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Keywords:

  • Aquatic macrophytes;
  • Atrazine;
  • Lemna minor;
  • Myriophyllum aquaticum;
  • Risk assessment

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. SUPPLEMENTAL DATA
  9. Acknowledgements
  10. REFERENCES
  11. Supporting Information

The relative sensitivity and recovery potential of two aquatic macrophyte species, Lemna minor and Myriophyllum aquaticum, exposed to atrazine (concentration ranges 80–1,280 µg/L and 40–640 µg/L, respectively) were evaluated using slightly adapted standard protocol for Lemna spp.: relative growth rates (RGR) and yield of both plants were measured in 3-d-long intervals during the exposure and recovery phase. Myriophyllum aquaticum was also exposed to atrazine-spiked sediment (0.1–3.7 µg/g) in a water-free system. The results of M. aquaticum sediment contact tests showed that root- and shoot-based growth parameters are equally sensitive endpoints. In the water (sediment-free) test system, L. minor recovered after short (3 d) and longer exposure (7 d) to all atrazine concentrations after only a 5- to 6-d-long recovery phase. The recovery of M. aquaticum after short exposure was slower and less efficient: after 12 d of recovery phase the final biomass of plants exposed to 380 and 640 µg/L was below the initial values. The last interval RGR provides a good indication of plant recovery potential regardless of species growth strategy. If compared to L. minor, the difference in growth rate, sensitivity, lag phase, recovery potential from water-column substances, and also suitability for studies investigating the effect of sediment-bound pollutants advocates the use of M. aquaticum as an additional macrophyte species in risk assessment. Environ. Toxicol. Chem. 2012;31:417–426. © 2011 SETAC


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. SUPPLEMENTAL DATA
  9. Acknowledgements
  10. REFERENCES
  11. Supporting Information

In the last few years, two concepts have been highlighted as tools for refining ecotoxicological risk assessment and adding more ecological realism: the trait-based approach, built on the hypothesis that sensitivity is a function of biological characteristics, and the vulnerability analysis, which combines susceptibility to exposure, sensitivity to a stressor, and recovery potential. In recent years, the risk assessment for aquatic macrophytes, particularly rooted submersed species, has triggered increased scientific attention 1. Currently, the only aquatic macrophyte test used in risk assessment of plant protection products under the European Regulation 2 is the standardized growth inhibition test with duckweed 3, despite the great variety of macrophyte species, differences in morphology, physiology, and life form, and their important role in aquatic ecosystems. Additional data with other aquatic plant species as well as studies on recovery potential were required on a case-by-case basis in the context of regulations concerning placement of plant protection products on the market 4. Macrophytes are not obligatory in the risk assessment of chemicals in the context of the Water Framework Directive 5. The Guidelines for Ecological Risk Assessment of the U.S. Environmental Protection Agency (U.S. EPA), however, suggest not only considering the nature and intensity of potential effects, including spatial as well as temporal scales, but also the potential for recovery 6.

Although Lemna minor and L. gibba are often regarded as being representative of all aquatic macrophytes, concerns have arisen that risk assessments based on Lemna spp. and their endpoints may not be protective of other macrophyte species due to different life history, growth strategy, exposure route, and potential differences in recovery rate or sensitivity to chemicals with specific toxic modes of action 7–9. In January 2008, the current state and potential options for refinement of aquatic macrophyte risk assessment procedures were discussed at the workshop Aquatic Macrophyte Risk Assessment for Pesticides, which was organized under the auspices of the Society of Environmental Toxicology and Chemistry Europe (SETAC Europe). According to the proposed modified aquatic macrophyte Tier 1 Risk Assessment scheme for Plant Protection Products in the European Union, Myriophyllum spp. is suggested as a test species when additional macrophyte species must be tested 1.

No internationally accepted guidelines exist for laboratory or field tests for rooted submersed or emerged macrophytes. Although guidelines are available for Canada and the U.S. 10, they are not accepted as a standard by regulators and other stakeholders 11. Therefore, various test systems with Myriophyllum spp. have been developed recently, such as a sediment contact test with M. aquaticum12, water (sediment-free) system with M. aquaticum8, and water-sediment test with M. spicatum13. At the moment, two ring tests are under way: M. aquaticum and M. spicatum growth inhibition tests in water-sediment system (P. Dohmen, BASF, Limburgerhof, Germany, personal communication) and M. aquaticum sediment contact test in a water-free system (U. Feiler, German Federal Institute of Hydrology, Koblenz, Germany, personal communication).

Many comparative studies of relative sensitivity of various macrophyte species to pesticides—particularly herbicides—have been conducted over the last 10 years 9, 11, 14–20, but only a few studies 21 have provided data on the sensitivity of M. aquaticum; most studies dealt with other species of the genus Myriophyllum. In most cases, relative sensitivity of rooted macrophyte species was compared to Lemna sp. Furthermore, most studies have focused on toxic effects of pesticides, but recovery after exposure is another important, ecologically relevant factor to be considered. A limited number of laboratory studies 22–25 focus on recovery potential of Lemna sp., and the open literature provides little evidence of recovery potential and patterns of Myriophyllum sp. after exposure to potentially toxic substances under laboratory conditions.

Approximately 50% of herbicides in use act by inhibiting photosynthesis at the photosystem I (PSI) and photosystem II (PSII) levels either by replacing PSI's ultimate electron acceptor or by blocking PSII-catalyzed photosynthetic electron transport 26. Atrazine is still one of the most frequently used triazine herbicides worldwide and is a good representative of the entire class of PSII inhibiting herbicides. With the short generation times of most algae and the presumed rapid growth rates of aquatic macrophytes, atrazine would not be expected to cause secondary effects on higher trophic levels unless exposure was of sufficiently long duration to irrevocably damage organisms 27. However, in most cases empirical evidence of rapid recovery from triazines and other inhibitors of photosynthesis have been provided only for Lemna sp. 22–24. The short life cycle of Lemna spp., however, is not representative of more slowly growing macrophyte species 28.

