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Risk posed by the antifouling agent Irgarol 1051 to the seagrass, Zostera marina
Aquatic Toxicology 45 (1999) 159 – 170
Risk posed by the antifouling agent Irgarol 1051 to the seagrass, Zostera marina A. Scarlett a, P. Donkin a,*, T.W. Fileman a, S.V. Evans a, M.E. Donkin b b
a Plymouth Marine Laboratory, West Hoe, Plymouth, De6on PL1 3DH, UK Plymouth En6ironmental Research Centre, Uni6ersity of Plymouth, Drake Circus, Plymouth, De6on PL4 8AA, UK
Received 2 April 1998; received in revised form 16 July 1998; accepted 21 July 1998
Abstract Irgarol 1051 (2-(tert-butylamino)-4-cyclopropylamino)-6-(methylthio)-1,3,5-triazine) is a triazine herbicide that is increasingly being used to boost the effectiveness of antifouling paints. Estuarine plants, such as the marine angiosperm Zostera marina L. (eelgrass) may accumulate, and be affected by, Irgarol 1051, in locations with high boat densities. Bioconcentration of Irgarol 1051 within Zostera tissue was determined in field plants and laboratory semi-static exposure experiments. Effects of Irgarol 1051 upon the growth rate and photosystem II photosynthetic efficiency of Zostera were examined over a concentration range of 0 to 25 mg dm − 3. Growth rate was assessed by comparison of leaf specific biomass ratios, and was found to be reduced at and above an Irgarol 1051 concentration of 10 mg dm − 3. Photosynthetic efficiency was assessed using fluorescence induction kinetics: efficiency was significantly reduced at 0.18 mg dm − 3 (0.4 mg g − 1 dry weight leaf tissue) and a 10-day EC50 value of 2.5 mg dm − 3 (1.1 mg g − 1) calculated. Longer-term exposure revealed a 36-day EC50 value of 0.2 mg dm − 3. Uptake of Irgarol 1051 was rapid within the Zostera leaves: tissue concentrations (dry weight basis) in excess of 300 times the water concentration were found within 2 days of exposure. Leaf concentrations in excess of 14 times root tissue concentration were found. Estuaries sampled in S.W. England had low aqueous Irgarol 1051 contamination, typically B 0.003 mg dm − 3, but Zostera leaf tissue concentrations (dry weight basis) were up to 25000 times the aqueous values; this was only 15 times below the 10-day EC50 value. The reported results will enable the level of risk to isolated Zostera meadows from Irgarol 1051 to be assessed based on leaf tissue concentration and also have implications for the siting of marinas. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Irgarol; Triazine; Bioconcentration; Zostera; Photosynthesis; Fluorescence
* Corresponding author. Tel.: +44 1752 633458; fax: + 44 1752 633101; e-mail: [email protected] 0166-445X/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII S0166-445X(98)00098-8
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1. Introduction Many antifouling paints currently contain the herbicide Irgarol 1051, 2-(tert-butylamino)-4-cyclopropylamino)-6-(methylthio)-1,3,5-triazine, the use of which has risen over the past decade since the application of paints containing tri-n-butyltin (TBT) was restricted to vessels over 25 m. Recent studies (Readman et al., 1993; Gough et al., 1994; Zhou et al., 1996; Tolosa et al., 1996; Scarlett et al., 1997) have shown that Irgarol 1051 is now widely distributed in European estuarine and coastal waters and sediments; reported estuarine aqueous concentrations are in the range B 0.001 – 0.19 mg dm − 3 and concentrations within ports and marinas of up to 1.7 mg dm − 3. Degradation of Irgarol 1051 in sea- and freshwater sediment is slow, with half lives of 100 and 200 days, respectively; under anaerobic conditions, the degradation is considerably slower and Irgarol 1051 is not readily biodegraded (Ciba Geigy, 1995). Irgarol 1051 is a particularly effective algicide: a reduction in photosynthetic activity EC50 value of 0.82 nM (0.205 mg dm − 3) was reported by Dahl and Blanck (1996) for marine microalgae, and a photosystem II (PSII) efficiency inhibition EC50 value of 2.5 mg dm − 3 was reported by Scarlett et al. (1997) for the marine green macroalga Enteromorpha intestinalis L. Accumulation of Irgarol 1051 has been shown to occur in freshwater macrophytes (To´th et al., 1996) with concentrations of up to 30000 times that within the surrounding water. Seagrasses are marine angiosperms that occur in dense beds in both temperate and tropical shallow-water coastal environments. Worldwide, seagrasses encompass only about 50 species, but are of great ecological importance (den Hartog, 1977). Seagrass meadows stabilise the seabed, creating habitats with high biodiversity and productivity (Edgar and Shaw, 1995). In the UK, Zostera marina L. (eelgrass) is the most common species of seagrass and is locally frequent around most of the coast, typically within estuaries, but has declined in recent decades (den Hartog, 1977; Rose, 1981). As a species of high conservation importance, Zostera is included within a number of recent international nature conservation desig-
nations (Davison, 1998). Estuaries are extensively used for the mooring of pleasure craft and are increasingly being used for the siting of marinas; Z. marina is, therefore, likely to be exposed to contamination from antifouling paint ingredients including Irgarol 1051. To our knowledge, bioconcentration of Irgarol 1051 within marine angiosperms, and its effect upon them, has not been previously reported.
2. Methodology
2.1. Standards and samples Irgarol 1051 (\97% purity) was obtained from Ciba Geigy, Basel, Switzerland; ametryn (\ 99% purity) was obtained from Promochem UK. Stock standard herbicide solutions (1 mg cm − 3 each) were prepared in acetone and stored at −15°C. For calibration purposes, a portion of these stock solutions were diluted with isohexane. Working standard solutions containing 1–100 mg cm − 3 of test compounds in isohexane were used to spike the plant and water samples. An Irgarol 1051 stock standard of 1 mg cm − 3 was prepared in methanol (MeOH), and dosing concentrations of 20 mg cm − 3 to 1 mg cm − 3 derived by dilution in MeOH for use in the exposure trials. All solvents were glass distilled or HPLC grade; all glassware was cleaned by ‘Decon 90’ washing followed by heating at 170°C for 24 h and finally solvent rinsed. Z. marina plants were collected from the Yealm estuary UK (50° 18.55% N, 4° 3.85% W) at low water spring tides (: midday) from Summer 1997 to Spring 1998 for use within exposure experiments. In situ measurement of photosynthetic efficiency and water sampling (at 0.5 m depth) was carried out concurrently with all tissue plant collection occasions. The Yealm estuary is a flooded ria system with low freshwater input and has relatively low Irgarol 1051 contamination (Scarlett et al., 1997), however, it has been used by International Paints for the testing of antifouling agents for a number of years and the environmental quality standard (EQS) was exceeded in the Yealm for TBT in 1994 and 1995, also for copper
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in 1995 (Environment Agency, 1996); additional Zostera beds within the Salcombe estuary (a similar but larger nearby estuary) were therefore monitored for comparative purposes. Both of the estuaries sampled have numerous small craft moorings but do not have marinas and are not used by large vessels. Prior to extraction and analyses, plant tissue was stored at − 15°C; water samples were stored at between 2 and 5°C for no longer than 3 days prior to processing.
2.2. Plant tissue extraction and analyses Z. marina leaf tissues, including sheath material (4.0 g wet weight), were cut into small sections; rhizomes and root tissues (4.0 g wet weight) were ground using a pestle and mortar for 5 min. Plant tissues were placed in cellulose extraction thimbles (20×50 mm, Whatman International UK), they were then mixed with :8 g anhydrous sodium sulphate (Fisher Scientific UK) and internal standard spikes (ametryn) added. Plant tissues were solvent extracted with 50 cm3 of 1:1 acetone/ dichloromethane (DCM), for 20 h using 25 mm bore soxhlets. The extracts were concentrated to near dryness under nitrogen; 2 cm3 of isohexane was added and the extract evaporated down as before, this was repeated three times to ensure that all the acetone was removed. The samples were purified using alumina column chromatography eluted with solvents of increasing polarity. Alumina was prepared from neutral aluminium oxide (100 – 250 mesh, Camag). Chromatographic glass columns (10× 100 mm) were filled with 3 g of alumina (5% de-activated with Milli-Q water) and topped with 1 cm of anhydrous sodium sulphate. The sample extracts were loaded onto the columns in 2 cm3 of isohexane and eluted with 20 cm3 of isohexane followed by 20 cm3 of isohexane/DCM (1:1). The last 20 cm3 was collected and concentrated to 0.3 cm3 under nitrogen. Extracts were analysed by capillary gas chromatography with mass selective detection using a Hewlett Packard 5890 GC (HP5MS capillary column: 30 m ×0.25 mm ×0.25 mm; the carrier gas was helium) coupled to a Hewlett Packard 5972A mass selective detector (electron impact,
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quadrupole). Other GC conditions were as described by Zhou et al. (1996). Irgarol 1051 and ametryn concentrations were determined using selective ion monitoring, and quantified using masses m/z 253 (additional qualifier ions m/z 182 and 238) and m/z 212 (additional qualifier ions m/z 184 and 227), respectively.
