137
Journal of Applied Phycology 9: 137–146, 1997. c 1997 Kluwer Academic Publishers. Printed in Belgium.
Changes in atrazine toxicity throughout succession of stream periphyton communities Helena Guasch , Isabel Mu˜noz, N´uria Ros´es & Sergi Sabater Departament d’Ecologia, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona, Spain ( Author for correspondence) Received 5 February 1997; revised 25 April 1997; accepted 4 May 1997
Key words: atrazine toxicity, periphyton, stream, succession
Abstract A study was made to describe atrazine toxicity and its changes throughout succession of periphyton communities of an undisturbed Mediterranean stream. Toxicity was assessed by short-term physiological tests (concentration-effect curves of photosynthesis to atrazine) in the laboratory using artificial substrates colonized in one stream site during winter, and two stream sites (one open and the other shaded) during summer. In the winter experiment, when environmental conditions were relatively steady and chlorophyll content was low, toxicity increased according to the increases in cell density and chlorophyll content throughout colonization. EC50 (concentration inhibiting photosynthesis by 50%) was above 0.8 M atrazine until day 16 and below 0.4 M atrazine after three weeks. In the summer experiment, under more variable environmental conditions, the differences between the EC50 at the beginning and the end of the colonization experiments were not significant (one factor ANOVA) at the two sites. EC50 was on average 0.89 M atrazine in the shaded site and 0.29 M atrazine in the open site. A significant negative correlation between irradiance and EC50 was observed when all the experiments were considered together (r = 0.464, n = 20, p<0.05), suggesting that light history may have an important role in the response to atrazine. This investigation reveals that the response of stream periphyton to atrazine is likely to be influenced by colonization time and the corresponding changes in algal density and community composition as well as by environmental conditions (e.g. light regime) throughout succession. Introduction Increased run-off of natural and man-made substances from catchments has marked effects on river communities. Natural periphyton communities are among the components of lotic ecosystems which may be used to monitor impacts (Whitton & Kelly, 1995). Within the methods applied to assess the effect of toxicants using freshwater algae, most research has been focused on single-species tests, conducted in the laboratory. Batch-culture toxicity methods have been reviewed critically by Nyholm and K¨allqvist (1989). In the last decade, several methods using complete periphyton communities have been used to assess the toxicity of chemicals on the structure and function of aquatic sys-
*140602
tems (e.g. Niederlehner & Cairns, 1993; Lewis et al., 1993; Blanck et al., 1988). The herbicide atrazine (6-chloro-N-ethil-N0-(1methylethyl)-1,3,5-triazine-2,4-diamine) is found in many surface and groundwaters and its ecological effects are a possible concern for aquatic ecosystems. Such concerns have generated much research on its toxicity to aquatic organisms (e.g. Huber, 1993; Solomon et al., 1996). Concentrations above 100 g L 1 cause very marked effects on the photosynthesis, growth, chlorophyll content and biomass of most aquatic primary producers (e.g. Plumley & Davis, 1980; Brockway et al., 1984; Kosinski & Merkle, 1984). Long-term exposure to these high atrazine concentrations performed in lentic mesocosms can change the algal community composition towards a
Article: japh 477 GSB: Pips nr 140602 BIO2KAP japh477.tex; 9/07/1997; 12:22; v.7; p.1
138 diatom-dominated community (Hamilton et al., 1988; Hoagland et al., 1993). The effect of atrazine on aquatic ecosystems when applied at environmental concentrations (15–25 g L 1 ) has been reported in a few studies (e.g. Krieger et al., 1988; Detenbeck et al., 1996). The effects of atrazine on lotic periphyton communities are even less clear. Lotic environments are usually highly dynamic systems. Linked to this natural variability, changes in the physiology of periphyton which may in turn affect or even obscure the response to toxic substances, may occur. Jurgensen and Hoagland (1990) introduced short-term pulses of atrazine (at 0– 100 g L 1) in a low order stream. In this study, runoffrelated sediment deposition and associated fluctuations in stream flow had a