Although M. aquaticum is a candidate for an additional species in refined risk assessment, toxicity data concerning the sensitivity of this species is scarce and available recovery data is largely nonexistent. The sediment contact tests with M. aquaticum12 have been implemented in several case studies 29–31. Although the test protocol has been suggested for spiked sediment toxicity tests as well, no results of such studies (if any) have been published thus far. Therefore, the objective of the present study was to compare relative sensitivity and recovery potential/patterns of M. aquaticum and L. minor to atrazine in laboratory conditions. To enable direct comparison, M. aquaticum was tested in a sediment-free test system, while the endpoints included only fresh weight biomass (expressed as total plant biomass, growth rate, and yield). To assess the applicability of a proposed protocol for spiked sediments as well as the sensitivity of M. aquaticum exposed to atrazine in sediments, a contact test with atrazine-spiked sediment in a water-free system using a range of various endpoints (plant biomass and growth rate, shoot and root length and biomass, chlorophyll a content) was also conducted.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. SUPPLEMENTAL DATA
  9. Acknowledgements
  10. REFERENCES
  11. Supporting Information

Plant cultivation

Lemna minor and M. aquaticum were grown inside a controlled-environment room in open 500-ml beakers, in sterilized Steinberg medium 3 at 25 ± 2°C, illuminated with continuous cool white fluorescent lighting (100–135 µE · m−2 s−1 equivalent to 8,500–1,0000 lux, checked regularly with a Digital Lux meter LX 101OB, Sinometer Instruments). A parallel culture of M. aquaticum was cultivated on standard 32 artificial sediment (5% dried sphagnum peat, 74% quartz sand, 20% kaolin clay, and 1% CaCO3) in 500-ml flower pots without drain holes, and watered to saturation with semiconcentrated Steinberg solution (1:1 v/v Steinberg medium: distilled water).

Toxicity tests

Three tests were run nonsimultaneously: standard 7-d L. minor growth inhibition test in semistatic conditions with test solution renewal after 72 h; 10-d M. aquaticum growth inhibition semistatic test (test solution renewal every 72 h) in a sediment-free system; and 10-d M. aquaticum sediment contact test in a water-free system.

The L. minor test was run according to the standard protocol 3: Colonies consisting of two to four visible fronds were transferred from the inoculum culture to a Steinberg medium and randomly assigned to the test vessels (200-ml beakers with 150-ml test solution). Each test vessel contained a total of nine fronds, three replicates per treatment, and six per control. Total fresh weight of gently dried fronds was measured before placing the fronds into treatment and control vessels. Total frond area was determined in each control and treatment vessel at the beginning of the test by image analysis (digital photographs were analyzed with Adobe Photoshop CS3 software). A randomized design for location of the test vessels was applied to minimize the influence of spatial differences in light intensity and temperature. The fronds were then carefully moved to freshly prepared test solutions of corresponding atrazine concentration or pure Steinberg medium (controls) after 3 d. The number of fronds, total frond area, and total fresh weight per test vessel were counted and measured at the end of the test. Technical-grade atrazine (CAS 1912-24-9, purity 98%, manufacturer Oxon SpA) was provided by Galenika Fitofarmacija. A preliminary range-finding test was run in a logarithmic series of five nominal atrazine concentrations ranging from 0.1 to 10,000 µg/L in three replicates per treatment and three per control. A definitive test was set in the following nominal concentration series: 80, 160, 320, 640, and 1,280 µg/L as three replicates per treatment and six per control.

The M. aquaticum 10-d growth inhibition test in a water column (sediment-free system) was run in semistatic conditions. At the beginning of the test, the plants from 21 ± 3-d-old precultures cultivated in Steinberg medium were cut into the whorls needed for testing (two to four maximum per shoot). The whorls (the parts of a plant consisting of a nodium and two adjoining parts of internodi) used for the test showed no signs of side shoots. The cut whorls were then collected in a glass vessel in water for randomization. Before weighing, the whorls were dried gently. The fresh weight of each whorl was in the range of 25 ± 6 mg. Three whorls per replicate, three replicates per treatment, and six per control were placed into the test vessel (500-ml beakers with 250-ml test solution), while a thin (3 mm) polystyrene floating cover with holes was used to keep the plants in an upright position. The plants were transferred to freshly prepared, corresponding atrazine solutions on the third, sixth, and ninth day of the test. A preliminary, range-finding test was run in a logarithmic series of five nominal atrazine concentrations ranging from 0.1 to 10,000 µg/L; the definitive test was set in the nominal concentration series: 10, 20, 40, 80, and 100 µg/L. The test duration was 10 d, and exposure conditions were the same as for the L. minor test.

The M. aquaticum contact sediment test (in a water-free system) was run in static conditions according to Feiler et al. 12. Three whorls per replicate, three replicates per treatment, and six per control were planted in 80 g of spiked and control sediment per test vessel at premarked positions (1–3), closed with translucent lids with openings for aeration. During the exposure period the plants were watered with semiconcentrated Steinberg solution and test vessels were randomized every 48–62 h. Artificial sediment, 65 g, was spiked with a 15-ml solution of technical-grade atrazine in a preliminary, range-finding test with a logarithmic nominal concentration range of 0.1, 1, 10, 100, 1,000, and 10,000 µg/L, calculated to sediment concentrations of 0.000023, 0.00023, 0.0023, 0.0231, 0.231, and 2.31 µg/g. The nominal concentration range for the definite test was 0.5, 1, 2, 4, 8, 10, and 16 mg/L atrazine, which makes 0.12, 0.23, 0.46, 0.92, 1.85, 2.31, and 3.69 µg/g sediment.

