity in periphyton from the three control aquaria (30000 dpm 15 r a i n - i disc-1). Error bars indicate standard deviations (n = 4). Dotted lines indicate the 95% confidence limits of the controls. Observations significantly different from the pooled controls (/9<0.05) are indicated by stars. Nominal Irgarol 1051 concentrations are given.

concentrations, whereas a peak in abundance at intermediate concentrations was observed for others. Obviously, there was a shift favouring more tolerant algae. However, all species decreased their abundances at concentrations higher than 1 riM. Species richness was significantly reduced at 4 nM Irgarol 1051, with a calculated ECs0 value of 15 nM (Fig. 7). The species richness index failed to detect the species shifts that occurred at intermediate concentrations. However, the Bray-Curtis dissimilarity index (Fig. 8) did detect this shift at 1 nM. The non-metric MDS analysis of community structure included Bray-Curtis indices for paired comparisons of all community samples and could thus reveal both within-treatment and between-treatment similarities (Fig. 9). Altered community structure of periphyton was indicated at Irgarol 1051 concentrations above 0.25 riM. The very low Kruskal's stress coefficient (0.04) indicated that the display gave an excellent

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trend in the ratio throughout the concentration range tested, with the first sigr~ficant effects at 0.25 nM. The lower photosynthetic activity found in periphyton exposed to Irgarol 1051 was clearly not only a result of a lower algal biomass but also due to decreased efficiency of photosynthesis. In addition to primary productivity, the community structure of periphyton was also affected by exposure to Irgarol 1051. The dynamic responses of each species to Irgarol 1051 can be seen in Fig. 6. Some taxa showed a decreased a b u n d a n c e even at the lowest Irgarol 1051

346

Fig. 6 The dynamic response of the relative abundances of algal species to the long-term Irgarol 1051 exposure. Symbols represent different algal species. Each point represents the average of three replicates. The samples from the three controls were pooled together (n = 9). Nominal Irgarol 1051 concentrations are given.

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Volume 32/Number 4/April 1996 1.00

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when the boating activities were high. In the outer archipelago (station 5), no Irgarol 1051 was found. The highest concentrations (0.39-1.6 riM) were detected at the end of June 1994 in the gradient from the marina to the fjord (stations 1-3). Also, diuron was detected (0.04-0.4 nM) close to the marina (stations 1-3) in both

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Irgarol 1051 is s h o w n to be very toxic to p e r i p h y t o n photosynthesis in the s h o r t - t e r m tests, with a n ECso value o f 5 nM a n d a L O E C of 3.2-10 nM (Fig. 2, T a b l e 3). I n fact, it was s h o w n to be m o r e toxic t h a n other PSII i n h i b i t o r s such as d i u r o n , m e t r i b u z i n , b r o m a c i l a n d atrazine that have been previously investigated in the s h o r t - t e r m test for p e r i p h y t o n photosynthesis (Blanck & M o l a n d e r , 1991; M o l a n d e r & Blanck, 1992). Based o n ECs0 values for s h o r t - t e r m exposure, Irgarol 1051 is 4 - 8 times m o r e toxic t h a n d i u r o n , m e t r i b u z i n a n d b r o m a c i l a n d a b o u t 70 times more toxic t h a n atrazine. G o l d s b o r o u g h & R o b i n s o n (1983) stated that terbutryn--2-(tert-butylamino)-

64

TABLE 2 Irgarol 1051 and diuron concentrations in water samples from the Swedish west coast taken in 1993 and 1994. Detection limits varied between analysing occasions and were 0.044).4 nu for Irgarol and 0.04--0.2 nu for diuron. See Fig. 1 for location of sampling stations.

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5 May 26 May 26 June 26 June 26 June 26 June 26 June 26 June 29 July 29 August 29 September

1 1 1 2 3 4 5 6 1 1 1

0.16 0.16 0.79 1.6 0.39 ND ND ND 0.79 0.20 0.12

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1

Fig. 9 Two-dimensional plot showing distances between long-term treatments after non-metric multidimensional scaling (MDS) of Bray-Curtis dissimilarity indices from all possible interdisc comparisons. Small distances between observations indicate high similarity in community structure. The loss function in the analysis (stress= 0.04) was very small, indicating an excellent representation of the relationship between observations. Open circles represents all observations (n=9) from the control aquarias. Periphyton established during Irgarol 1051 exposure (n = 3 for each level) are denoted by their respective exposure level. Observations from the 0.0625 and 0.25 rmi aquaria were inseparable from the controls and are not indicated in the plot.

representation of the similarity pattern between samples. The display also revealed an ordered change in c o m m u n i t y structure following the exposure gradient a n d that the w i t h i n - t r e a t m e n t similarities decreased with increasing I r g a r o l exposure levels. N o full p r o g r a m m e was c o n d u c t e d to estimate the e n v i r o n m e n t a l c o n c e n t r a t i o n s o f I r g a r o l 1051. I n spite o f the limited effort, I r g a r o l 1051 was detected in water samples from the Fiskeb~ickskil area (see Fig. 1, stations 1-3) d u r i n g the b o a t i n g seasons o f 1993 a n d 1994 (Table 2). L o w c o n c e n t r a t i o n s (0.12-0.16 nM) could be f o u n d close to the m a r i n a in spring a n d a u t u m n , whereas a p e a k (0.79 nM) was detected in J u n e a n d July

Irgarol 1051

Diuron

ND, not detected.