much more important impact on stream periphyton than short-term pulses of atrazine. Lynch et al. (1985) found that effects associated with seasonal changes had a greater influence on model stream periphyton communities than 30-day treatments with atrazine. Therefore, toxicity tests performed with periphyton communities of lotic ecosystems are necessary in order to assess the natural variability in the response to chemicals. In order to reduce the heterogeneity occurring on natural substrata (Townsend, 1989) and to obtain high numbers of replicates, samples with periphyton may be obtained from colonized artificial substrates. It is not sufficiently known whether changes associated with colonization might influence the sensitivity of the community to toxicants. Throughout colonization, successional processes might be accompanied by changes in the physiology, species composition, as well as biomass accrual of the community (Hill & Boston, 1991; Sabater & Roman´ı, 1996), which may also influence the response to toxic substances. The current study was performed in an undisturbed stream without traces of herbicides in its waters, and the toxicity of the herbicide atrazine was followed with short-term tests. Toxicity tests were performed in the laboratory with intact periphyton communities sampled during two periods (winter and summer) of contrasting environmental characteristics. During summer, two sites of differing ambient light conditions (one open and the other shaded) were simultaneously sampled. Artificial substrates (etched glass) were used for colonization and atrazine toxicity was followed throughout succession. The objectives of this study were twofold: 1. to describe the response of periphyton communities to atrazine; 2. to follow variations in tolerance throughout
succession, under different environmental conditions of light and temperature.
Materials and methods Description of study site The study was carried out in L’Avenc´o, an undisturbed Mediterranean stream. It is located at 41 460 N 2 190 E, 45 km N of Barcelona (NE Spain) and drains a well forested watershed in the Montseny Natural Reserve, virtually free of human disturbance. The Avenc´o is an undisturbed 3rd order stream characterized by high alkalinity and nutrient-poor waters (Table 1). The stream has a well-preserved riparian vegetation mainly made up of alder (Alnus glutinosa). Water velocity was measured at each sampling date by performing short-term injections of sodium chloride (Mart´ı & Sabater, 1996). Incident light at the stream bottom was measured with a LiCor quantum sensor with an underwater cell (Li-192SB). Dissolved oxygen, pH, water temperature and conductivity were measured with selective electrodes. Total alkalinity was determined by potentiometric (pH) titration followed by antilogarithmic Gran (Kramer, 1982). Dissolved nutrients (phosphorus as orthophosphate and nitrate) were analyzed following APHA (1989). Analysis of stream water residues of triazine herbicides Analysis of stream water residues of triazine herbicides were carried out at two occasions during this period. This investigation was focused on atrazine toxicity, but simazine was also analyzed. The motor action of simazine is similar to atrazine and its application as herbicide is common in the area of study. Both herbicides were analyzed in order to know whether the periphyton community was preexposed to this group of herbicides or not. Samples were filtered with Whatman GF/F filters and processed by solid-phase extraction applying 500 mg C18 cartridges (J & W Scientific Fisons) and determined using high performance liquid chromatography (Durand & Barcel´o, 1990). Atrazine and simazine were identified by comparing their specific retention times with known reference standards (pure atrazine and simazine). The detection level for both herbicides was 0.1 g L 1 .
japh477.tex; 9/07/1997; 12:22; v.7; p.2
139 Table 1. Environmental conditions in the Avenc´o stream during the experimental period. Mean values and standard deviation (in parentheses), minimum and maximum recorded values of irradiance, water temperature ( C), velocity, dissolved oxygen (DO)(% sat), conductivity, alkalinity, pH (at 25 C) and nutrient concentration during winter (20 February–24 March) and summer (26 June–16 August). Variable
Irradiance Temperature Velocity DO Conductivity Alkalinity pH NO3 -N SRP
Unit
Winter mean (SD)
mol m
2s 1
C 1
cm s % sat S cm meq L 25 C M M
1 1
345
min.
