Toxic and Accumulative Potential of the Antifouling Biocide and TBT

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Environ. Sci. Technol. 2009, 43, 6838–6843

Toxic and Accumulative Potential of the Antifouling Biocide and TBT Successor Irgarol on Freshwater Macrophytes: A Pond Mesocosm Study ¨ DIGER BERGHAHN, SILVIA MOHR,* RU WOLFGANG MAILAHN, RONNY SCHMIEDICHE, MICHAEL FEIBICKE, AND RALF SCHMIDT Umweltbundesamt, Schichauweg 58, 12307 Berlin, Germany

Received February 26, 2009. Revised manuscript received June 18, 2009. Accepted June 22, 2009.

After the ban of tributyltin (TBT) for vessels not longer than 25 m in 1986, Irgarol has become a commonly used antifouling biocide. Irgarol is highly toxic to autotrophic organisms and has the potential to accumulate in organic material. In the literature, environmental concentrations of Irgarol up to 2.4 µg L-1 were reported for freshwater. Within a comprehensive freshwater mesocosm study, experiments were conducted to gain more information on the effects of Irgarol on macrophytes. Six indoor pond mesocosms were contaminated once with concentrations between 0.04 and 5 µgL-1 Irgarol and monitored for 150 days; two mesocosms served as controls. The mesocosm study revealed that all macrophytes were directly affected by this single application. Myriophyllum verticillatum was the most sensitive macrophyte with an EC50 (Day 150) of 0.21 µg L-1 Irgarol. The duckweed Spirodela polyrhiza was the least sensitive species tested in the mesocosms and number of fronds even increased with increasing Irgarol concentrations. Timeweighted average calculations yielded high BCF values of up to 10,560 L kg-1 dry weight for M. verticillatum indicating a high potential for accumulation. The results give cause for concern that natural macrophyte communities are impaired at actual environmental concentrations.

Introduction Irgarol (Irgarol 1051, N-tert-butyl-N′-cyclopropyl-6-methylthio-1,3,5-triazine-2,4-diamine) is a highly effective biocide used in antifouling coatings to prevent the growth of autotrophic organisms on ship hulls. After the ban of tributyltin (TBT) and vessels not longer than 25 m in 1986, the use of TBT-free paints containing copper compounds and organic booster biocides such as Irgarol increased considerably and became more widespread (1, 2). The impact of these alternatives to TBT on the aquatic environment has become a topic of increasing importance in recent years, because they are also toxic and in most cases persistent (2). A further increase of biocides in the environment other than TBT is expected in the next years due to the definitive international ban of TBT in January 2008. Booster biocides like Irgarol are used in free association antifouling paints and are thereby continuously released to * Corresponding author tel: +49-30-89034220; fax: +49-3089034200; e-mail: [email protected]. 6838