The test duration was 10 d and laboratory conditions were the same as for the L. minor and M. aquaticum water-column (sediment-free) tests. The whole plants were weighed again after the test to calculate specific growth rate and inhibition and yield effect, but a number of other growth parameters were also recorded, such as total length of the shoots (the sum of the main and side shoots), total length of the roots (the sum of the main and side roots), total weight of the main and side shoots and the main and side roots, total content of chlorophyll a in shoots (as proxy to biomass), and concentration of chlorophyll a per gram of shoot. The root-to-shoot ratio based on length and weight was also calculated.

Recovery study

The experimental setup was similar to that described for the toxicity tests. The difference was that after 72 h of static exposure of L. minor and M. aquaticum, and after 7 d of static-renewal exposure in the second experiment with L. minor to a series of atrazine concentrations, the plants were transferred to clean Steinberg medium for the recovery phase of the experiment. In the recovery phase the medium was completely renewed every 72 h.

The experiments were run nonsimultaneously: the recovery test with L. minor after short-term exposure to atrazine (72-h exposure followed by a 6-d recovery phase in clean Steinberg medium); the recovery test with M. aquaticum after short-term exposure to atrazine (72-h exposure followed by a 12-d recovery phase); and the recovery test with L. minor after longer exposure to atrazine (7-d exposure followed by a 5-d recovery phase in clean Steinberg medium). Myriophyllum aquaticum was exposed for 72 h to the following range of nominal concentrations: 40, 80, 160, 320, and 640 µg/L of atrazine, while L. minor was exposed for 3 and 7 d to the following nominal atrazine concentrations: 80, 160, 320, 640, and 1,280 µg/L.

Response variables

The average relative growth rate (RGR) in L. minor toxicity tests and recovery experiments was calculated on the basis of change in the logarithms of frond numbers, total frond area, and fresh weight over time (expressed per day) in the controls and each treatment group. In all the tests with M. aquaticum, relative growth rates for each plant were calculated from the measured total plant fresh weights to enable calculation of the arithmetic mean of the RGR per test and control vessel using the following equations

  • equation image(1)

where RGRi-j is the average specific growth rate from time i to j, Ni is the measurement variable in the test or control vessel at time i, Nj is the measurement variable in the test or control vessel at time j, and t is the time period from i to j.

The percentage of inhibition of growth rate (Ir) was calculated for each test concentration (treatment group) according to the following equation

  • equation image(2)

where %Ir is the percentage of inhibition in average specific growth rate, RGRc is the mean value for RGR in the control, and RGRr is the mean value for RGR in the treatment group.

The effect on yield was assessed on the basis of number of fronds, fresh weight biomass, and total frond area for L. minor test and total plant fresh weight for M. aquaticum tests, according to the following equation.

  • equation image(3)

where %Iy is the percentage of reduction in yield, bc is the final biomass minus starting biomass for the control group, and bt is the final biomass minus starting biomass in the treatment group.

Chlorophyll a concentrations were measured at the end of each toxicity test and recovery experiment. Individual shoots in the case of M. aquaticum and total fronds from each test vessel in the case of L. minor were incubated in 7 ml methanol for 24 h in the dark. The samples were centrifuged for 15 min at 800 rpm and chlorophyll a was measured (Beckman DU-65 Spectrophotometer) in supernatant at two wavelengths, 653 and 666 nm.

The content of chlorophyll aCa in the extract (mg) was calculated as:

  • equation image(4)

where A666 and A653 are the absorbance at 666 and 653 nm, respectively.

And as chlorophyll a concentration – C – mg/g of plant material as

  • equation image(5)

where V (ml) is the solvent volume, m (g) is the plant material, and R is the dilution factor.

Analytical verification and water quality analyses

Temperature, dissolved oxygen content, oxygen saturation, electrical conductivity, and pH were measured electrochemically using the multiparameter instrument Ino Lab 3 (Wissenschaftlich–Technische Werkstatten), while biological and chemical oxygen demand, total organic carbon, total suspended solids, nitrates (NO3), and surfactants were analyzed using the portable multiparameter analyzer Pastel UV (Secomam).

Atrazine concentrations were checked in initial solutions of the M. aquaticum definitive test in water (sediment-free) system. The gas chromatography–mass spectrometry system (Agilent Technologies 7890A/5975C MSD) was used for quantification. Preconcentration of water samples was performed with Superclean ENVI-18 cartridges based on the operating procedure described by the U.S. EPA 33. The cartridges were conditioned with a 1:1 mixture of ethylacetate and methylene chloride, then with methanol, and finally with water. The components retained were eluted with ethylacetate and methylene chloride. Anhydrous sodium sulfate was added in eluates to remove residual water. The organic phase thus obtained was evaporated to dryness under a slow stream of nitrogen. The dry residue was dissolved in 1 ml of a 1:1 mixture of hexane:ethylene chloride. The sample of 1 µl was injected into a gas chromatography–mass spectrometry system (splitless) equipped with DB-5 MS column (30 m × 0.25 mm × 0.25 µm). The injector temperature was 250°C; the carrier gas was helium with a flow-rate of 1 ml/min. The initial temperature was 70°C, which was maintained for 2 min. The temperature was then increased to 150°C at 25°C/min, to 200°C at 3°C/min, and finally to 280°C at 20°C/min, which was maintained for 1 min. The selected ion monitoring mode was used for quantitative analysis of atrazine (m/z 200, m/z 215, m/z 73). The recovery of the method for atrazine analysis at the lower concentration level of 3 µg/L was 116% (RSD = 5.4%, n = 5) and for the higher concentration level of 90 µg/L it was 102% (RSD = 2.9%, n = 3). For nominal concentrations of 1, 91, and 100 µg/L, the measured values in initial solutions were 0.8, 103, and 96 µg/L, respectively. For initial solutions with nominal concentrations of 4 and 16 mg/L prepared for atrazine-spiked sediment contact test with M. aquaticum, significantly lower values were obtained by measurement: 2.1 and 5.4 mg/L, respectively.