(242)1
5.1 (1.1)1 5.6 (3.2)1 99.7 (6.1)1 236 (44)1 2.05 (0.25)1 8.2 (0.1)1 14.7 (9.2)3 0.134
39 4.0 4.1 93 203 1.85 8.1 4.7
Summer mean (SD)
max.
(216)2
650 6.2 10.5 105 286 2.30 8.2 16.8
1257 103 (89);2 21.9 (4.6)2 2.6 (1.9)2 127 (65)2 357 (91)2 3.83 (1.75)2 8.0 (0.3)2 19.54 0.15
min.
max.
1000 16 16.2 0 66 217 2.46 7.3
1600 271 30.2 5.8 240 505 6.90 8.4
0.06
0.27
Values of irradiance in a nearby shaded site. 1 n = 5; 2 n = 6; 3 n = 2; 4 n = 1. Experimental set-up and sampling Colonization experiments were carried out during winter and summer 1995. During these two periods small etched glass substrates (1.2 cm2 surface area) were glued to stream boulders and placed in the stream bed. Colonization sequences are summarized in Figure 1. During winter, glass substrates were allowed to colonize in an open site. Colonization was followed from early February and glass substrates were collected on days 9, 16, 23, 30 and 41. The summer experiment started on July in the same site and simultaneously in a nearby shaded site. Sampling at the two sites was performed on days 6, 14, 29 and 36. In order to obtain replicate experiments, other sets of etched glass substrates were allowed to colonize and were sampled at similar colonization dates. Two sets of substrates were left to colonize at the end of February and in mid-March and collected after 20 and 11 days, respectively, in the winter experiment, and another was left to colonize in late June and sampled after 13 days in the summer experiment (Figure 1). Colonized substrates were sampled in early morning and transported to the laboratory in cool-boxes filled with site water, thereby maintaining in-situ temperature. Incubations for the measurement of photosynthetic activity and the effect of atrazine on photosynthesis were carried in the laboratory within 3–6 h of sampling. Samples for the determination of chlorophyll content were frozen and those for the quantification of algal density and species composition determination were preserved in 4% formaldehyde.
Figure 1. Colonization series during the winter (top) and summer (bottom) experiments in the open site (straight line) and shaded site (broken line). Arrows indicate the time when substrates were left colonize (left limit) and the time when colonized substrates were sampled (right end). The length of each colonization period is given to the right of each arrow.
Photosynthesis measurement Photosynthesis was measured using a 14 C technique in a similar way to that described in Blanck and W˚angberg
japh477.tex; 9/07/1997; 12:22; v.7; p.3
140 (1988). Intact glass substrates were incubated in glass vials, each containing 4 mL filtered stream water (Whatman GF/F). 50 L aliquots of NaH14 CO3 (2 Ci, 1.9 g L 1 ) were added at 10-s intervals. Incubations were performed at light intensity which was considered to be near saturation: 150 mol photon m 2 s 1 when environmental light was above 100 mol photon m 2 s 1 or 50 mol photon m 2 s 1 when environmental light was below this value (for the shaded site in summer). Previous investigations in which the photosynthesis irradiance relationships of intact stream periphyton were studied (Guasch & Sabater, 1995), showed that irradiance of saturation was 30–100 and 100–380 mol photon m 2 s 1 for open and shaded sites, respectively. Water temperature was set at the range of 2 C of that in the stream at the time the samples were collected. Samples were continuously shaken to avoid the formation of diffusion gradients. After 60 min for incorporation of the 14 C, photosynthesis was stopped by adding 100 L formaldehyde. Previous incubations indicated linearity in the 14 C incorporation between 30–120 min, the minimum variability between replicates being at 60 min. The non incorporated carbon dioxide was eliminated by rinsing each substrate in clean stream water for 10–20 s. The glass substrates were then transferred to scintillation vials containing 1 mL stream water and sonicated until all periphyton was removed. The photosynthetic activity per surface area and per chlorophyll concentration were calculated throughout the colonization experiments. Tolerance quantification Periphyton photosynthetic activity (carbon uptake per sample) was measured in a concentration series of atrazine. Test solutions were prepared by diluting a stock solution of atrazine in acetone with filtered stream water. Intact glass substrates were preincubated for 120 min in glass vials, each containing 4 mL test solution in filtered stream water (Whatman GF/F). A series of eight concentrations from 0 to 20 mol L 1 atrazine was used in each test with five replicates for each concentration. From the concentration-effect curves generated, the atrazine tolerance could be estimated, i.e. as the effective concentrations inhibiting 50% (EC50 ) of the photosynthetic activity. EC50 values were quantified by loglinear interpolation giving the photosynthetic activity in atrazine-treated samples as a percentage of the average activity of the controls which was set to 100%.