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the surrounding water posing a potential threat to non-target organisms in the environment (3). Although Irgarol was predicted to easily dissipate under natural conditions (4), it is the most frequently detected antifouling biocide worldwide (2). Published values of the half-life (DT50) of Irgarol in water are between 24 and 200 days (5-7). In ports and marinas in coastal waters it was detected in relevant effect concentrations up to 4.2 µg L-1 (8). Until its restriction in the UK, Irgarol was one of the most commonly used booster biocides. Levels up to 1.4 and 2.4 µg L-1 have been reported from UK marinas and freshwater sites (9, 10). To date, most studies on Irgarol focused on marine areas and toxicity testing with marine organisms. In comparison to other triazines like atrazine and simazine, Irgarol is a more potent photosystem II inhibitor of algal photosynthesis and therefore highly toxic to macrophytes, phytoplankton, and periphyton (7, 11-13). The main metabolite M1 is also toxic to autotrophic organisms but in many cases much more than 10 times less toxic than Irgarol. Like other triazines, the biocide Irgarol inhibits the electron transfer in the photosystem II (PS II). Irgarol binds with high affinity to the plastoquinone (Qb) site of PS II, displacing the Qb quinone and preventing electron transfer (14). This results in oxidative stress, including photo-oxidation of chlorophyll and cell necrosis (15). For macrophytes, toxicity data are very scarce. The fools watercress Apium nodiflorum is the most sensitive macrophyte found in literature with a 14 d-EC50 for root length of 0.013 µg L-1 exposed to Irgarol and of 0.128 µg L-1 exposed to M1 (10). In comparison, the marine species Zosteria marina was less sensitive with a 10 d-EC50 of 1.1 µg L-1 (16).The standard test organism Lemna spp. is less sensitive with EC50 ranging from 1.6 to 11 µg L-1 (12, 17). Algae of the group of prymnesiophytes were found to be the most sensitive algal group in the marine environment with a 72 h-EC50 of 0.07 µg L-1 (1) while the diatom Navicula pelliculosa is the most sensitive freshwater species with a 5 d-EC50 of 0.1 µg L-1 (18). Irgarol is likely to be much less toxic to animals than flora (18, 19). Periphyton, not macrophytes, is the target group of Irgarol when applied to ship hulls. However, environmental concentrations of Irgarol in freshwater are in the range of the effect concentrations for macrophytes and they are therefore endangered under natural conditions. Macrophytes constitute an important functional and structural element in freshwater and their reduction may cause severe and longlasting effects on the whole ecosystem (20-23). Furthermore, accumulation in macrophytes and sediments is most likely due to the relatively low water solubility of 7 mg L-1, a high log Pow of 4.07, and log Koc of 3.14 (17). Detailed information on these issues, however, is lacking up to now. In consequence of the frequent occurrence of Irgarol in the environment and its high phytotoxicity, a pond mesocosm study was conducted by the German Federal Environment Agency to study long-term effects of Irgarol on the whole pond community. This also included the analysis of Irgarol in water and sediment, the effects of plankton, periphyton, and snails (7, 24) under near-natural conditions. This paper deals with the effects of the toxic and accumulative potential of Irgarol on macrophytes. The end points total biomass and Irgarol concentrations of the macrophyte standing stock at the end of the study were investigated as well as the leaf development of Potamogeton nodosus and the duckweed Spirodela polyrhiza. Biomass, growth, and length of potted sprouts of Myriophyllum verticillatum were also analyzed. 10.1021/es900595u CCC: $40.75

 2009 American Chemical Society

Published on Web 07/07/2009

Materials and Methods Experimental Design and Application of Irgarol. Eight indoor ponds (length 6.90 × width 3.25 × height 2.50 m) had been set up one year prior to the start of the experiment. A littoral zone was formed with sand leaving a volume of 15 m3 of open water at a maximum water depth of 1.0 m. The littoral zone of the ponds was covered with a thin layer of fine sediment and stocked identically with the macrophytes P. nodosus and M. verticillatum. Moreover, Carex spec., Iris pseudacorus, and Myosotis palustris were planted at the shore. For more technical information see Mohr et al. (7, 24). The introduced plants are common species in mesotrophic European waters and they have been used in toxicity studies (20, 22). In addition, M. verticillatum shoots and the duckweed S. polyrhiza were introduced separately into the pond systems. Two × 7 shoots of M. verticillatum (15-cm cuttings without side shoots) were planted into 2 pots which had been filled with a mixture (1:2) of sand and soil for water plants (100 mg/L N, 100 mg/L P; Stender Inc.) 4 days prior to Irgarol dosing. The plant pots were placed in the deepest zone of the pond mesocosms prior to the start of the experiment. In addition, 4 floating silicon rings were exposed in the littoral zone of the ponds and then stocked with 20 fronds of S. polyrhiza. A wire net of stainless steel was fixed at each ring in order to close the ring-opening toward the pond bottom and thus prevent the invasion and grazing of snails. Controlled illumination of each pond was obtained using 2 × 2000 W and 2 × 400 W high-pressure mercury-vapor lamps corresponding to a mean light intensity of 13,000 L × or ∼240 µE m-2 s-1 at the water surface. Each month, light/ dark ratios were adjusted to the outdoor conditions. Nitrate, phosphate, silicate, and trace elements were dosed in the water of all systems prior to the start and two times in the course of the experiment (50 and 100 days after Irgarol application). It was intended to keep the concentrations in water at relatively moderate levels (P-total c. 0.05 mg L-1, N-total c. 1.5 mg L-1) comparable to mesotrophic conditions. All systems were equally stocked with plankton and macrozoobenthos taken from a mesotrophic lake (Lake Britzer Garten, Germany). For more stocking details and nutrient level see Mohr et al. (7). Irgarol was dosed once at nominal concentrations of 0.04, 0.2, 1, and 5 µg L-1. The control, the 0.2 µg, and the 5 µg ponds were replicated, but not the 0.04 µg and 1 µg ponds. After Irgarol dosing, the pond water was completely mixed for 5 min by means of an electric outboard motor. The routine sampling program, including physicochemical parameters, periphyton and plankton analysis, Irgarol and its main metabolite M1 in water and sediment, was conducted daily at the beginning of the study to biweekly for a period of 6 months (150 d; (7)). Macrophyte Sampling. During the experiment, the development of the macrophyte standing stock was photodocumented regularly by taking fortnightly photos of each pond surface with a digital camera (Canon Powershot S40, Japan). The development of P. nodosus was analyzed by computerassisted counting of all floating leaves with more than 50% of green leaf area by means of analySIS software (25). Thus, leaves with damage greater than 50% caused by necrosis, chlorosis, or herbivorous activities were excluded from the analysis. The potted shoots of M. verticillatum (7 sprouts per sampling date) were harvested in each pond after 43 and 51 days and the following parameters were determined for each shoot separately: length of the shoots, length of side shoots, total length of plant (i.e., length of main shoot plus total length of side shoots), total length of roots as well as fresh and dry weight (at least 24 h at 105 °C). Frond number and area of S. polyrhiza in the ponds was documented for each