2.3. Water extraction Extraction of Yealm and Salcombe water was carried out as described by Zhou et al. (1996) and analysed by GC-MS as above. In summary, water samples were filtered (Whatman GF/F filter) and spiked with ametryn internal standard; the filtrate was passed through solid phase extraction cartridges (Jones Chromatography Isolute C18 trifunctional, 1 g, preconditioned with ethyl acetate, methanol and water) which were each eluted with 3 cm3 of ethyl acetate to recover the Irgarol 1051. Extraction of water samples from the laboratory exposure trials was by solvent extraction. Water taken from beakers during water changing was transferred into 500 cm3 volumetrics, these were shaken for 2 min with 5-cm3 aliquots of isohexane (×3) and rinsed with a further 5 cm3 to extract the Irgarol 1051. Ametryn internal standards were added to the extracts, which were then placed in a freezer at −15°C to facilitate separation of the solvent from the water. Extracts were blown down under nitrogen to small volumes and analysed by GC-MS as described above.
2.4. Exposure experiments Laboratory exposure trials utilised 2 dm3 Pyrex beakers, each containing five plants, equivalent to a field density of : 1200 blades m − 2; field values of up to this value occur within the estuaries sampled (personal observation). Only non-flowering plants with no apparent disease or damage were selected for the trials; these were standardised by removing all but the youngest three leaves, which were trimmed to 20 cm above the basal leaf meristem and wiped with paper towel to remove epiphytes and epifauna. Rhizomes were standardised to three segments. Plants were acclimated for 6 days in filtered (Whatman GF/F prefilter + 0.45
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mm cellulose nitrate filter,) seawater collected off the UK Southwest coast, close to the Eddystone lighthouse, and kept at a constant temperature of 15°C under ‘cool light’ fluorescent tubes giving a photon fluency rate of 25 mmol quanta m − 2 s − 1 photosynthetically active radiation (PAR), as measured by a standard PAR meter (Skye Instruments). A semi-static dosing system was used to expose the plants to Irgarol 1051; nominal concentrations were 25.0, 10.0, 5.0, 2.5, 1.0, 0.5 and 0.0 mg dm − 3 plus MeOH controls (n= 3). This experiment was carried out to establish concentration/response relationships. Concentrations were achieved by injecting 0.5 cm3 of Irgarol 1051 working standard into 20 dm − 3 of Eddystone water which was thoroughly vortex mixed for 2 h prior to dosing; carrier controls were treated similarly, i.e. 0.0025% MeOH. Water was changed in the beakers after 24 and 48 h, and thereafter every 48 h until the trial was stopped after 10 days. A time course experiment was also performed to monitor the uptake of Irgarol 1051 over a 10-day period. Plants were exposed to an Irgarol 1051 concentration of 2.5 mg dm − 3 plus controls under conditions as above (start of experiment: n=8). Water was changed in the beakers every 48 h. During each water change, one replicate beaker from each of the treatments was removed and the plant tissues and water analysed for the presence of Irgarol 1051 (by the end of experiment: n =4). Z. marina leaves from both the dose response and time course exposure experiments were subject to growth analyses and tested for two parameters of photosynthetic efficiency. In order to investigate the effects of longerterm low-level exposure to Irgarol 1051, i.e. similar to concentrations reported in UK estuaries (Gough et al., 1994), a 36-day experiment was performed. Plants were exposed to a nominal concentration of 0.225 mg dm − 3 plus controls (treatment replication: n = 3); experimental conditions were as above, except that only three plants per beaker were used. Photosynthetic efficiency parameters were monitored but growth analysis was not performed.
2.5. Exposure experiments: photosynthetic efficiency parameters The parameters used to assess plant stress were the dark adapted fluorescence induction ratio Fv:Fm, which is an indicator of the maximum efficiency of PSII (Krugh and Miles, 1996), and ‘Area’, which is proportional to the pool size of the primary quinone electron acceptor (QA) on the reducing side of PSII (Hall et al., 1993). The key fluorescence parameters of F0 (time zero fluorescence), Fm (maximum fluorescence) and Fv (variable fluorescence Fm − F0), were also recorded. Analyses were performed during water changes by a plant efficiency analyser (PEA; Hansatech Instruments UK). Dark adaptation time was determined to be optimum at 20 min; 50% light intensity (1500 mmol quanta m − 2 s − 1 at 650 nm peak wavelength) of 1-s exposure was sufficient for light saturation. During laboratory 10-day exposure trials, six readings were taken from leaves from each of three replicate beakers per treatment: a total of 18 readings per treatment level. During in situ monitoring of field plants, three readings were recorded from each of six plants per site.
2.6. Exposure experiments: growth analyses Growth was assessed using an adaptation of the leaf-marking techniques of Zieman (1974) and Dennison (1990). Leaves were puncture marked by syringe at 1 cm above the basal leaf meristem, after 10 days the plant tissue was divided into old and new growth; only the inner, youngest, leaves were measured. Leaf tissue above the puncture scar was considered old biomass, tissue below the scar but above the meristem, plus young leaves without a scar were considered new biomass. Following separation, tissues from each beaker were pooled and were placed in preweighed aluminium pans, dried for 1 week at 80°C, and weighed. The samples were then combusted at 500°C for 8 h, ash weights were obtained, and ash free dry weights were calculated. The ratio of new to old biomass provides a biomass specific ratio which was used as an indicator of plant growth. Growth rate analysis was not performed during the 36-day experiment.
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2.7. Statistical methods A preliminary analysis of the data was performed using Bartlett’s test for homogeneity of variance. Plant growth and Fv:Fm data was found to be homogenous: differences between doses (treatments) were tested for significance by a oneway analysis of variance (ANOVA), with an alpha of 0.05. Significant differences between treatments were presumed to exist at P-values less than or equal to 0.05. Tests for least significant differences were performed between individual treatments (PB0.05) when there was a significant P-value. The Area parameter data was found not to be homogenous: differences between doses were tested for significance by the Krustall – Wallis test and differences between individual treatments were assessed using heteroscedastic t-tests. Statistical analyses were performed using Statgraphics vers. 7.1 and Excel vers. 7.
3. Results
3.1. Analytical performance The calibration curve for Irgarol with GC-MS was linear over the range 0 – 10 mg dm − 3 (r = 0.9998, PB0.01). Precision of data and limits of detection are given in Table 1. Recovery was determined by spiking reference substrates with known volumes of the working standard solution of Irgarol 1051; plant tissue was also subject to exhaustive extraction, i.e. until further extraction produced no significant increase in Irgarol 1051
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concentration. Replicate analyses of spiked Z. marina revealed reasonable precision with good recovery. The concentrations reported are the mean values of triplicate determinations and were corrected for recovery. Tissue concentrations are based on dry weights unless stated otherwise. Leaf tissue dry:wet ratio=0.15; root/rhizome dry:wet ratio=0.13). Bioconcentration factors and EC50 values are based on measured concentrations.
3.2. Field sampling stations Aqueous Irgarol 1051 concentrations were monitored prior to, and throughout, the Z. marina collection period; concentrations were typically B0.003 mg dm − 3 at both the Yealm and Salcombe estuary sampling stations, a maximum concentration of 0.010 mg dm − 3 occurred at the Yealm in January 1998. Salinity was determined to be 35‰ on all sampling occasions, confirming that the plants were not subject to brackish water conditions at either site. Plant tissue concentrations were in the range 0.002–0.050 mg g − 1 at the Yealm and 0.004–0.071 mg g − 1 at Salcombe, maximum concentrations occurred in August 1997 at both sites. In situ monitoring of plant efficiency parameters revealed typical Fv:Fm values in the range 0.80–0.84, although a mean value of 0.719 0.04 S.E. was recorded at Salcombe in August, values below 0.80 also occurred at both estuaries in March 1998. The Area parameter values were typically in the range 14000–30000 bits ms and were highly variable at both estuaries. No overall significant differences were found between the two estuaries.
Table 1 Analytical performance data Matrix
LODa
Recoveryb (%)
R.S.D.c (%)
Water (solid phase extraction) (ng dm−3) Water (solvent extraction) (ng dm−3) Plant tissue (ng g−1 dry wt.)