Figure 2. Relationship between atrazine concentration (M) and photosynthesis (% control) for periphyton after 13 days of colonization during summer in the open site ( ) and shaded site ( ). Effective atrazine concentration (EC50 ) is calculated by log-linear interpolation.
#
Figure 2 shows an example of two concentration-effect curves. Chlorophyll analysis. Chlorophyll a was measured in triplicate after extraction in 90% acetone and sonication for 4 min. Chlorophyll a concentration was measured spectrophotometrically following Jeffrey and Humphrey (1975). The percentage of pheo-pigments was calculated after the addition of HCl up to a concentration of 10 3 M (Lorenzen, 1967). Abundance of algal species Algal density was estimated by triplicate after sonicating the colonized glass substrates. For each colonized glass, successive sonications (20 s each) were performed until the algae had been removed completely and become suspended. After each sonication, the sonicated material was removed and 2 mL distilled water added. The success of the procedure was checked by microscopy observations of the glass. The suspended material was collected in a known volume (e.g. 10 mL) of 4% formaldehyde. Subsequently, an aliquot was sedimented using a phytoplankton cuvette following the technique of Uterm¨ohl (1958) and counted using an inverted microscope at 600 magnification. Cell numbers are normalized per surface area (1.2 cm2 ).
japh477.tex; 9/07/1997; 12:22; v.7; p.4
141 Species composition was determined under a light microscope at 1000 magnification by permanent slides of diatom frustules complemented with the observation on aliquots of the suspended material for the determination and quantification of non-diatom cells. Diatom frustules were obtained after the digestion of the samples following the procedure of Barber and Haworth (1981). Permanent slides were mounted with a high refractive resin (Naphrax). 200–300 cells were counted per sample. Counted taxa were referred as percentage of the total number counted. The diversity of the algal community (i.e. including all the groups) was calculated with the Shannon-Wiener index using logarithms to base 2. Data treatment Differences in chlorophyll content and algal densities between sites were analyzed by one-factor analysis of variance (ANOVA). Relationships between the community response to atrazine (EC50 values) and colonization time (in days), biological variables (photosynthetic activity, chlorophyll content, algal densities, percentage of algal groups and species diversity) and environmental conditions (water temperature, light, pH, alkalinity and conductivity) were examined using Pearson correlation analysis. Differences between the effect of atrazine on the photosynthesis of different communities incubated simultaneously were established by two-factor analysis of variance (ANOVA). The analysis compared percentages of the control (five replicates per concentration) for five concentrations of atrazine (from 0.2–20 M) between the two communities.