exposed floating ring separately using digital equidistant top view photography (Canon Powershot S 40, Japan) taken once a week. Images were analyzed employing analySIS (cp. (25)). At the end of the experiment after 150 days, ponds were drained and all macrophytes and filamentous algae were harvested, sorted by species, washed, and centrifuged for 5 min at 2800 rpm with a spin drier. Then, fresh weight (FW), dry weight (at least 24 h at 105 °C), and ash free dry weight (AFDW; 6 h at 550 °C and 2 h at 900 °C) were determined for each species and each system. Irgarol in Macrophytes. Subsamples of macrophytes and filamentous algae, which were harvested after 150 days of exposure, were frozen immediately after sampling and analyzed for residues of Irgarol and M1. Duplicates of 8 g of fresh weight were freeze-dried, weighed, and then ground up with 15 g of sea sand (Merck, Germany). The mixture was spiked with 200 ng of propazine (100 µL of 2 mg L-1 in acetone), and extracted with acetone at 120 °C/14 MPa (Accelerated Solvent Extractor ASE 200, Dionex, USA). About 40 mL of acetone extract was concentrated to 5 mL in a rotary evaporator and dissolved in 500 mL of high-purity grade water. This solution was extracted and analyzed in the same way as the water samples (7, 24). For quality assurance, 8-g aliquots of control samples of each macrophyte species were spiked by dropping 1 mL of Irgarol and M1 in acetone onto the surface of the macrophyte. The solvent evaporated within 30 min and then the material was frozen at -18 °C at least overnight and later freezedried. Further analysis was performed as described above. Irgarol recoveries ranged from 81 to 110% and the limits of quantification were from 0.37 to 0.63 µg kg-1 FW. M1 recoveries ranged from 79 to 103% and the limits of quantification were between 0.31 and 0.86 µg kg-1 FW. The coefficient of variation was between 1.1 and 10.7% for Irgarol and between 1.4 and 14.9% for M1. Statistical Analyses. EC50 values were calculated for nominal concentrations and for time-weighted average concentration (TWA) using a sigmoidal dose-response model (4-parameter logistic equation or Hill equation) with a sigmoid shape (GraphPad Prism V 4.00 for Windows, GraphPad Software, San Diego, CA, www.graphpad.com) including an add-on module (EC10, EC50) designed by the working group of Prof. Oehlmann, University Frankfurt, Germany: Y ) Bottom +