Statistical analysis and presentation of the data

Mean, standard deviation (SD), and coefficient of variation (CV, %) were calculated for specific growth rates, yield, and concentration of chlorophyll a per test treatment in all toxicity tests and recovery experiments. No observable effect concentrations (NOEC) and lowest observable effect concentrations (LOEC) were calculated by comparing the treatments and controls using Dunnett's procedure, which integrates one-way analysis of variance (ANOVA) and t test (or t test with Bonferroni's adjustment in case of nonequal number of replicates per treatment) as a post-hoc test 34. The significance was assigned uniformly at p = 0.05. Statistical power of the test was assessed by the minimal significant difference (MSD) and sensitivity by the percentage of decrease compared to the corresponding control (MSD, %). Inhibitory concentrations causing 50 and 25% inhibition versus control (IC50 and IC25) based on both growth rate and yield were calculated using the linear interpolation method. A linear interpolation method (method of curve fitting using linear polynomials, developed by the U.S. EPA) for calculating inhibition concentrations in sublethal toxicity tests has been incorporated into TesTox software 34 used in the present study.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. SUPPLEMENTAL DATA
  9. Acknowledgements
  10. REFERENCES
  11. Supporting Information

Initial water quality parameters (temperature, dissolved oxygen, oxygen saturation, electrical conductivity, and pH, as well as biological oxygen demand, chemical oxygen demand, total organic carbon, total suspended solids, nitrates [NO3], and surfactants) remained within the recommended values 3 and did not change more than 20% over the 72-h renewal period. Because the measured values of atrazine did not differ considerably from the nominal values in the range of 1 to 100 µg/L, the results were calculated on a nominal concentrations basis.

The validity criteria 3 for L. minor—the doubling time of frond number in the control less than 2.5 d (60 h) and an average specific growth rate of 0.275 d—were met in the control treatment under semistatic conditions. The official validity criteria for M. aquaticum do not exist. Still, the growth rate of M. aquaticum cultivated in the sediment-free system was 0.04 to 0.05 mg/d, as reported earlier 35.

Toxicity tests

The results of toxicity tests in the sediment-free system with L. minor and M. aquaticum, expressed as IC50, IC25, NOEC, and LOEC values (µg/L) based on fresh weight biomass growth rates and yield for both plants, as well as the number of fronds and total frond area for L. minor, are summarized in Table 1. When growth rates calculated on a fresh weight basis were compared, IC50 values for M. aquaticum and L. minor were 94 µg/L and 122 µg/L, respectively, while NOEC/LOEC values differed considerably: 20/40 µg/L for M. aquaticum and 80/160 µg/L for L. minor. Inhibitory concentrations calculated on the yield basis were lower; that is, 77 µg/L and 62 µg/L, respectively. In the case of L. minor, based on the number of fronds and total frond area, IC50 and IC25 values were higher than the corresponding ones calculated on a biomass basis for growth rates as well as for yield.

Table 1. Toxicity of atrazine to Lemna minor and Myriophyllum aquaticum in sediment-free system, expressed as IC50, IC25, NOEC, and LOEC values (µg/L)a
Species/exposure (d)EndpointRGRYield
IC50IC25NOECLOECIC50IC25NOECLOEC
  • a

    Inhibition versus control were calculated using linear interpolation method; confidence intervals are given in brackets; NOEC and LOEC were determined by comparing each treatment and controls using Dunnett procedure (one-way ANOVA and t test as a post test); significance was assigned uniformly at p = 0.05.

    RGR = relative growth rate; IC50 = inhibitory concentrations causing 50%; IC25 = inhibitory concentrations causing 25%; NOEC = no observable effect concentrations; LOEC = lowest observable effect concentrations.

Myriophyllm aquaticum, 10 dFresh weight93.51 (85, 98)35.16 (27, 82)204076.42 (56, 95)32.56 (9, 80)4080
Lemna minor, 7 dFresh weight121.85 (102, 136)82.07 (51, 107)8016061.71 (53, 84)30.87 (26, 42)<8080
Number of fronds215.45 (172, 234)122.8 (84, 142)<8080125.23 (78, 47)109.7 (41, 486)<8080
Total frond area188.75 (162. 210)112.86 (92, 127)80160105.08 (69, 134.06)50.97 (35, 93.2)<8080

The results of the M. aquaticum contact test (in the water-free system) with sediment spiked with atrazine, expressed as IC50, IC25, NOEC, and LOEC values (µg/g), are summarized in Table 2. Coefficients of variation in control treatments (CV, %) as well as the percentage of decrease compared to the corresponding control (MSD, %) are provided for each of the measured endpoints as follows: growth rate and yield (based on the total plant fresh biomass weight basis), weight (mg) and length (mm) of the root and shoot, total chlorophyll a in a shoot (as a proxy for total shoot biomass), and chlorophyll a concentration (as mg/g shoot). The concentration response curves are presented in Figure 1. For each of the listed endpoints, the inhibition (%) versus control treatment was calculated for all test concentrations. Clear concentration response was observed for all endpoints except for root-to-shoot ratio. The lowest IC50 value of 0.4 µg/g was calculated for shoot fresh weight, while the highest value of 3.1 µg/g was recorded in the case of chlorophyll a concentration. Apart from the latter, the IC50 value calculated for total shoot chlorophyll a content was also relatively high compared to other endpoints, at 2 µg/g. With the exception of two chlorophyll a based endpoints, all other IC50 values fell into a narrow range from 0.4 (shoot fresh weight) to 0.7 µg/g (relative growth rate based on the total plant fresh weight biomass). Similarly, the NOEC/LOEC values for all endpoint except for those based on chlorophyll a measurements were 0.23 and 0.46 µg/g, respectively.