colonizers on the glass substrates were the diatoms Achnanthes spp. and Cymbella spp. and the encrusting green alga Gongrosira sp. Cyanobacteria became abundant (51%) after 40 days of colonization (Figure 1a). Shannon-Wiener’s index of diversity was 3.2 in the first days of colonization indicating the existence of an initial inoculum of colonizers, and declined to 1.5–1.8 up to day 41. There was a linear increase in cell abundance, chlorophyll a content and photosynthetic activity throughout the colonization experiment (Figures 3b, 3d). This pattern was also observed for the two sets of colonization experiments started later. This was shown by the significant correlation between colonization day and algal density and between colonization day and chlorophyll content when all winter data were reported together (r = 0.91, r = 0.97, n = 7, p<0.01, respectively). Photosynthetic activity per chlorophyll unit had slightly increased at day 16 and continued to do so to the end of the experiment (Figure 3e). EC50 values for atrazine ranged between 0.19 and 1.04 M. Values above 0.8 M were determined in the first days of colonization (until day 16) and below 0.4 M after three weeks of colonization (Figure 3f). The lowest value was reached on day 23. There was a significant negative correlation between the time of colonization and EC50 (r = 0.774, n = 7, p<0.05). EC50 was also negatively correlated with the chlorophyll a content (r = 0.815, p<0.05) and algal density (r = 0.759, p<0.05). However, the EC50 was not correlated with any of the environmental variables (water temperature and light), or with any of the variables related with the algal community composition (species diversity and percentage of different algal groups).
Results
Summer experiment
No traces of atrazine or simazine were detected in the stream water of the Avenc´o.
Environmental conditions during the summer experiment were highly variable (Table 1). Reduction in water flow was accompanied by changes in water chemistry. The main physical and chemical difference between the open and shaded sites was ambient irradiance (Table 1). In the open site, diatoms accounted for the 54% of the cell abundance in the first days of colonization. A slight increase in the percentage of green algae was reported in later stages (Figure 4a). Several cyanobacteria appeared after four weeks of colonization. The diatom community was dominated by Cocconeis spp. during the first days of colonization, and subsequently by rosette-like colonies of Synedra sp. Navicula
Winter experiment Water quality characteristics during winter are given in Table 1. The hydrological (velocity), physical (water temperature) and chemical (conductivity and alkalinity) variables remained relatively steady during this period. Irradiance showed a progressive increase from 100 to 650 mol photon m 2 s 1 . Diatoms usually accounted for half of the total cell number (40–48%). Green algae and cyanobacteria shared the remaining percentage (52–66%). Early
japh477.tex; 9/07/1997; 12:22; v.7; p.5
142
Figure 3. (a) Changes in the percentage numbers of diatoms green algae and cyanobacteria after 9, 11, 16, 20, 23, 30 and 41 days; (b) cell abundance; (c) chlorophyll content; (d) photosynthetic activity (carbon uptake per surface area); (e) photosynthetic efficiency (carbon uptake per unit chlorophyll) and (f) EC50 values in three colonization series. One series (vertical bars) started on 1 January and was sampled after 9, 16, 23, 30 and 41 days; another series ( ) started on 28 February and was sampled after 20 days and a third series ( ) started on 14 March and was sampled 11 days after.
N
japh477.tex; 9/07/1997; 12:22; v.7; p.6
143 spp., Nitzschia spp. and Gomphonema spp. were also common throughout the colonization. The green alga community was composed of crusts of Gongrosira sp., small mats of Spirogyra sp., Stigeoclonium tenue K¨utz. and Oedogonium sp., as well as Scenedesmus colonies that were common during some periods of low flow. In the shaded site, diatoms accounted for 52% of total algal cells on day 6, while green algae (mostly Stigeoclonium tenue and Oedogonium) became dominant up to the end of the colonization experiment (Figure 4). The Shannon-Wiener index of diversity showed an initial high value (2.6 and 3.4 in the open and shaded sites respectively), then declined (2.4 and 2.8 on day 13) and subsequently increased again in both sites (3.4 and 3.8 on day 36). Chlorophyll a content maintained values below 1 g cm 2 in early days of colonization (up to day 15) in both sites (Figure 4c). Values up to 2.8 g cm 2 were recorded after 29 and 36 days of colonization in the open site, while it was between 0.9–1.4 g cm 2 in the shaded site. The photosynthetic activity per unit area ranged between 2–7 gC cm 2 h 1 in the open site and between 1–3 gC cm 2 h 1 in the shaded site. It experienced a continued rise up to day 29, and later on decreased in both sites (Figure 4d). The photosynthetic activity per chlorophyll unit reached ca. 5–8 gC g chl a 1 h 1 at the beginning of the colonization sequence, but then declined after day 36 at both sites (Figure 4e). In the open site, EC50 was below 0.3 M atrazine until day 29 and showed a slight increase at the end of the experiment (Figure 4f). In the shaded site, EC50 values above 1.5 M were observed in two occasions in the early days of colonization (days 13 and 14), while EC50 values below 0.4 M were obtained at later stages of the colonization sequence. Comparison between sites showed that after one week (day 6) and two weeks (days 13 and 14), atrazine toxicity was slightly higher in the open than in the shaded site (ANOVA, F = 4.27, p<0.05; F = 4.15, p<0.01, and F = 4.26, p<0.005, respectively). Differences were not significant on day 29, and EC50 was slightly higher in the open than the shaded site on day 36 (ANOVA, F = 4.0, p<0.05). No correlation was obtained between EC50 and the environmental or biological variables.