1.0 5.0 2.0
95 101 97 (92)
4.5 8.0 7.9
a
Limit of detection of the whole analytical procedure (signal-to-noise ratio =10). Derived from the slope of the regression line of standard addition at five different levels. Value in parenthesis: exhaustive extraction of plant tissue demonstrated that bioaccumulated Irgarol 1051 was less easily recovered than spikes. The lower value was used to correct tissue concentration. c Relative standard deviation was derived from replicate analysis of matrix (n = 3). b
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3.3. Uptake and bioconcentration of Irgarol 1051 The monitoring of tissue burden during the 2.5 mg dm − 3 exposure time course experiment revealed that uptake of Irgarol 1051 was rapid with a mean leaf tissue concentration of 0.223 9 0.077 mg g − 1 (S.D.) achieved within 2 days, this was over 300 times the measured water concentration of 0.660 mg dm − 3. Measured water concentrations were close to nominal concentrations by day 10 of the experiment. Accumulation was highly variable with a maximum range of 0.140 – 0.639 mg g − 1 occurring after seven days exposure; high variation was found both within and between replicates. The final mean concentration after 10 days exposure of 0.2439 0.053 mg g − 1 (S.D.) was not significantly higher than that after 2 days. Analyses of plant tissues from the 10-day concentration-response experiment revealed a linear relationship between Zostera leaf tissue concentration and water concentration (r =0.973, P B 0.01), but due to the accumulation within the tissue being relatively greater at the lower exposure concentrations, it was better described by the log – log transformation log y = 0.588 log x + 0.966 (r=0.998), where y =leaf concentration (ng g − 1 dry wt.) and x = water concentration (ng dm − 3). Accumulation of Irgarol 1051 within the roots and rhizomes was described by the linear function y= 0.026x + 26 (r= 0.999, units as above). Bioconcentration within roots/rhizomes was less variable but significantly lower (P B 0.001) than within the leaves: concentrations within the leaves were greater than five times that within the roots/rhizomes at the highest nominal exposure of 25 mg dm − 3 and \ 14 times at 0.5 mg dm − 3. No significant difference was observed between the leaves and rhizomes of the controls or field Zostera. From the field data for the estuaries sampled (see above), a BCF (tissue concentration }water concentration) of up to 25000 times was calculated for Zostera accumulation of Irgarol 1051.
3.4. Short-term exposure experiments: photosynthetic efficiency parameters During the acclimation period, Fv:Fm and Area
parameter values increased significantly (PB 0.001). Fv:Fm values increased from a mean of 0.832 to 0.855 and stabilised to a very low relative standard deviation (R.S.D.) of 1%. Area values increased from a mean of 16206 to 19950 bits ms, but retained high variation with a R.S.D. of 20%. During the exposure period, no significant difference was observed between the controls and MeOH controls; Fv:Fm values remained stable with very low variation; Area values fluctuated and maintained a high degree of variation. Monitoring of water concentrations revealed that with the exception of the 0.5 and 1.0 mg dm − 3 treatments, which had final measured concentrations of 0.178 and 0.619 mg dm − 3, respectively, steady state conditions had been achieved within the 10-day exposure period, with analysed values close to nominal values. No Irgarol 1051 was detectable within the water of the controls. All plants retained a healthy appearance throughout the experiment and epiphytic growth was minimal. After 1 day’s exposure to an Irgarol 1051 concentration of 2.5 mg dm − 3 and above, Fv:Fm values dropped significantly (PB 0.01, Fig. 1). The Area parameter values only fell significantly (PB 0.01) at treatment levels of 10 mg dm − 3 and above on day one of the experiment (Fig. 2). The variation in measured parameters within the Irgarol 1051 treatments increased during the initial exposure; this variation was also observed in measurements taken from the same plant. By day 4 of the trial, the Fv:Fm values for all treatments were significantly lower (PB 0.05) than the controls, but only treatments of ] 2.5 mg dm − 3 had affected the Area values (Figs. 1 and 2). By the end of the experiment, Fv:Fm values had fallen further: the 0.5 mg dm − 3 treatments had declined by about 10% (Fv:Fm = 0.774) during the 10 days and the 25 mg dm − 3 was reduced by nearly 80% (Fv:Fm = 0.219). From the Fv:Fm dose–response curve (Fig. 1), a 10-day EC50 value of 2.5 mg dm − 3 was interpolated. Area values at the end of the experiment had dropped to near zero for treatments ] 5.0 mg dm − 3, but the lowest treatment of 0.5 mg dm − 3 was unaffected. Plants exposed to 1.0 mg dm − 3 had Area values signifi-
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Fig. 1. Effect of aqueous Irgarol 1051 on the Zostera marina leaf Fv:Fm values (expressed as a % of the methanol control value) after 1, 4 and 10 days exposure (error bars = + 1 standard error).
cantly higher (P B0.05) than the controls. From the Area dose–response curve (Fig. 2) a 10-day EC50 of 2.6 mg dm − 3 was interpolated. Fv:Fm values were significantly reduced (P B0.001) at a leaf tissue concentration of 0.395 mg g − 1 (Fig. 3). The EC50 value based on leaf dry tissue was interpolated to be 1.1 mg g − 1 for the Fv:Fm response (Fig. 3). As stated above, the accumulation of Irgarol 1051 within the root/rhizome tissue was substantially less than within the leaves and this is reflected in the concentration/response relationships (Fig. 3); however, it would be inappropriate to calculate EC50 values for a photosynthetic response based on root tissue concentration. Photosynthetic efficiency parameters obtained during the time course experiment were in accord with that of the dose–response experiment; the mean leaf tissue concentration of 0.223 mg g − 1 after 2 days exposure, caused a significant (P B0.001) reduction in Fv:Fm of over 15%.
3.5. Short-term exposure experiments: growth rate Substantial leaf growth occurred at all treatment levels during the exposure period. Results
comparing specific biomass ratios were similar when calculated using wet, dry and ash free dry weights. Significant reduction in growth rate (PB 0.05) only occurred at and above the 10 mg dm − 3 treatment level (Fig. 4). No significant reduction in growth occurred during the time course experiment, carried out at an exposure concentration of 2.5 mg dm − 3.
3.6. Long-term exposure experiments Due to the lack of steady state conditions achieved at the lower exposure concentrations during the 10-day exposure experiments, it was deemed necessary to carry out a longer-term exposure experiment at an environmentally realistic Irgarol 1051 concentration. Plants were exposed to a nominal concentration of 0.225 mg dm − 3 for 36 days, measured concentration at the end of the experiment was 0.20090.025 mg dm − 3. The Irgarol 1051 treated plants showed a decreasing PSII efficiency throughout the experiment (linear relationship y= 107–1.56x; r= 0.983, PB 0.001; where y is the % of the control PSII efficiency and x is the exposure period in days). The final mean Fv:Fm value of the plants exposed to Irgarol 1051
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was 0.49 0.03 S.E. compared to 0.89 0.02 S.E. for the controls, i.e. 36-day PSII efficiency inhibition EC50 values of 0.20 mg dm − 3 and 2.6 mg g − 1 (measured aqueous and leaf tissue dry wt. concentrations, respectively). By the end of the trial the Irgarol 1051 treated plants were beginning to exhibit a dramatic drop in the Area parameter, although this was not statistically significant.
4. Discussion The PSII photosynthetic efficiency of Z. marina leaves, as measured by the decline in Fv:Fm, was significantly reduced (P B 0.01) by about 10% at a measured Irgarol 1051 concentration of 0.18 mg dm − 3 (Fig. 1); this water concentration is below that reported within the marine environment in the UK (Gough et al., 1994; Zhou et al., 1996), the Mediterranean (Readman et al., 1993; Tolosa et al., 1996) and Bermuda (Readman, personal communication). Estuarine concentrations tend to be lower than the above concentration, although Gough et al. (1994) reported Irgarol 1051 concentrations in the Hamble estuary (Solent, UK) of up to 0.19 mg dm − 3. The 10-day concentration – re-
sponse experiment revealed that the Area parameter was either unaffected, or stimulated, at current environmental concentrations. The Area parameter is potentially a very useful indicator of stress caused by triazine herbicides, as it is dramatically reduced when electron transfer from the chlorophyll reaction centres to the quinone pool is blocked (Hall et al., 1993), as caused by the mode of action of triazines. The results indicate that a time lag exists between the initial fall in Fv:Fm and the subsequent dramatic fall in Area (Figs. 1 and 2). It is therefore unclear whether long term chronic exposure to concentrations below 1 mg dm − 3 would result in a measurable decline in the Area parameter. The high variation in Area, observed in both field Zostera and exposure experiment plants, make this parameter difficult to assess statistically: its use as in indicator of herbicide induced stress in the field is therefore questionable. The long-term exposure experiment did indicate that the Irgarol 1051 treated plants were beginning to exhibit a dramatic drop in the Area parameter by the end of the trial; although this was not statistically significant, it suggests that the turnover rate of the PSII subunit D1 protein, the binding site of triazine herbicides (He and
Fig. 2. Effect of aqueous Irgarol 1051 on the Zostera marina leaf Area parameter values (expressed as a % of the methanol control value) after 1, 4 and 10 days exposure (error bars = + 1 standard error).
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Fig. 3. Effect of Irgarol 1051 tissue concentration upon Zostera marina leaf Fv:Fm values (expressed as a % of the methanol control value) after 10 days exposure (error bars = + 1 standard error).