Table 2. Summary results of Myriophyllm aquaticum contact test with atrazine-spiked sedimenta
EndpointIC50 (µg/g)IC25 (µg/g)NOEC (µg/g)LOEC (µg/g)CV (%) in control treatmentMSD (%)
  • a

    Inhibition versus control were calculated using linear interpolation method; confidence intervals are given in brackets; NOEC and LOEC were determined by comparing each treatment and controls using Dunnett procedure (one-way ANOVA and t test as a post test); significance was assigned uniformly at p = 0.05.

    RGR = relative growth rate; IC50 = inhibitory concentrations causing 50%; IC25 = inhibitory concentrations causing 25%; NOEC = no observable effect concentrations; LOEC = lowest observable effect concentrations; CV (%) = coefficients of variation in control treatments; MSD (%) = percentage of decrease in comparison to corresponding control.

RGR (total plant fresh weight)0.70 (055–1.02)0.40 (0.22–0.52)0.230.4641.635.76
Yield (total plant fresh weight)0.51 (0.39–0.64)0.34 (0.16–0.38)0.230.4658.734.25
Total shoot chlorophyll a content (mg)1.99 (1.74–2.34)1.02 (0.65–1.42)0.921.8534.735.92
Chlorophyll a concentration (mg/g shoot)3.13 (2.53–3.56)2.19 (1.73–2.85)2.313.6918.326.17
Root fresh weight (mg)0.55 (0.41–1.16)0.34 (0.22–0.38)0.230.4635.137.18
Root length (mm)0.45 (011–1.17)0.11 (0.06–0.42)0.230.4649.952.96
Shoot fresh weight (mg)0.42 (0.26–0.57)0.27 (0.09–0.36)0.230.4680.249.22
Shoot length (mm)0.69 (0.12–0.84)0.40 (0.06–0.59)0.460.9248.138.44
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Figure 1. Myriophyllum aquaticum contact test with atrazine-spiked sediment: concentration response curves. (A) Percentage of inhibition of relative growth rates (RGR), yield (based on fresh weight biomass), chlorophyll a content in shoot, and chlorophyll a concentration per gram of shoot. (B) Percentage of inhibition of weight and length of root and shoot. (C) Percentage of inhibition of root-to-shoot ratio (based on weight and length).

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Recovery studies

The results of the recovery studies with L. minor and M. aquaticum after short-term (3 d) and 7-d exposure of L. minor to water-column atrazine are summarized in the Supplemental Data. Toxicity of water-column atrazine to L. minor and M. aquaticum after exposure and recovery phase is expressed as IC50, IC25, NOEC, and LOEC values (µg/L).

Relative growth rate and yield (both based on fresh weight biomass) IC50 values immediately after a 3-d-long exposure to atrazine could not be estimated for M. aquaticum, as both were above 640 µg/L, that is, the highest applied test concentration. The value of IC50 for L. minor after a 3-d exposure was 74 and 67 µg/L when estimated for RGR and yield based on fresh weight biomass, respectively. The values of IC50 estimated for RGR as well as yield based on the number of fronds and total frond area were considerably higher, at 285 to 416 µg/L. However, after 9- and 12-d-long recovery phases, the IC50 values for M. aquaticum estimated for RGR and yield based on fresh weight basis were 66 to 92 µg/L, with NOEC/LOEC values of 40/80 µg/L. As far as L. minor is concerned, after 4- and 6-d recovery phases, neither IC50 values nor NOEC/LOEC values could be estimated for any of the RGR-based endpoints (fresh weight biomass, number of fronds, and total frond area) as all the values increased above the highest applied test concentration at 1,280 µg/L.

The recovery patterns of L. minor and M. aquaticum after 3-d-long exposure to atrazine are shown in Figures 2 and 3, by presenting growth curves (Figs. 2A, 3A) and RGRs (Figs. 2B, 3B) based on fresh weight biomass basis in each treatment and control.

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Figure 2. Recovery patterns of Lemna minor after short-term (3-d) exposure to atrazine. (A) Total biomass (fresh weight, mg). (B) Relative growth rates (RGR) (based on fresh weight biomass).

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Figure 3. Recovery patterns of Myriophyllum aquaticum after short-term (3-d) exposure to atrazine. (A) Total biomass (fresh weight, mg). (B) Relative growth rates (RGR) (based on fresh weight biomass).

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The growth curves of L. minor exposed to all test concentrations followed the pattern of the control (Fig. 2A), while in the case of M. aquaticum the increase of the final compared to the initial biomass was observed only in treatments with the two lowest applied atrazine concentrations, 40 and 80 µg/L (Fig. 3A). Whole plant biomass in treatments with 160, 320, and 640 µg/L was lower than the initial biomass even after a 12-d-long recovery period (Fig. 3A).