Discussion This investigation shows that changes in the physiology and taxonomic structure of periphyton communities
throughout succession may influence their response to atrazine. A negative relationship between the age of the community (colonization time) and tolerance to atrazine (EC50 values) was observed in winter. The lowest EC50 value was reached after 23 days of colonization and remained below 0.5 M up to the end of the experiment. During the experiment the algal community (made up of early colonizers-diatoms) was replaced by less diverse and more mature community in which green algae and cyanobacteria had a higher percentage. Differences in tolerance to atrazine among different groups of periphytic algae may be inferred from long-term effects on the species composition. In general, exposure of periphyton to atrazine tends to shift community composition from dominance by filamentous green-algae or cyanobacteria to dominance by diatoms (e.g. Hamala & Kollig, 1985; J¨uttner et al., 1995). These results indicate that tolerance profiles of early colonizers probably do not reflect the response of the mature community. Studies aiming to describe the response of the natural community to toxicants should include colonization times of sufficient length to develop a community similar to the natural one. Microbenthic communities in L’Avenc´o had not been pre-exposed to atrazine (no traces of atrazine or simazine were detected in the stream water) but their EC50 values for atrazine ranged from 0.19 M (31 g L 1 ) for the old winter community, to 2.08 M (447 g L 1 ) for the shade-adapted summer community. Similar ranges of effective atrazine concentrations have been described for phytoplankton and periphyton communities (Solomon et al., 1996). A low level effect of atrazine (15–25 g L 1 ) on periphyton productivity and biomass have been reported in some field studies (e.g. Krieger et al., 1988; Detenbeck et al., 1996). However, other experiments performed in mesocosms and artificial streams have shown no effect at 25 g L 1 (Lynch et al., 1985) and the first effect at 130–180 g L 1 (J¨uttner et al., 1995; Krieger et al., 1988). Differences in tolerance could be explained partially by differences in environmental conditions during growth. During the summer experiment, environmental conditions in the stream (water temperature, dissolved oxygen and alkalinity) fluctuated according to variations in the water flow. These variations probably affected the physiological behaviour of the algal communities and hence contributed to mask their ecotoxicological responses to atrazine. This could partially explain why differences in atrazine toxicity between young and more mature communities were not detected at that time. However, it is difficult to distinguish which
japh477.tex; 9/07/1997; 12:22; v.7; p.7
144
Figure 4. Changes in (a) percentage numbers of diatoms and green algae after 6, 13, 14, 29 and 36 days of colonization in the open (left bar) and shaded (right bar) sites; (b) cell abundance; (c) chlorophyll content; (d) photosynthetic activity (carbon uptake per surface area); (e) photosynthetic efficiency (carbon uptake per unit chlorophyll) and (f) EC50 values of two colonization series. One series was started in mid-June and sampled on days 6, 14, 29 and 36 (vertical bars) and the other started in late June and was sampled on day 13 in the open ( ) and shaded ( ) sites.