Malkin, 1998), is barely sufficient to allow continued electron-transport at a concentration of 0.20 mg dm − 3. The low Fv:Fm values observed at Salcombe in August 1997 coincided with the highest recorded Irgarol 1051 tissue concentration. The low PSII efficiency can, however, be attributed to elevated F0 values associated with photoinhibition (Bolha`r-Nordenkampf et al., 1989) caused by exposure to high light intensity. Although the Fv:Fm parameter provides a useful indicator of general stress of plants in situ, a longer period of dark adaptation, or neap tide sampling, may be advisable when monitoring normally sub-tidal plants, such as Z. marina, in order to help distinguish between natural photoinhibition stress and damage from phytotoxic chemicals. Zostera beds typically contain macroalgae; under certain environmental conditions, rapid growing algae such as Enteromorpha spp. can smother and eliminate the Zostera through shading (den Hartog, 1994). Mature E. intestinalis has a reported 3-day Fv:Fm response EC50 value of 2.5 mg dm − 3 (Scarlett et al., 1997), i.e. similar to that of
Z. marina; in addition, the zoospore reproductive stage of E. intestinalis was shown to be highly responsive to Irgarol 1051, with a 6-day growth response EC50 value of 0.5 mg dm − 3. The data indicates that the presence of Irgarol 1051 would not provide Enteromorpha with a competitive advantage. An important function of Zostera habitats is the provision of substrates for a high biodiversity of epiphytic algae. Dahl and Blanck (1996) reported a reduction in photosynthetic activity EC50 value of only 0.82 nM (0.205 mg dm − 3) for marine periphyton exposed to Irgarol 1051 over a 3-week period. It is therefore likely that aqueous Irgarol 1051 concentrations of below 0.2 mg dm − 3 will impact the flora associated with Zostera beds as well as the seagrasses. Accumulation of Irgarol 1051 was found to be far greater within the leaves than the roots/rhizomes during the laboratory exposure experiment, but this difference was not evident within the field Zostera sampled. It is therefore likely that the uptake of the herbicide is merely slower into the rhizome, this may be due to the smaller surface
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area:volume ratio and/or greater resistance to diffusion across the rhizome epidermis. Although these results show that it is preferable to sample the leaves of field plants in order to assess potential stress resulting from tissue burden, it would be acceptable to measure rhizome concentrations if it were not possible to sample the leaves: such as where Zostera populations are not winter green, as is the case for most intertidal populations in the UK. Bioconcentration of Irgarol 1051 has not been previously reported in marine macrophytes, but To´th et al. (1996) reported concentrations within freshwater macrophytes and algae in the range 0.053 – 0.103 mg g − 1 dry wt. (measured water concentrations were in the range 0.0025 – 0.145 mg dm − 3) at Port d’Ouchy (Lake Geneva, Switzerland) and 0.004–0.0052 mg g − 1 (water concentrations B 0.0003 mg dm − 3) at a Lake Geneva reference site at Buchillon (Switzerland). Z. marina mean tissue concentrations at the Yealm and Salcombe estuaries were in the range 0.002 – 0.071 mg g − 1 (water concentration 0.001 – 0.010 mg dm − 3), which suggests that accumulation of Irgarol 1051 is similar in Zostera and some freshwater macrophytes. BCF values were found to
be greater in plants at the lower exposure concentrations, and very high at the estuaries sampled: field Zostera BCF values (dry weight basis) were up to 25000× ; To´th et al. (1996) calculated values up to 30000×. It was noted in the exposure experiment, that control plants had similar Irgarol 1051 concentrations at the end of trial, i.e. up to 42 days in uncontaminated seawater, to that of the field population from which they were taken: this indicates that depuration is either minimal or does not occur. Plants in estuaries that receive regular tidal or freshwater flushing, are unlikely to loose their Irgarol 1051 tissue burden through simple diffusion. Despite the relatively low aqueous Irgarol 1051 contamination within the sampled estuaries, some Zostera plants had tissue concentrations that were only 15 times below the calculated 10-day PSII efficiency inhibition EC50 value of 1.1 mg g − 1, and less than three times below that shown to significantly reduce PSII efficiency. This represents only a very small ‘margin of safety’, especially as other phytotoxic chemicals are likely to be present within estuaries (House et al., 1997).
Fig. 4. Z. marina leaf biomass specific ratios from 10-day semi-static Irgarol 1051 exposure (error bars = +1 standard deviation). Treatments that are significantly different to the methanol control (P B0.05) are indicated by an asterisk (*).
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Reduction in growth rate, as assessed by leaf specific biomass ratios, was only observed at concentrations at or above 10 mg dm − 3 (Fig. 4). Schwarzschild et al. (1994), using a static 10-day atrazine exposure, reported that Z. marina leaf specific biomass ratios were not significantly reduced at a measured concentration of 80 mg dm − 3. These results corroborate the data of Dahl and Blanck (1996), which indicated that Irgarol 1051 was considerably more toxic than atrazine to coastal marine plants. The exposure period of 10 days used in the present growth rate study, may be too short for the reduction in photosynthetic efficiency to measurably affect the growth rate at low Irgarol 1051 concentrations. It is also possible that the plants compensate for the loss of efficiency by some means, e.g. increasing their chlorophyll content, density of chloroplasts or relative leaf area; these parameters were not assessed. Loss of photosynthetic efficiency must however, carry an energetic cost and therefore have implications for a plant’s ability to cope with additional stresses, including competition from other organisms and environmental factors, both natural and anthropogenic.
5. Conclusions Z. marina plants that occur close to marinas, or other areas where boat density is high, are likely to experience reduced photosynthetic efficiency of PSII, but the number of Zostera beds in the UK that may be affected is unknown due to the lack of up to date distribution data in the literature. Within the relatively clean estuaries of S.W. England, there is only a small margin of safety; elsewhere in the world, especially where plants are continuously exposed to high levels of Irgarol 1051 throughout the year, Zostera beds are likely to be damaged. The results indicate that the siting of new marinas in close proximity to Zostera beds, or an increase in the use of Irgarol 1051, is likely to cause stress to the plants. Multi-toxicant exposure experiments are required to fully assess the risk to estuarine macrophytes posed by phytotoxic chemicals.
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Acknowledgements The authors gratefully acknowledge Ciba Geigy (UK) Additives Division for the gift of the Irgarol 1051 used in this investigation. We also thank Aref Sheikh for his assistance during field sampling. The research was funded by the Natural Environment Research Council, grant reference: GR9/03123.
References Bolha`r-Nordenkampf, H.R., Long, S.P., Baker, N.R., O8 quist, G., Schreiber, U., Lechner, E.G., 1989. Chlorophyll fluorescence as a probe of the photosynthetic competence of leaves in the field: a review of current instrumentation. Func. Ecol. 3, 497 – 514. Ciba Geigy, 1995. Irgarol 1051 Material Safety Data Sheet 638/9501. Ciba Geigy Limited, Additives Division, Basle, Switzerland. Dahl, B., Blanck, H., 1996. Toxic effects of the antifouling agent Irgarol 1051 on periphyton communities in coastal water microcosms. Mar. Pollut. Bull. 32, 342 – 350. den Hartog, C., 1977. Structure, function and classification in sea grass communities. In: McRoy, C.P., Helfferich, C. (Eds.), Seagrass Ecosystems. Marcel Dekker, New York, pp. 89 – 121. den Hartog, C., 1994. Suffocation of a littoral Zostera bed by Enteromorpha radiata. Aquat. Bot. 47, 21 – 28. Davison, D. M., 1998. An overview of the dynamic and sensitivity characteristics of Zostera as relevant to the conservation management of marine SACs. SAMS, Edinburgh (in press). Dennison, W.C., 1990. Leaf production. In: Phillips, R.C., McRoy, C.P. (Eds.), Seagrass Research Methods. UNESCO, Paris, pp. 77 – 79. Edgar, G.J., Shaw, C., 1995. The production and trophic ecology of shallow-water fish assemblages in Southern Australia. 3. General relationships between sediments, seagrasses, invertebrates and fishes. J. Exp. Mar. Biol. Ecol. 194, 107 – 131. Environment Agency, 1996. Local environment agency plan, Tamar estuary and tributaries, p. 97. Gough, M.A., Fothergill, J., Hendrie, J.D., 1994. A survey of Southern England coastal waters for the s-triazine antifouling compound Irgarol 1051. Mar. Pollut. Bull. 28, 613 – 620. Hall, D.O., Scurlock, J., Bolha`r-Nordenkampf, H., Leegood, R., 1993. Photosynthesis and Production in a Changing Environment. Chapman & Hall, London, pp. 197 – 198. He, W.Z., Malkin, R., 1998. Photosystems I and II. In: Raghavendra, A.S. (Ed.), Photosynthesis. A Comprehensive Treatise. Cambridge University Press, Cambridge, pp. 29 – 43.