(Top - Bottom) (1 + 10)(logEC50-X)Hillslope

(1)

where Y is the response which starts at Bottom (0%), Bottom is lower plateau of effect (0%), Top is the upper plateau of effect (100%), EC50 is the effect concentration of 50%, X is the logarithm of concentration, and Hillslope is the form factor of slope. The bioconcentration factor (BCF) was calculated in accordance with the OECD guideline 305 (26) and is the quotient of the content of a chemical in the FW of biological material Cbiota (µg kg-1 FW) and the concentration Cwater (µg L-1) of the chemical in the surrounding water: BCF )

Cbiota Cwater

(2)

For the calculation of the BCF the time-weighted average (TWA) of the concentration of Irgarol in the water column was used: TWA )

1 t - t0

∫ C(t)dt ) t -1 t0 [- k e t

C1

to

1

-k1t

-

]

C2 -k2t e k2

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t

to

(3)

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FIGURE 1. Effect-concentration relationship for the end point total harvest of macrophytes (fresh weight) at the end of the mesocosm effect study. Bars indicate the range; n ) 2. where TWA is the time-weighted average concentration (µg L-1), t is the actual time (d), t0 is the time 0 (start at application), k1, k2 is the rate constant 1 and 2 (d-1), and C1, C2 is the theoretical start concentration 1 and 2. From the contents of Irgarol detected (µg kg-1 FW) and the FW (kg) determined right after the harvest, the total uptake Mup of Irgarol (µg) by the corresponding macrophyte species was calculated as: Mup ) C · f

(4)

where Mup is the total uptake of Irgarol by the corresponding macrophyte species (µg), C is the concentration of Irgarol in the biological material (µg kg-1 FW), and f is the fresh weight (kg). In the first studies on the occurrence of Irgarol in water, sediment, and biota in freshwater performed in Lake Geneva (27, 28), the BCF for Irgarol in macrophytes was calculated from the contents in dry weight (DW). For comparison, the contents of Irgarol and M1 in P. nodosus, M. verticillatum, and filamentous algae were also calculated for dry weight and the corresponding BCF. The total uptake of Irgarol in all macrophytes was calculated as the sum of the uptake by the macrophytes species as follows: Mtotal )

∑ (C · f ) i

i

(5)

i

where Mtotal is the total uptake of Irgarol by all macrophytes (µg), Ci is the Irgarol in macrophyte species i (µg kg-1 FW), and fi is the fresh weight of macrophyte species i (kg).

therefore not countable. The calculated EC50 from the leaf counts of P. nodosus (Figure 2) were higher than the EC50 for total biomass of this species at the end of the experiment (Table 1). The final counts had to be based on pictures taken on Day 120, i.e., almost one month prior to the end of the study due to increased development of filamentous green algae toward the end of the study. The filamentous algae overgrew the floating leaves of P. nodosus and made it impossible to count them at the last sampling date. The duckweed S. polyrhiza reacted inversely to Irgarol application. After 32 days of exposure, the number of fronds in this species increased with increasing Irgarol concentrations. For that reason, the EC50 could not be calculated. It is assumed that the EC50 is much higher than 5 µg L-1. S. polyrhiza was the least sensitive macrophyte in this study. Growth rates ranged from 0.06 d-1 in the low contaminated ponds up to 0.1 d-1 in the 1 and 5 µg L-1 contaminated mesocosms. Accumulative Potential of Irgarol in Macrophytes. The time-weighted average concentrations of Irgarol for 150 d in the water ranged from 0.006 to 1.41 µg L-1 Irgarol. They were 3.5-6.6 times lower than the nominal concentrations (Table 2). In filamentous algae the BCF was highest at the lowest Irgarol concentration of 0.04 µg L-1 with 2430 L kg-1. In contrast, the BCF of M. verticillatum and P. nodosus was highest in higher contaminated ponds (0.2 and 1 µg L-1) when calculated with fresh weight data. It was not possible to measure Irgarol in M. verticillatum in the highest contaminated ponds since there was too little biomass for analysis (Table 2). The BCF calculated from dry weight data was up to 7 times higher than that from fresh weight data (Table 2). With regard to dry weight, BCF of 10,560 was highest for M. verticillatum (Table 2). The ratio between Irgarol in macrophytes after 150 days and total Irgarol dosed into the ponds was highest in the 0.04 µg L-1 treatment (18%) and decreased down to 2% in the 5 µg L-1 ponds. There was no indication of bioaccumulation of the metabolite M1 in any of the investigated taxa. Highest BCF values of 1000 L kg-1 were calculated for M. verticillatum in the lowest contaminated pond. In the ponds with low Irgarol exposure, the metabolite M1 could either not be detected in macrophyte samples or was below the limit of quantification. For detectable contents the share of M1 in relation to Irgarol was between 3.7 and 6.8% for M. verticillatum, between 8.6 and 17% for P. nodosus, and between 5.8 and 23% for filamentous algae.