#
japh477.tex; 9/07/1997; 12:22; v.7; p.8
145 variable or variables may explain the temporal variation in atrazine toxicity reported here. Atrazine toxicity was higher in the open than in the shaded sites of the summer experiment (averages of 0.29 and 0.89 M respectively), and also it was lower in winter than in summer when the open sites were compared (averages of 0.63 and 0.29 M respectively). When the EC50 data from the winter and summer experiments were related with irradiance a negative correlation was detected (r = 0.464, n = 20, p<0.05). This suggests that light history may have an important role in influencing atrazine toxicity in periphyton communities. The relationship between the toxicity of triazine herbicides and light conditions during growth has been reported in algal cultures. Mayasich et al. (1986) reported differences in atrazine-induced inhibition of growth for two algal cultures grown under different light conditions. Millie et al. (1992) found a negative relationship between EC50 values of simazine (a triazine-herbicide) for oxygen evolution and growth rate and light conditions during growth of populations of Anabaena circinalis. They concluded that differences in sensitivity to photosystem II herbicides among photoacclimated populations were related to differences in pigment contents. These results indicate that environmental conditions may affect the ecotoxicological response of natural communities, specifically periphytic algae growing in lotic systems. A range in sensitivity to an induced stress is perhaps characteristic of natural assemblages of micro- organisms and emphasizes the importance of using natural communities to assess the effect of toxic chemicals in real-world environments.
Acknowledgements This work was supported financially by the EC project (EN AA 13496). The ‘Serveis cient´ıfico t`ecnics de la Universitat de Barcelona’ provided facilities and technical help with HPLC analysis. We especially thank Prof. W. Admiraal and V. Lehmann for reviewing an earlier draft of the manuscript. An anonymous referee provided very useful comments to the manuscript.
References American Public Health Association (APHA) (1989) Standard Methods for the Examination of Water and Wastewater. 17th edn, Washington DC. Barber HG, Haworth EY (1981) A Guide to the Morphology of the Diatom Frstule. Sci. Publs Freshwat. Biol. Ass. U.K. 44, 112 pp.
Blanck H, W˚angberg SA (1988) Induced community tolerance in marine periphyton established under arsenate stress. Can. J. Fish. aquat. Sci. 45: 1816–1819. Blanck H, W˚angberg SA, Molander S (1988) Pollution-Induced Community Tolerance – A New Ecotoxicological Tool. In Functional testing of aquatic biota for estimating hazards of chemicals. ASTM STP 988. Cairns J, Jr, Pratt JR (eds), American Society for Testing and Materials. Philadelphia, 219–230. Brockway DL, Smith PD, Stancil FE (1984) Fate and effects of atrazine in small aquatic microcosms. Bull. envir. Contam. Toxicol. 32: 345–353. Detenbeck EN, Hermanutz R, Allen K, Schwift MC (1996) Fate and effects of the herbicide atrazine in flow-through wetland mesocosms. Envir. Toxicol. Chem. 15: 937–946. Durand D, Barcel´o D (1990) Determination of chlorotriazines and their photolysis products by liquid chromatography with photodiode-array and thermospray mass spectrometric detection. J. Chromat. 502: 275–286. Guasch H, Sabater S (1995) Seasonal variation in photosynthesisirradiance responses by biofilms in Mediterranean streams. J. Phycol. 31: 725–735. Hamala JA, Kollig HP (1985) The effects of atrazine on periphyton communities in controlled laboratory ecosystems. Chemosphere 14: 1391–1408. Hamilton PB, Jackson GS, Kaushik NK, Solomon KR, Stephenson GL (1988) The impact of two applications of atrazine on the plankton communities of in situ enclosures. Aquat. Toxicol. 