Detailed analyses of RGR during exposure phase, recovery phase, cumulative RGR during the entire study, and the last interval RGR (RGR calculated for the final 3-d-long interval of the recovery phase) are presented in Figure 4. In the case of L. minor, a statistically significant decrease of RGR during the 3-d-long exposure phase (Fig. 4B) was observed in all test concentrations compared to control. However, relative growth rates of M. aquaticum during the 3-d-long exposure phase (Fig. 4A) decreased significantly compared to control only at two of the highest applied concentrations, 320 and 640 µg/L. During the recovery phase, RGR of L. minor exposed to all atrazine concentrations (Fig. 4B) exceeded the control treatment. In treatments with 320, 640, and 1,280 µg/L, the increase was statistically significant. Contrary to L. minor patterns, the RGR of M. aquaticum exposed to atrazine decreased significantly during the recovery phase (Fig. 4A) in all test concentrations except for the lowest applied—40 µg/L. When RGR was taken as a cumulative value for the whole duration of the present study (3-d exposure + 6-d recovery = 9 d in total), there were no statistical differences between any of the treatments and control in the case of L. minor (Fig. 4B), but in the case of M. aquaticum the cumulative (3-d exposure + 12-d recovery = 15 d in total) RGR pattern (Fig. 4A) was similar to the curve from the recovery phase. Figure 4A,B also shows the last interval RGR from days 6 to 9 in the L. minor study (Fig. 4B) and from days 12 to 15 in the M. aquaticum study (Fig. 4A). No significant differences were observed between control and test treatments in the last interval RGR in the M. aquaticum study, although a rather high variance in controls as well as most of the treatments should be noted. The last interval RGRs of L. minor exposed to all atrazine concentrations were higher than in the control. In 320 to 1,280 µg/L test concentrations, the differences versus control were statistically significant (Fig. 4B).

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Figure 4. Detailed analysis of recovery patterns of Lemna minor and Myriophyllum aquaticum after exposure to atrazine expressed as relative growth rates (RGR) (based on fresh weight biomass). (A) Myriophyllum aquaticum, 3-d exposure. (B) Lemna minor, 3-d exposure. (C) Lemna minor, 7-d exposure. Letters A through D denominate significant difference versus the corresponding control (one-way ANOVA, post-hoc test, t test, p ≤ 0.05). (A) Exposure phase RGR, (B) recovery phase RGR, (C) cumulative RGR, (D) final interval RGR (calculated for the last three experimental days).

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The recovery patterns of L. minor after 7-d-long exposure to atrazine are presented in Figure 5 as total fresh weight biomass (Fig. 5A) and RGR (Fig. 5B) based on fresh weight biomass. Detailed analyses of RGR based on fresh weight biomass during 7-d-long exposure phase, 5-d-long recovery phase, cumulative RGR during the entire study (7-d exposure + 5-d recovery = 12 d in total), and the last interval RGR (from day 9 to 12) are presented in Figure 4C. The stagnation of the final compared to the initial biomass was observed in treatments with 640 and 1,280 µg/L (Fig. 5A). After a significant decrease of RGR during the exposure phase (Fig. 4C), the recovery phase RGRs in all tests concentration exceeded the control treatment RGR. Cumulative RGRs in all treatments were significantly lower than in the control, but the last interval RGRs (from day 9 to 12) in all treatments exceeded the controls.

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Figure 5. Recovery pattern of Lemna minor after 7-d exposure to atrazine. (A) Total biomass (fresh weight, mg). (B) relative growth rates (RGR) based on fresh water biomass.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. SUPPLEMENTAL DATA
  9. Acknowledgements
  10. REFERENCES
  11. Supporting Information

M. aquaticum versus L. minor: sensitivity to atrazine

The results of toxicity tests carried out in the sediment-free system showed comparable sensitivity of M. aquaticum and L. minor to atrazine. Although the duration of the tests differed (7-d exposure for L. minor and 10-d exposure for M. aquaticum), IC50 and IC25 as well as NOEC/LOEC values based on fresh weight biomass were similar. However, if compared to other L. minor growth parameters (RGR and yield based on the number of fronds and total frond area), M. aquaticum appears to be the more sensitive species to atrazine (Table 1).

The presented results on toxicity of atrazine to L. minor are in agreement with literature data, which in most cases present the results based on RGR calculated on the number of fronds. Fairchild et al. 14 reported 96-h EC50 values for L. minor to be 92 to 152 µg/L. Drost et al. 25 exposed L. minor to different triazine herbicides, including atrazine for 72 h and reported a 72-h EC50 of 0.7 µM/L (155 µg/L) and 6-d EC50 0.8 µM/L (176 µg/L) based on the number of fronds. Mohammad et al. 23 reported 7-d EC50 values of 89 ppb atrazine for L. gibba.

The open literature on the effects of atrazine to aquatic macrophytes provides little information on its toxicity to M. aquaticum, so the results of the present study contribute to filling in the missing data on M. aquaticum sensitivity to herbicides. The comparative data on sensitivity of other Myriophyllum versus Lemna species to herbicides, including atrazine, can be frequently found in the literature. Coutris et al. 20 compared sensitivity of different macrophyte species to a mixture of atrazine, isoproturone, and alachlor (a mixture contained 50% atrazine); the reported EC50 values were 70 ± 16 µg/L for L. minor and 59 ± 44 µg/L for M. spicatum. Other studies also found Myriophyllum species to be among macrophyte species that were the most sensitive to pesticides 21, 36. However, the opposite results have also been frequently reported 14, 16, 17. It can therefore be concluded that species sensitivity depends on the compounds considered 11.

M. aquaticum contact test: atrazine-spiked sediment

Myriophyllum aquaticum subculture cultivated on water-saturated sediments used for the contact test in the present study fulfilled the proposed minimum growth rate of 0.09 mg for 10 d as suggested by Feiler et al. 12. Recent studies showed that the growth rate of M. aquaticum is higher if grown in water-sediment than in sediment-free systems 35. The present study, as well as the results published recently by Feiler et al. 12 and Wersal and Madsen 37 showed that, as an emerged macrophyte species (contrary to other species from the same genus, which are mainly submersed), M. aquaticum grew very well on water-saturated sediment in water-free systems.