TABLE 3 Endpoints for short-term and long-term effects of Irgarol 1051 on marine periphyton communities. NOEC and LOEC (p<0.05) values are expressed in nominal Irgarol 1051 concentrations. Irgarol 1051 concentration (nu) Effects

NOEC

LOEC

ECso

Short-term Photosynthesis

1-3.2

3.2-10

4.7- 5.5

Long-term Photosynthesis/Chl a Photosynthesis Bray~Zurds index Biomass (Chl a) Number of algal taxa

0.062 0.25 0.25 1.0 1.0

0.25 1.0 1.0 4.0 4.0

0.72 0.82 5.4 2.7 15

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Marine Pollution Bulletin

4-(ethylamino)-6-(methylthio)- 1,3,5-triazine, another s-triazine which is similar in structure to Irgarol 1051--possesses stronger algicidal properties than similar levels of other triazines. Irgarol 1051 and terbutryn both contain a methylthio-group instead of a chlorine, which might be the reason for their higher toxicity. Based on the short-term toxicity to marine periphyton, Irgarol 1051 must be considered as one of the most potent inhibitors of algal photosynthesis. The strong inhibition of photosynthesis by Irgarol 1051 is also indicated through the long-term tests, with effects occurring at even lower concentrations (Table 3). After long-term exposure to Irgarol 1051, the photosynthetic capacity of periphyton is reduced even at 1.0 riM, and photosynthesis per unit of chlorophyll a at 0.25 nM (LOECs). This indicates a malfunctioning of the photosynthetic apparatus that the algae are unable to compensate for. These effects are also expressed in a lowered net production as judged from the reduced chlorophyll a values of the periphyton communities at Irgarol 1051 concentrations above 1 nM. Also, the community structure was affected with a LOEC of I riM. The responses of all of these effect indicators consistently point to an effect threshold of Irgarol 1051 in marine periphyton communities at nanomolar or even sub-nanomolar concentrations. The results imply a very low functional redundancy in periphyton communities, since structural and functional effects of Irgarol 1051 occur at approximately the same exposure level (Table 3). When this effect threshold is compared to recently analysed concentrations of Irgarol 1051 in the marine environment (Table 2; Readman et al., 1993; Gough et al., 1994), it can be concluded that Irgarol 1051 is likely to damage microalgal communities in contaminated coastal waters. The observed Irgarol 1051 levels on the Swedish west coast are well in accordance with those of Readman et al. (1993) and Gough et al. (1994), who found Irgarol 1051 concentrations less than 1 nu (0.25 Ixg 1-1) outside, and as high as 6.7 nM (1.7 Ixg 1-1) inside, marinas and ports. In addition, diuron contamination was also observed, due to its use as an antifouling additive at that time (Table 2). Irgarol 1051 is shown to affect community structure of periphyton by inducing a shift of species (Fig. 6) at about 1 nM. This shift in species composition is an indication of selection processes taking place, favouring Irgarol 1051 tolerant species at the cost of sensitive ones. Similar shifts due to exposure to other triazine herbicides have been reported for periphyton (Kosinski, 1984; Goldsborough & Robinson, 1986; Hamilton et al., 1987) and phytoplankton (DeNoyelles et al., 1982; Hamilton et al., 1988; Gustavson & W/ingberg, 1995). This early effect on community structure at about 1 nM could not be detected by effect indicators like chlorophyll a content (Fig. 3) or species richness (Fig. 7). However, the species shifts were detected by the Bray-Curtis dissimilarity indices (Fig. 8) and also by the non-metric MDS analysis (Fig. 9), because these measures involve the identity of each species and their relative abundances. It appears that quantitative community structure data are more powerful than qualitative data for the detection of shifts in species 348