Results Effects on Macrophytes. Total harvest of all macrophyte species at the end of the experiment (Day 150) revealed a clear concentration response relationship (Figure 1) with an EC50 of 1.38 µg L-1 (Table 1). Myriophyllum verticillatum was the most sensitive macrophyte species in the pond mesocosms with an EC50 of 0.21 µg L-1. The EC50 for P. nodosus was higher and less reliable as indicated by the 95% confidence limits (Table 1). The potted M. verticillatum shoots were already harvested on Day 43 and 51 after Irgarol application. Due to the shorter exposure time, the effects were less pronounced for all parameters as compared to the specimens that had been planted directly in the littoral zone (Table 1). EC50 and EC10 could only be calculated for Day 43 since the concentration-effect relationship was not monotonous on Day 51. The end points main shoots and total plant length were more reliable parameters than weight as indicated by the CVs. The leaf development of P. nodosus increased over time in the pond mesocosms except in the 5 µg L-1 treatment (Figure 2). There were clear concentration response relationships on the sampling days 38 and 147. It was not possible to count leaves of P. nodosus prior to and shortly after Irgarol application since the leaves were still submerged and 6840

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Discussion Toxic Effects of Irgarol on Macrophytes. Macrophytes reacted strongly to a single application of Irgarol. In the course of the 150-day mesocosm study, recovery of macrophytes was not observed in the highest contaminated ponds (nominal start concentration 5 µg L-1). M. verticillatum was the most sensitive macrophyte species followed by P. natans and filamentous green algae. The duckweed S. polyrhiza was far less sensitive and even increased with increasing Irgarol concentrations. Published EC50 from laboratory results with the duckweed Lemna gibba are very heterogeneous ranging from 1.6 (14 d; (18)) to 11 µg L-1 Irgarol (7 d; (13)). The EC50 data for L. gibba as well as the results for S. polyrhiza indicate that the standard test organism Lemna sp. is less sensitive to Irgarol than other macrophyte species like Apium nodiflorum (10), Myriophyllum spp. (this study, (10)) or Elodea canadensis (28). The question whether the floating monocot duckweed Lemna sp. is a good representative for dicot macrophytes which are submerged and rooting in the sediment, was raised recently at the AMRAP (Aquatic Macrophyte Risk Assessment for Pesticides) workshop held in The Netherlands 2008. There was a general consensus among scientists, industry, and

TABLE 1. EC50 and EC10 for the End Point Fresh Weight (FW) of Total Macrophyte Biomass, Potamogeton nodosus, Filamentous Algae, and Myriophyllum verticillatum at the End of the Study (150 d) (EC50 and EC10 for Different End Points on Day 43 of the Potted M. verticillatum Plants)a total macrophytes

P. nodosus

filamentous algae

nominal (µg L )

150 d

150 d

150 d

150 d

main shoot (43 d)

total length (43 d)

FW (43 d)

EC50 C.I. 95% EC10 C.I. 95% R2

1.38 0.75-2.55 0.12 0.027-0.53 0.973

0.92

2.13 0.37-12.42 0.34 0.004-26.7 0.72

0.21 0.10-0.42 0.06 0.003-1.24 0.921

3.00 0.98-9.23 0.34 0.004-1.01 0.83

2.30 0.67-7.87 0.30 0.01-7.29 0.83

1.10 0.19-6.31 0.31 0.005-18.75 0.69

-1

a

0.76 0.902

M. verticillatum

Nominal ) nominal start concentration; C.I.95% ) 95% confidence interval; R2 ) curve fitting.