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House, W.A., Leach, D., Long, J.L.A., Cranwell, P., Smith, C., Bharwaj, L., Meharg, A., Ryland, G., Orr, D.O., Wright, J., 1997. Micro-organic compounds in the Humber rivers. Sci. Tot. Environ. 194/195, 357–371. Krugh, B.W., Miles, D., 1996. Monitoring the effects of five ‘nonherbicidal’ pesticide chemicals on terrestrial plants using chlorophyll fluorescence. Environ. Toxicol. Chem. 15, 495 – 500. Readman, J.W., Wee Kwong, L.L., Grondin, D., Bartocci, J., Villeneuve, J.P., Mee, L.D., 1993. Coastal water contamination from a triazine herbicide used in antifouling paints. Environ. Sci. Technol. 27, 1940–1942. Rose, R. 1981. The Wild Flower Key, British Isles–N.W. Europe. Penguin, London, p. 450. Scarlett, A., Donkin, M.E., Fileman, T.W., Donkin, P., 1997. Occurrence of the marine antifouling agent Irgarol 1051 within the Plymouth Sound locality: implications for the green macroalga Enteromorpha intestinalis. Mar. Pollut. Bull. 34, 645 – 651. Schwarzschild, A.C., Macintyre, W.G., Moore, K.A., Libelo,
E.L., 1994. Zostera marina L. growth-response to atrazine in root-rhizome and whole plant exposure experiments. J. Exp. Mar. Biol. Ecol. 183, 77 – 89. Tolosa, I., Readman, J.W., Blaevoet, A., Ghilini, S., Bartocci, J., Horvat, M., 1996. Contamination of Mediteranean (Cote d’Azur) coastal waters by organotins and Irgarol 1051 used in antifouling paints. Mar. Pollut. Bull. 32, 335 – 341. To´th, S., Becker-van Slooten, K., Spack, L., de Alencastro, L.F., Tarradellas, J., 1996. Irgarol 1051, an antifouling compound in freshwater, sediment, and biota of Lake Geneva. Bull. Environ. Contam. Toxicol. 57, 426 – 433. Zhou, J.L., Fileman, T.W., Evans, S., Donkin, P., Mantoura, R.F.C., Rowland, S.J., 1996. Seasonal distribution of dissolved pesticides and polynuclear aromatic-hydrocarbons in the Humber estuary and Humber coastal zone. Mar. Pollut. Bull. 32, 599 – 608. Zieman, J.C., 1974. Methods for the study of the growth and production of the turtle grass Thalassia testudinum Konig. Aquaculture 4, 139 – 143.
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Risk posed by the antifouling agent Irgarol 1051 to the seagrass, Zostera marina A. Scarlett a, P. Donkin a,*, T.W. Fileman a, S.V. Evans a, M.E. Donkin b b
a Plymouth Marine Laboratory, West Hoe, Plymouth, De6on PL1 3DH, UK Plymouth En6ironmental Research Centre, Uni6ersity of Plymouth, Drake Circus, Plymouth, De6on PL4 8AA, UK
Received 2 April 1998; received in revised form 16 July 1998; accepted 21 July 1998
Abstract Irgarol 1051 (2-(tert-butylamino)-4-cyclopropylamino)-6-(methylthio)-1,3,5-triazine) is a triazine herbicide that is increasingly being used to boost the effectiveness of antifouling paints. Estuarine plants, such as the marine angiosperm Zostera marina L. (eelgrass) may accumulate, and be affected by, Irgarol 1051, in locations with high boat densities. Bioconcentration of Irgarol 1051 within Zostera tissue was determined in field plants and laboratory semi-static exposure experiments. Effects of Irgarol 1051 upon the growth rate and photosystem II photosynthetic efficiency of Zostera were examined over a concentration range of 0 to 25 mg dm − 3. Growth rate was assessed by comparison of leaf specific biomass ratios, and was found to be reduced at and above an Irgarol 1051 concentration of 10 mg dm − 3. Photosynthetic efficiency was assessed using fluorescence induction kinetics: efficiency was significantly reduced at 0.18 mg dm − 3 (0.4 mg g − 1 dry weight leaf tissue) and a 10-day EC50 value of 2.5 mg dm − 3 (1.1 mg g − 1) calculated. Longer-term exposure revealed a 36-day EC50 value of 0.2 mg dm − 3. Uptake of Irgarol 1051 was rapid within the Zostera leaves: tissue concentrations (dry weight basis) in excess of 300 times the water concentration were found within 2 days of exposure. Leaf concentrations in excess of 14 times root tissue concentration were found. Estuaries sampled in S.W. England had low aqueous Irgarol 1051 contamination, typically B 0.003 mg dm − 3, but Zostera leaf tissue concentrations (dry weight basis) were up to 25000 times the aqueous values; this was only 15 times below the 10-day EC50 value. The reported results will enable the level of risk to isolated Zostera meadows from Irgarol 1051 to be assessed based on leaf tissue concentration and also have implications for the siting of marinas. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Irgarol; Triazine; Bioconcentration; Zostera; Photosynthesis; Fluorescence
* Corresponding author. Tel.: +44 1752 633458; fax: + 44 1752 633101; e-mail: [email protected] 0166-445X/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII S0166-445X(98)00098-8
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1. Introduction Many antifouling paints currently contain the herbicide Irgarol 1051, 2-(tert-butylamino)-4-cyclopropylamino)-6-(methylthio)-1,3,5-triazine, the use of which has risen over the past decade since the application of paints containing tri-n-butyltin (TBT) was restricted to vessels over 25 m. Recent studies (Readman et al., 1993; Gough et al., 1994; Zhou et al., 1996; Tolosa et al., 1996; Scarlett et al., 1997) have shown that Irgarol 1051 is now widely distributed in European estuarine and coastal waters and sediments; reported estuarine aqueous concentrations are in the range B 0.001 – 0.19 mg dm − 3 and concentrations within ports and marinas of up to 1.7 mg dm − 3. Degradation of Irgarol 1051 in sea- and freshwater sediment is slow, with half lives of 100 and 200 days, respectively; under anaerobic conditions, the degradation is considerably slower and Irgarol 1051 is not readily biodegraded (Ciba Geigy, 1995). Irgarol 1051 is a particularly effective algicide: a reduction in photosynthetic activity EC50 value of 0.82 nM (0.205 mg dm − 3) was reported by Dahl and Blanck (1996) for marine microalgae, and a photosystem II (PSII) efficiency inhibition EC50 value of 2.5 mg dm − 3 was reported by Scarlett et al. (1997) for the marine green macroalga Enteromorpha intestinalis L. Accumulation of Irgarol 1051 has been shown to occur in freshwater macrophytes (To´th et al., 1996) with concentrations of up to 30000 times that within the surrounding water. Seagrasses are marine angiosperms that occur in dense beds in both temperate and tropical shallow-water coastal environments. Worldwide, seagrasses encompass only about 50 species, but are of great ecological importance (den Hartog, 1977). Seagrass meadows stabilise the seabed, creating habitats with high biodiversity and productivity (Edgar and Shaw, 1995). In the UK, Zostera marina L. (eelgrass) is the most common species of seagrass and is locally frequent around most of the coast, typically within estuaries, but has declined in recent decades (den Hartog, 1977; Rose, 1981). As a species of high conservation importance, Zostera is included within a number of recent international nature conservation desig-
nations (Davison, 1998). Estuaries are extensively used for the mooring of pleasure craft and are increasingly being used for the siting of marinas; Z. marina is, therefore, likely to be exposed to contamination from antifouling paint ingredients including Irgarol 1051. To our knowledge, bioconcentration of Irgarol 1051 within marine angiosperms, and its effect upon them, has not been previously reported.
2. Methodology
2.1. Standards and samples Irgarol 1051 (\97% purity) was obtained from Ciba Geigy, Basel, Switzerland; ametryn (\ 99% purity) was obtained from Promochem UK. Stock standard herbicide solutions (1 mg cm − 3 each) were prepared in acetone and stored at −15°C. For calibration purposes, a portion of these stock solutions were diluted with isohexane. Working standard solutions containing 1–100 mg cm − 3 of test compounds in isohexane were used to spike the plant and water samples. An Irgarol 1051 stock standard of 1 mg cm − 3 was prepared in methanol (MeOH), and dosing concentrations of 20 mg cm − 3 to 1 mg cm − 3 derived by dilution in MeOH for use in the exposure trials. All solvents were glass distilled or HPLC grade; all glassware was cleaned by ‘Decon 90’ washing followed by heating at 170°C for 24 h and finally solvent rinsed. Z. marina plants were collected from the Yealm estuary UK (50° 18.55% N, 4° 3.85% W) at low water spring tides (: midday) from Summer 1997 to Spring 1998 for use within exposure experiments. In situ measurement of photosynthetic efficiency and water sampling (at 0.5 m depth) was carried out concurrently with all tissue plant collection occasions. The Yealm estuary is a flooded ria system with low freshwater input and has relatively low Irgarol 1051 contamination (Scarlett et al., 1997), however, it has been used by International Paints for the testing of antifouling agents for a number of years and the environmental quality standard (EQS) was exceeded in the Yealm for TBT in 1994 and 1995, also for copper
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in 1995 (Environment Agency, 1996); additional Zostera beds within the Salcombe estuary (a similar but larger nearby estuary) were therefore monitored for comparative purposes. Both of the estuaries sampled have numerous small craft moorings but do not have marinas and are not used by large vessels. Prior to extraction and analyses, plant tissue was stored at − 15°C; water samples were stored at between 2 and 5°C for no longer than 3 days prior to processing.