composition. In addition, it is possible to measure and visualize the absolute similarities in species composition, not only compared to the controls but also between all community samples, by using the MDS analysis. In this way it was possible to show that the change in community structure was ordered, following the successive increase in Irgarol 1051 concentrations. The similarity in community structure between replicate samples decreased with increasing Irgarol 1051 levels, most probably as an effect of the very low abundances of algae at the higher exposure levels. Periphyton photosynthesis decreased at 1 nM of Irgarol 1051 (Fig. 4) in spite of the maintained chlorophyll a content at this exposure level (Fig. 3). This indicates a malfunction of the photosynthetic apparatus and is clearly shown as a decrease in photosynthesis per unit of chlorophyll a (Fig. 5). Higher plants treated with PSII inhibitors resemble shade-adapted ones which increase their amount of light-harvesting pigments (the so-called greening effect) in order to maintain the conversion of light energy into chemical energy (Fedtke, 1982). This response has also been shown for cyanobacteria (Koenig, 1990). As a consequence, algae should decrease their rate of photosynthesis per unit of chlorophyll a when affected by triazines, an observation which has been reported earlier (Goldsborough & Robinson, 1983; Stay et al., 1985; Larsen et al., 1986; Pratt et al., 1988). Thus, the inhibition of photosynthesis and the increase in chlorophyll a will co-operate to amplify the response of the ratio of photosynthesis per unit of chlorophyll a. This would explain the high sensitivity of this effect indicator in this study (Table 3). Light promotes the damaging capacity of PSII inhibitors (Fedtke, 1982), because radicals and singlet oxygen are formed when chloroplasts with reduced electron transport capacity are irradiated. The normal production of these reactive species can be kept under control by protective systems, but when formed in excess they give rise to the destructive effects observed during and after herbicide exposure (Fedtke, 1982; Kunert & Dodge, 1989). Therefore, algae will suffer from a double assault from PSII inhibitors such as Irgarol 1051: a decrease in electron transport and an increased production of radicals. The greening effect described above, which compensates for lower electron transport by directing more light to the reaction centra, simultaneously promotes the formation of reactive molecular species leading to oxidative stress. These mechanisms might lead to an accelerated breakdown of periphyton community structure and function, as suggested by the steepness of the Irgarol 1051 concentration-effect curves (Figs 2-4, 7 and 8). The double mode of action of PSII inhibitors make it more difficult for algae to develop efficient tolerance mechanisms. This may explain why no tolerance increase was detected, in spite of shifts in species composition, for the PSII inhibitor atrazine (Kosinski, 1984; Gustavson & W/ingberg, 1995) and only a minor tolerance increase for diuron (Molander & Blanck 1992). Such an increased tolerance would be expected according to the PICT concept (Blanck et al., 1988).

Volume 32/Number 4/April 1996

Atrazine is probably the most rigorously tested s-triazine and therefore offers an interesting comparison to Irgarol 1051. Both short-term (e.g. Blanck & Molander, 1991) and long-term toxicity studies of microalgal communities are available (e.g. DeNoyelles et al., 1982; Kosinski, 1984; Pratt et al., 1988). The short-term toxicity of Irgarol 1051 (ECs0 value of 5 nl~I, Table 3) is about 70 times higher than that of atrazine (ECs0 value of 350 nM; Blanck & Molander, 1991) in the present test system. In a recent review of the environmental toxicity of atrazine, Huber (1993) concluded that initial ecotoxicological effects appear at or above concentrations of 90 riM. However, the lowest reported long-term (3-4 weeks) LOEC values for atrazine are 14-45 nM, for a significant increase in chlorophyll a and protein content (Pratt et al., 1988) and reduced abundance of several species (Kosinski, 1984). These studies are strictly comparable to the present study because of the similar approach using colonization of microalgae on artificial substrata. For the long-term toxicity, if an atrazine/Irgarol-1051 ratio of 70 is assumed, a predicted long-term LOEC of 0.2-0.6 nM (14-45 divided by 70) is obtained. This is similar to the lowest nominal long-term LOECs of 0.251.0 nM (Table 3) and very close to the actual LOECs (0.16-0.79 nM), i.e. using the analysed concentrations rather than nominal (Table 1). Hence, Irgarol 1051 is about 70 times more toxic to microalgal communities than atrazine, both in short-term and long-term experiments. This finding has the implications that the two triazines are similar both in their biochemical and their ecological mode of action, and furthermore that their long-term toxicity may be correctly estimated from short-term tests.

Conclusions It is shown that the antifouling algicide Irgarol 1051 is highly toxic to non-target marine algae and that it is sufficiently stable to reach toxic concentrations in certain areas in the marine environment. Therefore, it is concluded that the present use of Irgarol 1051 is likely to damage non-target microalgal communities in contaminated coastal waters. The fate and effects of Irgarol 1051 requires substantial further evaluation. However, these studies should consider the contamination of the coastal waters with other herbicides (Bester & Hiihnerfuss, 1993; Pereira & Hostettler, 1993; Gough et al., 1994; Law et al., 1994) that are similar to Irgarol 1051 in structure and mode of action, and which will contribute to combined effects (Altenburger et al., 1993). Anna Linusson and the staff at Kristineberg Marine Research Station are gratefully acknowledged for~ technical assistance and Mats Kuylenstierna for his skillful taxonomic analysis of periphyton algae. The authors thank Ciba-Geigy for supplying the Irgarol 1051. This work was financed by the Swedish National Chemicals Inspectorate and by the Swedish Natural Science Research Council (NFR). ]kkerblom, M., Thorrn, L. & Staffas, A. (1990). Detection of pesticides in drinking water. Vdr Fr"da. 4-5, 236-243 (in Swedish). Altenburger, R., BSdeker, W., Faust, M. & Grimme, L. H. (1993). Analysis of combination effects in aquatic toxicology. In Handbook

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