FIGURE 2. Leaf development of Potamogeton nodosus over time. Bars indicate the range; n ) 2. regulators to integrate other macrophyte species in the risk assessment process. The milfoil Myriophyllum sp. was proposed as additional standard test species (29-31). Results of this and other studies underline this suggestion. The increase of S. polyrhiza with increasing nominal Irgarol concentrations can be explained by increasing nutrient levels in the systems with high loads of Irgarol. Due to the reduction of almost all rooted macrophytes combined with both the decrease in Irgarol in the free water due to sorption and degradation and the lower sensitivity of S. polyrhiza, this species took advantage of this deserted situation in the highly contaminated ponds. As an ecological consequence for freshwater ecosystems experiencing high Irgarol contamination, strong duckweed development caused by high nutrient levels may hinder the recovery of rooting macrophytes by covering the water surface. Such species

shifts can be assumed to become prominent even in the subsequent growth season in view of the recent trend toward warmer winters in Europe (32, 33) and the resulting absence of breakdowns in standing stocks in duckweed. In the ditches of agricultural areas in The Netherlands there is indeed a trend of Lemna and related duckweed genera replacing submerged macrophytes with increasing nutrient levels (34). The exposed macrophytes were about 5 times less sensitive to Irgarol than the periphyton community in this study (7). This result supports the findings of Nystro¨m et al. (28) and Okamura et al. (13). Nystro¨m et al. (28) found that the phytoplankton community of Lake Geneva (EC20, CO2 uptake: 0.1 µg L-1) was about 200 times more sensitive to shortterm exposure than the macrophyte Elodea canadensis (EC20 (fluorescence): 20 µg L-1). Differences were less pronounced in the study of Okamura et al. (factor 100; (13)). The much longer exposure time of 150 d of the macrophytes, which had been directly planted into the ponds, may have been the reason for the higher sensitivity of the macrophytes in the study presented here as compared to short-term laboratory results (1-7 d) for macrophytes (13, 28). This assumption is underlined by the results for M. verticillatum. The EC50 of 1.1-3 µg L-1 for potted sprouts of M. verticillatum after 48 days of exposure was in the same range as found for M. spicatum in a 14 d laboratory experiment (2 µg L-1) carried out by Lambert et al. (10), whereas the EC50 for M. verticillatum, which had been directly planted in the ponds, was lower by factor 10 after 150 days of exposure. A decrease in EC50 concentration with longer exposure duration was also observed for P. nodosus leaf counts in this study and for Potamogeton natans and M. verticillatum exposed to the herbicide metazachlor in a previous mesocosm study (23).

TABLE 2. Time-Weighted Average Concentration (TWA) of Irgarol, Content of Irgarol in the Fresh Weight (FW) and Dry Weight (DW) of Different Macrophyte Species, and the Respective Bioaccumulation Factor (BCF) Myriophyllum verticillatum

Potamogeton nodosus

filamentous algae

pond nominal start concentration (µg L-1)

TWA of Irgarol 150 d (µg L-1)

content (µg kg-1)

BCF (L kg-1)

content (µg kg-1)

BCF (L kg-1)

content (µg kg-1)

BCF (L kg-1)

FW 0.04 0.20 (1) 0.20 (2) 1.00 5.00 (1) 5.00 (2)

0.006 0.030 0.032 0.211 1.444 1.406

7.4 26.5 48.7 157 0 0

1330 895 1520 744 n.d. n.d.

0.94 8.20 6.38 60.0 257 307

169 277 199 284 178 218

13.5 68.7 67.6 372 600 406

2430 2320 2110 1760 415 289

DW 0.04 0.20 (1) 0.20 (2) 1.00 5.00 (1) 5.00 (2)

0.006 0.030 0.032 0.211 1.444 1.406

58.1 201 338 1530 0 0

10400 6790 10560 7250 n.d. n.d.