2.2. Plant tissue extraction and analyses Z. marina leaf tissues, including sheath material (4.0 g wet weight), were cut into small sections; rhizomes and root tissues (4.0 g wet weight) were ground using a pestle and mortar for 5 min. Plant tissues were placed in cellulose extraction thimbles (20×50 mm, Whatman International UK), they were then mixed with :8 g anhydrous sodium sulphate (Fisher Scientific UK) and internal standard spikes (ametryn) added. Plant tissues were solvent extracted with 50 cm3 of 1:1 acetone/ dichloromethane (DCM), for 20 h using 25 mm bore soxhlets. The extracts were concentrated to near dryness under nitrogen; 2 cm3 of isohexane was added and the extract evaporated down as before, this was repeated three times to ensure that all the acetone was removed. The samples were purified using alumina column chromatography eluted with solvents of increasing polarity. Alumina was prepared from neutral aluminium oxide (100 – 250 mesh, Camag). Chromatographic glass columns (10× 100 mm) were filled with 3 g of alumina (5% de-activated with Milli-Q water) and topped with 1 cm of anhydrous sodium sulphate. The sample extracts were loaded onto the columns in 2 cm3 of isohexane and eluted with 20 cm3 of isohexane followed by 20 cm3 of isohexane/DCM (1:1). The last 20 cm3 was collected and concentrated to 0.3 cm3 under nitrogen. Extracts were analysed by capillary gas chromatography with mass selective detection using a Hewlett Packard 5890 GC (HP5MS capillary column: 30 m ×0.25 mm ×0.25 mm; the carrier gas was helium) coupled to a Hewlett Packard 5972A mass selective detector (electron impact,
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quadrupole). Other GC conditions were as described by Zhou et al. (1996). Irgarol 1051 and ametryn concentrations were determined using selective ion monitoring, and quantified using masses m/z 253 (additional qualifier ions m/z 182 and 238) and m/z 212 (additional qualifier ions m/z 184 and 227), respectively.
2.3. Water extraction Extraction of Yealm and Salcombe water was carried out as described by Zhou et al. (1996) and analysed by GC-MS as above. In summary, water samples were filtered (Whatman GF/F filter) and spiked with ametryn internal standard; the filtrate was passed through solid phase extraction cartridges (Jones Chromatography Isolute C18 trifunctional, 1 g, preconditioned with ethyl acetate, methanol and water) which were each eluted with 3 cm3 of ethyl acetate to recover the Irgarol 1051. Extraction of water samples from the laboratory exposure trials was by solvent extraction. Water taken from beakers during water changing was transferred into 500 cm3 volumetrics, these were shaken for 2 min with 5-cm3 aliquots of isohexane (×3) and rinsed with a further 5 cm3 to extract the Irgarol 1051. Ametryn internal standards were added to the extracts, which were then placed in a freezer at −15°C to facilitate separation of the solvent from the water. Extracts were blown down under nitrogen to small volumes and analysed by GC-MS as described above.
2.4. Exposure experiments Laboratory exposure trials utilised 2 dm3 Pyrex beakers, each containing five plants, equivalent to a field density of : 1200 blades m − 2; field values of up to this value occur within the estuaries sampled (personal observation). Only non-flowering plants with no apparent disease or damage were selected for the trials; these were standardised by removing all but the youngest three leaves, which were trimmed to 20 cm above the basal leaf meristem and wiped with paper towel to remove epiphytes and epifauna. Rhizomes were standardised to three segments. Plants were acclimated for 6 days in filtered (Whatman GF/F prefilter + 0.45
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mm cellulose nitrate filter,) seawater collected off the UK Southwest coast, close to the Eddystone lighthouse, and kept at a constant temperature of 15°C under ‘cool light’ fluorescent tubes giving a photon fluency rate of 25 mmol quanta m − 2 s − 1 photosynthetically active radiation (PAR), as measured by a standard PAR meter (Skye Instruments). A semi-static dosing system was used to expose the plants to Irgarol 1051; nominal concentrations were 25.0, 10.0, 5.0, 2.5, 1.0, 0.5 and 0.0 mg dm − 3 plus MeOH controls (n= 3). This experiment was carried out to establish concentration/response relationships. Concentrations were achieved by injecting 0.5 cm3 of Irgarol 1051 working standard into 20 dm − 3 of Eddystone water which was thoroughly vortex mixed for 2 h prior to dosing; carrier controls were treated similarly, i.e. 0.0025% MeOH. Water was changed in the beakers after 24 and 48 h, and thereafter every 48 h until the trial was stopped after 10 days. A time course experiment was also performed to monitor the uptake of Irgarol 1051 over a 10-day period. Plants were exposed to an Irgarol 1051 concentration of 2.5 mg dm − 3 plus controls under conditions as above (start of experiment: n=8). Water was changed in the beakers every 48 h. During each water change, one replicate beaker from each of the treatments was removed and the plant tissues and water analysed for the presence of Irgarol 1051 (by the end of experiment: n =4). Z. marina leaves from both the dose response and time course exposure experiments were subject to growth analyses and tested for two parameters of photosynthetic efficiency. In order to investigate the effects of longerterm low-level exposure to Irgarol 1051, i.e. similar to concentrations reported in UK estuaries (Gough et al., 1994), a 36-day experiment was performed. Plants were exposed to a nominal concentration of 0.225 mg dm − 3 plus controls (treatment replication: n = 3); experimental conditions were as above, except that only three plants per beaker were used. Photosynthetic efficiency parameters were monitored but growth analysis was not performed.
2.5. Exposure experiments: photosynthetic efficiency parameters The parameters used to assess plant stress were the dark adapted fluorescence induction ratio Fv:Fm, which is an indicator of the maximum efficiency of PSII (Krugh and Miles, 1996), and ‘Area’, which is proportional to the pool size of the primary quinone electron acceptor (QA) on the reducing side of PSII (Hall et al., 1993). The key fluorescence parameters of F0 (time zero fluorescence), Fm (maximum fluorescence) and Fv (variable fluorescence Fm − F0), were also recorded. Analyses were performed during water changes by a plant efficiency analyser (PEA; Hansatech Instruments UK). Dark adaptation time was determined to be optimum at 20 min; 50% light intensity (1500 mmol quanta m − 2 s − 1 at 650 nm peak wavelength) of 1-s exposure was sufficient for light saturation. During laboratory 10-day exposure trials, six readings were taken from leaves from each of three replicate beakers per treatment: a total of 18 readings per treatment level. During in situ monitoring of field plants, three readings were recorded from each of six plants per site.
2.6. Exposure experiments: growth analyses Growth was assessed using an adaptation of the leaf-marking techniques of Zieman (1974) and Dennison (1990). Leaves were puncture marked by syringe at 1 cm above the basal leaf meristem, after 10 days the plant tissue was divided into old and new growth; only the inner, youngest, leaves were measured. Leaf tissue above the puncture scar was considered old biomass, tissue below the scar but above the meristem, plus young leaves without a scar were considered new biomass. Following separation, tissues from each beaker were pooled and were placed in preweighed aluminium pans, dried for 1 week at 80°C, and weighed. The samples were then combusted at 500°C for 8 h, ash weights were obtained, and ash free dry weights were calculated. The ratio of new to old biomass provides a biomass specific ratio which was used as an indicator of plant growth. Growth rate analysis was not performed during the 36-day experiment.
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2.7. Statistical methods A preliminary analysis of the data was performed using Bartlett’s test for homogeneity of variance. Plant growth and Fv:Fm data was found to be homogenous: differences between doses (treatments) were tested for significance by a oneway analysis of variance (ANOVA), with an alpha of 0.05. Significant differences between treatments were presumed to exist at P-values less than or equal to 0.05. Tests for least significant differences were performed between individual treatments (PB0.05) when there was a significant P-value. The Area parameter data was found not to be homogenous: differences between doses were tested for significance by the Krustall – Wallis test and differences between individual treatments were assessed using heteroscedastic t-tests. Statistical analyses were performed using Statgraphics vers. 7.1 and Excel vers. 7.