13: 123–140. Hill WR, Boston HL (1991) Effects of community development on photosynthesis irradiance relations in stream periphyton. Limnol. Oceanogr. 36: 1375–1389. Hoagland KD, Drenner RW, Smith JD, Cross DR (1993) Freshwater community responses to mixtures of agricultural pesticides: Effects of atrazine and bifenthrin. Envir. Toxicol. Chem. 12: 627– 637. Huber W (1993) Ecotoxicological relevance of atrazine in aquatic systems. Envir. Toxicol. Chem. 12: 1865–1881. Jeffrey S, Humphrey GF (1975) New spectrophotometric equations for determining chorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochem. Physiol. Pflanzen 167: 191– 194. Jurgensen TA, Hoagland KD (1990) Effects of short-term pulses of atrazine on attached algal communities in a small stream. Arch. envir. Contam. Toxicol. 19: 617–623. J¨uttner I, Peither A, Lay JP, Kettrup A, Ormerod SJ (1995) An outdoor mesocosm study to assess ecotoxicological effects of atrazine on a natural plankton community. Arch. envir. Contam. Toxicol. 29: 435–441. Kosinski R, Merkle MG (1984) The effect of four terrestrial herbicides on the productivity of artificial stream algal communities. J. envir. Qual. 13: 75–82. Kramer JR (1982) Alkalinity and acidity. In Water Analysis, Academic Press, 131–134. Krieger KA, Baker DB, Kramer JW (1988) Effects of herbicides on stream Aufwuchs productivity and nutrient uptake. Arch. envir. Contam. Toxicol. 17: 299–306. Lewis MA, Pittinges CA, Davidson DH, Ritchie CJ (1993) In situ response of natural periphyton to an anionic surfactant and an environmental risk assessment for phytotoxic effects. Envir. Toxicol. Chem. 12:1803–1812. Lorenzen C (1967) Determination of chlorophyll and pheopigments: spectrophotometric equations. Limnol. Oceanogr. 12: 343–346.
japh477.tex; 9/07/1997; 12:22; v.7; p.9
146 Lynch T, Johnson HE, Adams WJ (1985) Impact of atrazine and hexaclorobiphenyl on the structure and function of model stream ecosystems. Envir. Toxicol. Chem. 4: 399–413. Mart´ı E, Sabater F (1996) High variability in temporal and spatial nutrient retention in Mediterranean streams. Ecology 77: 854– 869. Mayasich JM, Karlander EP, Terlizzi DE, Jr (1986) Growth responses of Nannochloris oculata Droop and Phaeodactylum tricornutum Bohlin to the herbicide atrazine as influenced by light intensity and temperature. Aquat. Toxicol. 8: 175–184. Millie DF, Harrsh CM, Dionigri CP (1992) Simazine-induced inhibition in photoacclimated populations of Anabaena circinalis (Cyanophyta). J. Phycol. 28: 19–26. Niederlehner BR, Cairns J, Jr (1993) Effects of previous zinc exposure on pH tolerance of periphyton communities. Envir. Toxicol. Chem. 12: 743–753. Nyholm N, K¨allqvist T (1989) Methods for growth inhibition toxicity tests with freshwater algae. Envir. Toxicol. Chem. 8: 689–703.
Plumley F, Davis DE (1980) The effect of a photosynthesis inhibitor, atrazine, on salt marsh edaphic algae, in culture, microecosystems, and in the field. Estuaries 3: 217–223. Sabater S, Roman´ı A (1996) Metabolic changes associated with biofilm formation in an undisturbed Mediterranean stream. Hydrobiologia 335: 107–113. Solomon KR, Baker DB, Richards RP, Dixon KR, Klaine SJ, La Point TW, Kendall RJ, Weiskopf CP, Giddings JM, Giesy JP, Hall LW, Marty Williams W (1996) Ecological risk assessment of atrazine in North American surface waters. Envir. Toxicol. Chem. 15: 31–76. Townsend CR (1989) The patch dynamics concept of stream community ecology. J. North am. Benth. Soc. 8: 36–60. Uterm¨ohl H (1958) Zur Vervollkommung der quantitativen Phytoplankton-Methodik. Mitt. int. Ver. Limnol. 9: 1–39. Whitton BA, Kelly MG (1995) Use of algae and other plants for monitoring rivers. Austral. J. Ecol. 20: 45–56.
japh477.tex; 9/07/1997; 12:22; v.7; p.10