A dose response was observed for all endpoints except for root-to-shoot ratios (Fig. 1). Shoot and root growth parameters seem to be similar in terms of sensitivity to atrazine exposure, as IC50 values fell into a rather narrow range from 0.4 (shoot fresh weight) to 0.7 µg/g (relative growth rate based on the total plant fresh weight biomass) (Table 2). An increase of root-to-shoot ratio (based on fresh weight), although not significant, was recorded in all test treatments compared to the control, but it must be noted that none of the plants exposed to the two highest test concentrations have developed roots. Wersal and Madsen previously reported no significant atrazine uptake difference between roots and shoots in a sediment-free system 38. Myriophyllum aquaticum develops adventitious roots that grow from each node of the stolon where growth will begin once the old emergent shoots are submersed in the water column. Adventitious roots can grow to lengths of approximately 30 to 50 cm, giving M. aquaticum greater access to water column substances than other macrophyte species. Myriophyllum aquaticum has sediment roots; however, they are highly cuticularized, which may limit the uptake from the sediment 39.

The results of the present study showed that when grown in a water-free system, M. aquaticum developed sediment but not adventitious roots, as all shoots remained emergent throughout the test. Therefore, the only possible uptake was directly from sediments. In a risk assessment, this can be seen as a comparative advantage to other aquatic macrophyte species—M. aquaticum can be used for sediment assessment and risk assessment of sediment bound substances exclusively, because the impact or interference of water column substances can be completely excluded.

Recovery patterns of L. minor and M. aquaticum

The differences between the two tested macrophyte species are even more evident from the results of the recovery study. The response of L. minor after short exposure was fast and intense. Immediately after the exposure period, the decrease of RGR calculated on fresh weight basis was observed in all treatments compared to the control (Fig. 2B). The IC25 and the IC50 were lower than after 7 d of continuous exposure (Supplemental Data). However, after only 5 d of recovery phase the IC50 values for L. minor could not be estimated, as RGR inhibition (calculated for all three growth parameters) was below 50% compared to the control. After 6 d of recovery, IC25 values could not be estimated either, as the growth inhibition versus controls was below 25%. Relative growth rates increased in all treatments versus control during the recovery phase of the present study. No statistically significant difference was observed in cumulative RGRs (calculated for the whole duration of the present study, exposure + recovery phase) of any of the measured growth parameters, in any of the treatments compared to the control (Fig. 4B). The overall increase of total biomass compared to the initial values was observed in all treatments (Fig. 2A).

The recovery patterns of L. minor after 7-d-long exposure to the same range of atrazine concentration are rather similar to those after short exposure. Again, a significant decrease of RGRs observed in all treatments compared to the control during the exposure phase was followed by rapid significant increase in recovery phase. The only difference is that the cumulative RGRs after longer exposure remain lower in all treatments compared to the control, whereas the cumulative RGRs after short exposure did not differ significantly between the control and treatments (Fig. 4C). Again, no negative growth trend was observed: colonies grew slowly but continuously; therefore, decreases in biomass compared to the initial values were not recorded at any interval of the present study (Fig. 5A).

Myriophyllum aquaticum seems to respond to atrazine with a considerable delay. The initial response was mild. After the 3-d-long exposure phase, the IC50 value could not be estimated, as growth inhibition was below 50%. However, RGR decreased during the recovery phase compared to the exposure phase and reached the minimum 6 d after transfer to a clean Steinberg medium (day 12 of the present study) (Fig. 3B). At the same time, the total biomass of plants exposed to 320 and 640 µg/L decreased below initial values (Fig. 3A). The increase of RGR was observed during the last two intervals of the recovery phase, from day 9 to 12 and from day 12 to 15 in all treatments. However, the final biomass of plants exposed to 320 and 640 µg/L did not reach the initial biomass until the end of the recovery phase. A continuous increase of biomass during the whole study was observed only in the control, 40 and 80 µg/L exposed plants. At the end of the present study, after a 3-d exposure phase and a 12-d-long recovery phase, the cumulative IC50 value was 92 µg/L, which is close to IC50 estimated after 10 d of continuous exposure to the same atrazine concentrations. Still, atrazine did not cause irreversible changes to M. aquaticum exposed even to the highest applied concentration, 640 µg/L. Visual inspection of the plants after the present study showed that the nodes remained in several cases, although without leaves. It might be expected that the growth would slowly continue in all treatments and that after a longer recovery period even plants that have exhibited negative growth trend during the whole recovery phase might start growing again. Aquatic plants are known for their high regeneration capacity. Interestingly, a characteristic of some aquatic weeds is the ability to grow from small fragments. Myriophyllum aquaticum was able to form new shoots from a single node (with or without leaves) and showed a high potential for regeneration by developing new shoots from single leaves, although such capacity was significantly lower than the regeneration from stem fragments with nodes with or without leaves 40.

The presented results indicate that the exposure duration seems not to affect sensitivity of either L. minor or M. aquaticum considerably, but the recovery of M. aquaticum even after short exposure is rather slow and less efficient than in the case of L. minor, which is capable of efficient recovery even after prolonged exposure. The effects of short exposure (3 d) to s-triazines (ametryn and prometon) on the growth of L. minor are easily reversible 24. The concentration-dependent growth inhibition was clearly visible; however, after the recovery phase the growth rate was nearly back to the control levels. This remained true even if the plants were exposed to extraordinarily high concentrations of ametryn (up to more than 15 times the EC50) 24. Similar results have been observed earlier 24 for L. gibba, which also showed a comparable recovery after a 5-d exposure to atrazine. In a 28-d-long study by Mohammad et al. 22, L. gibba growth was significantly inhibited after a 7-d exposure at 200 µg/L atrazine, and the comparable inhibition continued during a 28-d exposure. The RGR decreased slightly after 7 d of exposure and was almost constant from day 14 to 28 of exposure. The authors 23 concluded that phytostatic concentrations of atrazine to L. gibba were 1,600 and 800 µg/L in the exposure periods of 14 and 28 d, respectively, and that the phytocidal concentration was greater than 3,200 µg/L for a 28-d exposure. Atrazine inhibited growth completely but was not lethal for L. gibba even at a concentration of 3,200 ppb with 28-d exposure, and the RGR was 43% in recovery. In another study the same authors showed that chemicals that act as inhibitors of photosynthesis in PSII are moderately toxic to L. gibba, and that moderate recovery (RGR, 76%) was observed after exposure to 1,000 µg/L of atrazine for 7 d 23. No available literature data exist on the recovery potential of M. aquaticum after exposure to herbicides under laboratory conditions.