3. Results
3.1. Analytical performance The calibration curve for Irgarol with GC-MS was linear over the range 0 – 10 mg dm − 3 (r = 0.9998, PB0.01). Precision of data and limits of detection are given in Table 1. Recovery was determined by spiking reference substrates with known volumes of the working standard solution of Irgarol 1051; plant tissue was also subject to exhaustive extraction, i.e. until further extraction produced no significant increase in Irgarol 1051
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concentration. Replicate analyses of spiked Z. marina revealed reasonable precision with good recovery. The concentrations reported are the mean values of triplicate determinations and were corrected for recovery. Tissue concentrations are based on dry weights unless stated otherwise. Leaf tissue dry:wet ratio=0.15; root/rhizome dry:wet ratio=0.13). Bioconcentration factors and EC50 values are based on measured concentrations.
3.2. Field sampling stations Aqueous Irgarol 1051 concentrations were monitored prior to, and throughout, the Z. marina collection period; concentrations were typically B0.003 mg dm − 3 at both the Yealm and Salcombe estuary sampling stations, a maximum concentration of 0.010 mg dm − 3 occurred at the Yealm in January 1998. Salinity was determined to be 35‰ on all sampling occasions, confirming that the plants were not subject to brackish water conditions at either site. Plant tissue concentrations were in the range 0.002–0.050 mg g − 1 at the Yealm and 0.004–0.071 mg g − 1 at Salcombe, maximum concentrations occurred in August 1997 at both sites. In situ monitoring of plant efficiency parameters revealed typical Fv:Fm values in the range 0.80–0.84, although a mean value of 0.719 0.04 S.E. was recorded at Salcombe in August, values below 0.80 also occurred at both estuaries in March 1998. The Area parameter values were typically in the range 14000–30000 bits ms and were highly variable at both estuaries. No overall significant differences were found between the two estuaries.
Table 1 Analytical performance data Matrix
LODa
Recoveryb (%)
R.S.D.c (%)
Water (solid phase extraction) (ng dm−3) Water (solvent extraction) (ng dm−3) Plant tissue (ng g−1 dry wt.)
1.0 5.0 2.0
95 101 97 (92)
4.5 8.0 7.9
a
Limit of detection of the whole analytical procedure (signal-to-noise ratio =10). Derived from the slope of the regression line of standard addition at five different levels. Value in parenthesis: exhaustive extraction of plant tissue demonstrated that bioaccumulated Irgarol 1051 was less easily recovered than spikes. The lower value was used to correct tissue concentration. c Relative standard deviation was derived from replicate analysis of matrix (n = 3). b
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3.3. Uptake and bioconcentration of Irgarol 1051 The monitoring of tissue burden during the 2.5 mg dm − 3 exposure time course experiment revealed that uptake of Irgarol 1051 was rapid with a mean leaf tissue concentration of 0.223 9 0.077 mg g − 1 (S.D.) achieved within 2 days, this was over 300 times the measured water concentration of 0.660 mg dm − 3. Measured water concentrations were close to nominal concentrations by day 10 of the experiment. Accumulation was highly variable with a maximum range of 0.140 – 0.639 mg g − 1 occurring after seven days exposure; high variation was found both within and between replicates. The final mean concentration after 10 days exposure of 0.2439 0.053 mg g − 1 (S.D.) was not significantly higher than that after 2 days. Analyses of plant tissues from the 10-day concentration-response experiment revealed a linear relationship between Zostera leaf tissue concentration and water concentration (r =0.973, P B 0.01), but due to the accumulation within the tissue being relatively greater at the lower exposure concentrations, it was better described by the log – log transformation log y = 0.588 log x + 0.966 (r=0.998), where y =leaf concentration (ng g − 1 dry wt.) and x = water concentration (ng dm − 3). Accumulation of Irgarol 1051 within the roots and rhizomes was described by the linear function y= 0.026x + 26 (r= 0.999, units as above). Bioconcentration within roots/rhizomes was less variable but significantly lower (P B 0.001) than within the leaves: concentrations within the leaves were greater than five times that within the roots/rhizomes at the highest nominal exposure of 25 mg dm − 3 and \ 14 times at 0.5 mg dm − 3. No significant difference was observed between the leaves and rhizomes of the controls or field Zostera. From the field data for the estuaries sampled (see above), a BCF (tissue concentration }water concentration) of up to 25000 times was calculated for Zostera accumulation of Irgarol 1051.
3.4. Short-term exposure experiments: photosynthetic efficiency parameters During the acclimation period, Fv:Fm and Area
parameter values increased significantly (PB 0.001). Fv:Fm values increased from a mean of 0.832 to 0.855 and stabilised to a very low relative standard deviation (R.S.D.) of 1%. Area values increased from a mean of 16206 to 19950 bits ms, but retained high variation with a R.S.D. of 20%. During the exposure period, no significant difference was observed between the controls and MeOH controls; Fv:Fm values remained stable with very low variation; Area values fluctuated and maintained a high degree of variation. Monitoring of water concentrations revealed that with the exception of the 0.5 and 1.0 mg dm − 3 treatments, which had final measured concentrations of 0.178 and 0.619 mg dm − 3, respectively, steady state conditions had been achieved within the 10-day exposure period, with analysed values close to nominal values. No Irgarol 1051 was detectable within the water of the controls. All plants retained a healthy appearance throughout the experiment and epiphytic growth was minimal. After 1 day’s exposure to an Irgarol 1051 concentration of 2.5 mg dm − 3 and above, Fv:Fm values dropped significantly (PB 0.01, Fig. 1). The Area parameter values only fell significantly (PB 0.01) at treatment levels of 10 mg dm − 3 and above on day one of the experiment (Fig. 2). The variation in measured parameters within the Irgarol 1051 treatments increased during the initial exposure; this variation was also observed in measurements taken from the same plant. By day 4 of the trial, the Fv:Fm values for all treatments were significantly lower (PB 0.05) than the controls, but only treatments of ] 2.5 mg dm − 3 had affected the Area values (Figs. 1 and 2). By the end of the experiment, Fv:Fm values had fallen further: the 0.5 mg dm − 3 treatments had declined by about 10% (Fv:Fm = 0.774) during the 10 days and the 25 mg dm − 3 was reduced by nearly 80% (Fv:Fm = 0.219). From the Fv:Fm dose–response curve (Fig. 1), a 10-day EC50 value of 2.5 mg dm − 3 was interpolated. Area values at the end of the experiment had dropped to near zero for treatments ] 5.0 mg dm − 3, but the lowest treatment of 0.5 mg dm − 3 was unaffected. Plants exposed to 1.0 mg dm − 3 had Area values signifi-
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Fig. 1. Effect of aqueous Irgarol 1051 on the Zostera marina leaf Fv:Fm values (expressed as a % of the methanol control value) after 1, 4 and 10 days exposure (error bars = + 1 standard error).
cantly higher (P B0.05) than the controls. From the Area dose–response curve (Fig. 2) a 10-day EC50 of 2.6 mg dm − 3 was interpolated. Fv:Fm values were significantly reduced (P B0.001) at a leaf tissue concentration of 0.395 mg g − 1 (Fig. 3). The EC50 value based on leaf dry tissue was interpolated to be 1.1 mg g − 1 for the Fv:Fm response (Fig. 3). As stated above, the accumulation of Irgarol 1051 within the root/rhizome tissue was substantially less than within the leaves and this is reflected in the concentration/response relationships (Fig. 3); however, it would be inappropriate to calculate EC50 values for a photosynthetic response based on root tissue concentration. Photosynthetic efficiency parameters obtained during the time course experiment were in accord with that of the dose–response experiment; the mean leaf tissue concentration of 0.223 mg g − 1 after 2 days exposure, caused a significant (P B0.001) reduction in Fv:Fm of over 15%.
3.5. Short-term exposure experiments: growth rate Substantial leaf growth occurred at all treatment levels during the exposure period. Results
comparing specific biomass ratios were similar when calculated using wet, dry and ash free dry weights. Significant reduction in growth rate (PB 0.05) only occurred at and above the 10 mg dm − 3 treatment level (Fig. 4). No significant reduction in growth occurred during the time course experiment, carried out at an exposure concentration of 2.5 mg dm − 3.
3.6. Long-term exposure experiments Due to the lack of steady state conditions achieved at the lower exposure concentrations during the 10-day exposure experiments, it was deemed necessary to carry out a longer-term exposure experiment at an environmentally realistic Irgarol 1051 concentration. Plants were exposed to a nominal concentration of 0.225 mg dm − 3 for 36 days, measured concentration at the end of the experiment was 0.20090.025 mg dm − 3. The Irgarol 1051 treated plants showed a decreasing PSII efficiency throughout the experiment (linear relationship y= 107–1.56x; r= 0.983, PB 0.001; where y is the % of the control PSII efficiency and x is the exposure period in days). The final mean Fv:Fm value of the plants exposed to Irgarol 1051
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was 0.49 0.03 S.E. compared to 0.89 0.02 S.E. for the controls, i.e. 36-day PSII efficiency inhibition EC50 values of 0.20 mg dm − 3 and 2.6 mg g − 1 (measured aqueous and leaf tissue dry wt. concentrations, respectively). By the end of the trial the Irgarol 1051 treated plants were beginning to exhibit a dramatic drop in the Area parameter, although this was not statistically significant.