5.9 52.5 41.7 393 1950 2590

1050 1770 1310 1860 1640 1830

51.8 212 216 1110 1950 1350

9250 7160 6750 5260 1350 960

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It is still under debate which reference concentration should be used for EC50 calculations generated from longterm mesocosm experiments (23, 35). The BCF data show that Irgarol has bioaccumulative potential in macrophytes and the rapid decrease of Irgarol in the water phase directly after application has to be attributed to strong adsorption effects (7). Therefore, nominal concentrations seem to better reflect the toxic potential of Irgarol on the macrophyte community in the pond mesocosm than the TWA concentrations. Bioaccumulation in Macrophytes. According to OECD guideline 305 (26) for bioaccumulation in fish, the BCF can be related to the contents in both fresh weight and fat. There is yet no convention how to calculate the BCF in plant tissue. If the calculation with dry weight is applied, the range of BCF, which is considered to indicate bioaccumulation, should be redefined. Furthermore, steady state conditions are a prerequisite for the calculation of BCF in laboratory tests. These conditions could not be achieved in the mesocosm studies at hand. Nevertheless, it is considered appropriate to calculate the BCF in this study since the TWA approach was used. According to the guidance document on aquatic ecotoxicology, BCF values exceeding 1000 give reason for concern (36) since bioaccumulation is indicated. The BCF (dry weight) for P. nodosus ranged from 1050 to 1860 L/kg, which is in the same order of magnitude as the BCF (dry weight) for Potamogeton pectinatus in Lake Geneva at Irgarol water concentrations between 0.105 and 0.135 µg L-1 (28). The BCF for Elodea canadensis at another sampling site in Lake Geneva with Irgarol concentrations in the free water of 0.039 µg L-1 even amounted to 4500 (28). The BCF for Irgarol in macrophytes was high especially in M. verticillatum and filamentous algae, which both have a large surface to volume ratio. It seems that plants with a larger surface have higher uptake rates as demonstrated for P. nodosus < M. verticillatum < filamentous algae with regard to the BCF for fresh weight. Highest contents of Irgarol have been reported for the green alga Tetraselmis suecica amounting to 108,600 µg kg-1 DW and the BCF ranged between 15,500 and 84,800 L kg-1 (37). In that experiment both scenario and analysis were different compared to the study presented here: 3 tanks of 400 L containing the green alga had been spiked with Irgarol to a start concentration of 1 µg L-1. After one week, Irgarol levels were raised again to 1 µg L-1. The algae collected on dried glass fiber filters were extracted and analyzed by means of high-performance liquid chromatography-mass spectrometry (HPLC-MS). The BCF was calculated from the contents of Irgarol related to dry weight since fresh weight of the algae cannot be determined on wet fiberglass filters. The noticeable differences between the BCF related to either fresh or dry weight in this study make it clear that a harmonized test procedure for bioaccumulation in macrophytes is needed. Irgarol concentration in the exposed macrophytes was highest in the 0.04 µg L-1 dosed pond (18%) and lowest in the 5 µg L-1 contaminated ponds (2%). The macrophytes in the highest treatments were strongly affected which resulted in reduced biomass and less uptake. Therefore, the uptake of Irgarol by macrophytes is an important contribution to the mass balance of the compound. In particular slightly impacted macrophytes and algae seem to be a considerable sink for Irgarol in the water column. The loss of macrophytes can cause long-term ecosystem changes (20, 22, 38). TBT has been claimed to be responsible for macrophyte decline in the Norfork Broads (39). Since its ban, TBT concentrations decreased but the macrophytes did not recover in this area, mainly due to the increase of other biocides such as Cu and Irgarol (39, 40). The results of this study indicate that Irgarol is likely to have a serious impact 6842

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on natural macrophyte communities at environmentally relevant concentrations. In our own field monitoring measurements in streams and lakes in North Germany Irgarol was detected in all samples with concentrations up to 0.224 µg L-1 (24) which is in the EC50 range of M. verticillatum. The fact that Irgarol accumulates in macrophytes especially at lower concentrations may even lead to an underestimation of the expected toxicity at actual water concentrations of Irgarol in freshwater.

Acknowledgments We thank department IV 2.5 of the Federal Environment Agency for the technical assistance. Bernhardt Katona, Sabine Rust, and Dagmar Schnee are especially acknowledged for their help in the analytical measurements. The manuscript benefited from valuable comments of three anonymous reviewers.

Note Added after ASAP Publication There were minor text errors in the version of this paper published ASAP July 7, 2009; the corrected version published ASAP July 17, 2009.

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