M. aquaticum and recovery potential

Differences in sensitivity between macrophytes and experiments might be related to growth, as faster-growing plants are sometimes more sensitive to toxicants than slower-growing ones 15. The results of the present study showed, however, that faster-growing species such as L. minor reacted faster than slower-growing M. aquaticum, but in total, seems to be less sensitive to atrazine, particularly if its fast and efficient recovery after exposure is taken into account. Because no macrophyte species proved to be consistently the most sensitive, the choice should be based on taxonomic considerations and likelihood of exposure, but also on some other, practical criteria such as performance and growth in the laboratory, suitability as test species, and geographical distribution. Many of these issues would be resolved by adopting a multispecies approach in risk assessment 11.

The results of the present study advocate the use of Myriophyllum sp. as an additional macrophyte species in risk assessment. The sediment contact test (in water-free system) can be appropriate if the study objective is to assess the risk of contaminated sediments (especially at hazardous waste sites), to establish the relative contribution of sediment-bound residues to the overall toxicity, or for risk assessment of compounds that partition strongly to sediment. Although atrazine is not the best representative of such compounds, the present study still showed some advantages of using a water-free system for M. aquaticum spiked-sediment contact test. Given that realistic exposure for many compounds would largely be by way of the water column, a water-sediment exposure might be more relevant for submerged aquatic macrophytes. The outcome of the ongoing water-sediment and contact-sediment ring tests with Myriophyllum (P. Dohmen, BASF, Limburgerhof, Germany, and U. Feiler, German Federal Institute of Hydrology, Koblenz, Germany, personal communication) would therefore be valuable for assessing applicability of different Myriophyllum species and test designs in risk assessment.

The necessity to include the recovery studies in an ecological hazard assessment—not only for plant protection products but also for other toxic environmental pollutants—has already been emphasized 25. Such important and ecologically relevant information does not necessarily have to be obtained only in higher-tier risk assessment, but also from suitable minor adaptations of standard laboratory tests. The results of the present and a few studies published earlier 23–25 indicate that in the case of fast-growing Lemna species, standard test protocols 3 that specify a 7-d exposure period could be adapted by adding a 7-d-long recovery phase. In the case of a slowly growing species, such as Myriophyllum, considering their longer lag phase and slow recovery, it would be advisable for a recovery phase to last at least twice, preferably three times longer than the exposure phase. Another issue to be specified is the choice of the most appropriate endpoint. Standard protocols 3 rely on RGR (or yield) inhibition in treatments versus control at the end of the test. Should the recovery phase be included in the test protocol, a cumulative RGR (or yield) inhibition might be a suitable endpoint for fast-growing species such as Lemna, but can easily overestimate the hazard to species with a long lag phase and slow recovery. The present study showed that it was worth dividing exposure and recovery phases into short, 3-d-long intervals. Frequent observations and measurements during the whole study, particularly during the recovery phase, can provide a better understanding of response and recovery patterns of different macrophyte species. The last interval RGR (calculated for the final 3-d-long interval of the recovery phase) seems to provide a good indication of plant recovery potential regardless of the growth strategy of selected test species. However, more studies that would focus on aquatic plant recovery after exposure to chemicals with a different mode of action are necessary to suggest the most appropriate test design and endpoint.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. SUPPLEMENTAL DATA
  9. Acknowledgements
  10. REFERENCES
  11. Supporting Information

The results of the present study showed that although L. minor reacted faster and more intensely to atrazine than slower-growing M. aquaticum, it seems to be less sensitive, particularly if its fast and efficient recovery after exposure is taken into account.

The differences in growth strategy, route of exposure, lag phase, and particular recovery patterns, advocate the use of Myriophyllum sp. as an additional macrophyte species in risk assessment. It can be tested in a water (sediment-free) system, and the test protocol might easily include recovery potential studies. The results of the present study also provided some evidence that in a spiked-sediment contact test (in a water-free system), M. aquaticum could be suitable for the risk assessment of sediment-bound substances.

Ecologically relevant information about aquatic plant recovery potential and patterns can be obtained from laboratory tests by adding a recovery after exposure phase. The duration of exposure and recovery phases in the case of fast-growing species (e.g., Lemna) could be the same, but for slowly growing species, such as Myriophyllum, the recovery phase should be twice or three times longer than the exposure phase. The last interval RGR (calculated for the final 3-d-long interval of the recovery phase) seems to provide a good indication of plant recovery potential regardless of the life history of selected test species.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. SUPPLEMENTAL DATA
  9. Acknowledgements
  10. REFERENCES
  11. Supporting Information

We thank the editor and two anonymous reviewers for constructive criticism. The present study was funded by the Ministry of Education and Science, Republic of Serbia, grants 17337 and 172028.

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  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. SUPPLEMENTAL DATA
  9. Acknowledgements
  10. REFERENCES
  11. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. SUPPLEMENTAL DATA
  9. Acknowledgements
  10. REFERENCES
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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