4. Discussion The PSII photosynthetic efficiency of Z. marina leaves, as measured by the decline in Fv:Fm, was significantly reduced (P B 0.01) by about 10% at a measured Irgarol 1051 concentration of 0.18 mg dm − 3 (Fig. 1); this water concentration is below that reported within the marine environment in the UK (Gough et al., 1994; Zhou et al., 1996), the Mediterranean (Readman et al., 1993; Tolosa et al., 1996) and Bermuda (Readman, personal communication). Estuarine concentrations tend to be lower than the above concentration, although Gough et al. (1994) reported Irgarol 1051 concentrations in the Hamble estuary (Solent, UK) of up to 0.19 mg dm − 3. The 10-day concentration – re-
sponse experiment revealed that the Area parameter was either unaffected, or stimulated, at current environmental concentrations. The Area parameter is potentially a very useful indicator of stress caused by triazine herbicides, as it is dramatically reduced when electron transfer from the chlorophyll reaction centres to the quinone pool is blocked (Hall et al., 1993), as caused by the mode of action of triazines. The results indicate that a time lag exists between the initial fall in Fv:Fm and the subsequent dramatic fall in Area (Figs. 1 and 2). It is therefore unclear whether long term chronic exposure to concentrations below 1 mg dm − 3 would result in a measurable decline in the Area parameter. The high variation in Area, observed in both field Zostera and exposure experiment plants, make this parameter difficult to assess statistically: its use as in indicator of herbicide induced stress in the field is therefore questionable. The long-term exposure experiment did indicate that the Irgarol 1051 treated plants were beginning to exhibit a dramatic drop in the Area parameter by the end of the trial; although this was not statistically significant, it suggests that the turnover rate of the PSII subunit D1 protein, the binding site of triazine herbicides (He and
Fig. 2. Effect of aqueous Irgarol 1051 on the Zostera marina leaf Area parameter values (expressed as a % of the methanol control value) after 1, 4 and 10 days exposure (error bars = + 1 standard error).
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Fig. 3. Effect of Irgarol 1051 tissue concentration upon Zostera marina leaf Fv:Fm values (expressed as a % of the methanol control value) after 10 days exposure (error bars = + 1 standard error).
Malkin, 1998), is barely sufficient to allow continued electron-transport at a concentration of 0.20 mg dm − 3. The low Fv:Fm values observed at Salcombe in August 1997 coincided with the highest recorded Irgarol 1051 tissue concentration. The low PSII efficiency can, however, be attributed to elevated F0 values associated with photoinhibition (Bolha`r-Nordenkampf et al., 1989) caused by exposure to high light intensity. Although the Fv:Fm parameter provides a useful indicator of general stress of plants in situ, a longer period of dark adaptation, or neap tide sampling, may be advisable when monitoring normally sub-tidal plants, such as Z. marina, in order to help distinguish between natural photoinhibition stress and damage from phytotoxic chemicals. Zostera beds typically contain macroalgae; under certain environmental conditions, rapid growing algae such as Enteromorpha spp. can smother and eliminate the Zostera through shading (den Hartog, 1994). Mature E. intestinalis has a reported 3-day Fv:Fm response EC50 value of 2.5 mg dm − 3 (Scarlett et al., 1997), i.e. similar to that of
Z. marina; in addition, the zoospore reproductive stage of E. intestinalis was shown to be highly responsive to Irgarol 1051, with a 6-day growth response EC50 value of 0.5 mg dm − 3. The data indicates that the presence of Irgarol 1051 would not provide Enteromorpha with a competitive advantage. An important function of Zostera habitats is the provision of substrates for a high biodiversity of epiphytic algae. Dahl and Blanck (1996) reported a reduction in photosynthetic activity EC50 value of only 0.82 nM (0.205 mg dm − 3) for marine periphyton exposed to Irgarol 1051 over a 3-week period. It is therefore likely that aqueous Irgarol 1051 concentrations of below 0.2 mg dm − 3 will impact the flora associated with Zostera beds as well as the seagrasses. Accumulation of Irgarol 1051 was found to be far greater within the leaves than the roots/rhizomes during the laboratory exposure experiment, but this difference was not evident within the field Zostera sampled. It is therefore likely that the uptake of the herbicide is merely slower into the rhizome, this may be due to the smaller surface
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area:volume ratio and/or greater resistance to diffusion across the rhizome epidermis. Although these results show that it is preferable to sample the leaves of field plants in order to assess potential stress resulting from tissue burden, it would be acceptable to measure rhizome concentrations if it were not possible to sample the leaves: such as where Zostera populations are not winter green, as is the case for most intertidal populations in the UK. Bioconcentration of Irgarol 1051 has not been previously reported in marine macrophytes, but To´th et al. (1996) reported concentrations within freshwater macrophytes and algae in the range 0.053 – 0.103 mg g − 1 dry wt. (measured water concentrations were in the range 0.0025 – 0.145 mg dm − 3) at Port d’Ouchy (Lake Geneva, Switzerland) and 0.004–0.0052 mg g − 1 (water concentrations B 0.0003 mg dm − 3) at a Lake Geneva reference site at Buchillon (Switzerland). Z. marina mean tissue concentrations at the Yealm and Salcombe estuaries were in the range 0.002 – 0.071 mg g − 1 (water concentration 0.001 – 0.010 mg dm − 3), which suggests that accumulation of Irgarol 1051 is similar in Zostera and some freshwater macrophytes. BCF values were found to
be greater in plants at the lower exposure concentrations, and very high at the estuaries sampled: field Zostera BCF values (dry weight basis) were up to 25000× ; To´th et al. (1996) calculated values up to 30000×. It was noted in the exposure experiment, that control plants had similar Irgarol 1051 concentrations at the end of trial, i.e. up to 42 days in uncontaminated seawater, to that of the field population from which they were taken: this indicates that depuration is either minimal or does not occur. Plants in estuaries that receive regular tidal or freshwater flushing, are unlikely to loose their Irgarol 1051 tissue burden through simple diffusion. Despite the relatively low aqueous Irgarol 1051 contamination within the sampled estuaries, some Zostera plants had tissue concentrations that were only 15 times below the calculated 10-day PSII efficiency inhibition EC50 value of 1.1 mg g − 1, and less than three times below that shown to significantly reduce PSII efficiency. This represents only a very small ‘margin of safety’, especially as other phytotoxic chemicals are likely to be present within estuaries (House et al., 1997).
Fig. 4. Z. marina leaf biomass specific ratios from 10-day semi-static Irgarol 1051 exposure (error bars = +1 standard deviation). Treatments that are significantly different to the methanol control (P B0.05) are indicated by an asterisk (*).
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Reduction in growth rate, as assessed by leaf specific biomass ratios, was only observed at concentrations at or above 10 mg dm − 3 (Fig. 4). Schwarzschild et al. (1994), using a static 10-day atrazine exposure, reported that Z. marina leaf specific biomass ratios were not significantly reduced at a measured concentration of 80 mg dm − 3. These results corroborate the data of Dahl and Blanck (1996), which indicated that Irgarol 1051 was considerably more toxic than atrazine to coastal marine plants. The exposure period of 10 days used in the present growth rate study, may be too short for the reduction in photosynthetic efficiency to measurably affect the growth rate at low Irgarol 1051 concentrations. It is also possible that the plants compensate for the loss of efficiency by some means, e.g. increasing their chlorophyll content, density of chloroplasts or relative leaf area; these parameters were not assessed. Loss of photosynthetic efficiency must however, carry an energetic cost and therefore have implications for a plant’s ability to cope with additional stresses, including competition from other organisms and environmental factors, both natural and anthropogenic.
5. Conclusions Z. marina plants that occur close to marinas, or other areas where boat density is high, are likely to experience reduced photosynthetic efficiency of PSII, but the number of Zostera beds in the UK that may be affected is unknown due to the lack of up to date distribution data in the literature. Within the relatively clean estuaries of S.W. England, there is only a small margin of safety; elsewhere in the world, especially where plants are continuously exposed to high levels of Irgarol 1051 throughout the year, Zostera beds are likely to be damaged. The results indicate that the siting of new marinas in close proximity to Zostera beds, or an increase in the use of Irgarol 1051, is likely to cause stress to the plants. Multi-toxicant exposure experiments are required to fully assess the risk to estuarine macrophytes posed by phytotoxic chemicals.
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Acknowledgements The authors gratefully acknowledge Ciba Geigy (UK) Additives Division for the gift of the Irgarol 1051 used in this investigation. We also thank Aref Sheikh for his assistance during field sampling. The research was funded by the Natural Environment Research Council, grant reference: GR9/03123.
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