Growth of Halodule wrightii in culture and the effects of cropping, light, salinity and atrazine

Aquatic Botany, 28 (1987) 25-37

25

Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

GROWTH EFFECTS

OF HALODULE OF CROPPING,

WRIGHTIIIN LIGHT,

CULTURE SALINITY AND

AND THE ATRAZINE

CHRISTINE A. MITCHELL

P.O. Box 16028, Baton Rouge, LA 70893 (U.S.A.) (Accepted for publication 15 October 1986)

ABSTRACT Mitchell, C.A., 1987. Growth of Halodule wrightii in culture and the effects of cropping, light, salinity and atrazine. Aquat. Bot., 28: 25-37. The interaction of three environmental variables, light, salinity and cropping, with the effects of the herbicide atrazine on Halodule wrightii Ascherson was investigated in the laboratory. Atrazine at 30 ppm caused a significant reduction in survival of ramets, production of new ramets, above-ground biomass and growth, when compared to Halodule wrightii not exposed to atrazine. The three levels of each environmental condition did not alter the toxicity of atrazine to Halodule

wrightii.

INTRODUCTION Seagrass meadows are one of the most productive ecosystems on earth (Phillips, 1978; McRoy and Helfferich, 1980; Phillips, 1980a). The productivity of Texas bays and estuaries is due, in part, to the presence of extensive seagrass meadows (Hellier, 1962; Pulich, 1980a; Rabalais, 1980), with Halodule wrightii Ascherson being the most widespreadspecies (Merkord, 1978). In Texas (Merkord, 1978), as in other parts of the U.S.A. (Bayleyet al., 1978) seagrass meadows seem to be disappearing. Many causes and contributing factors for this decline have been investigated and include dredging (Odum, 1963; Rickner, 1979), thermal stress (Roessler, 1971), salinity (McMillan and Moseley,1967; Zieman,1975), oil pollution (Foster et al., 1971 ) and heavy metal exposure (Pulich, 1980b; Schroeder and Thorhaug, 1980; Van der Werff and Pruyt, 1982). One possible factor that has received little attention is the role that herbicides are playing in the loss of seagrass habitat. One commonlyused herbicide in the U.S.A. is atrazine (2-chloro-4-ethylamino-6-isopropylamino-l,3,5triazine). Atrazine is persistent and is known to remain active for up to i year, or sometimes longer, after application ( Smith et al., 1975; Ashton, 1982). It also 0304-3770/87/$03.50

© 1987 Elsevier Science Publishers B.V.

26 is capable of being transported in water away from its initial application site (Rohde et al., 1981; Olsen et al., 1982). Atrazine ( < 1 ppb) was found in estuarine waters of Chesapeake Bay during every month of the year, even though it was applied only during May ( Correll et al., 1978; Wu, 1981 ). Atrazine, along with other herbicides, is probably entering other bays and estuaries. The interpretation of herbicide toxicity data is complicated because the effects of the toxicant can be modified by the phenological stage of the plant (Da Silva and Warren, 1976; Paley and Radosevich, 1984), time of day (Rikin et al., 1984 ), the presence of other chemicals (Jana and Choudhuri, 1981; Pieterse and Roorda, 1982), temperature (Freyman et al., 1982; Rubin and Benjamin, 1983) and light conditions (Anderson, 1981; Kay et al., 1984). The natural habitat of H. wrightii is variable with regard to salinity, light and cropping, so it is important to know how environmental variables interact with a toxicant in order to evaluate its possible effects. The object of this study was to determine if varying levels of salinity, irradiance or cropping interacted synergistically, antagonistically or additively with the effects of atrazine on Halodule wrightii. METHODS Separate interaction trials were designed to investigate the effects of three different environmental conditions on the toxicity of atrazine to H. wrightii. Each trial was a six-celled experiment with three different levels for each environmental condition, each either with or without one level of atrazine. The sample size was 10 aquaria per cell. The environmental conditions that were varied were light intensity, salinity and the degree to which ramets were cropped. Halodule wrightii was cultured in the laboratory using standard methods (McMillan, 1976, 1980a). At the beginning of each trial, 60-70 plugs of H. wrightii were collected at a site in Redfish Bay, 5.1 km east of the junction of Highways 35 and 361 in Aransas Pass, Texas (Wiginton and McMillan, 1979). The plugs, measuring about 55 m m in diameter and 60 mm deep, were removed from the ground with the bay sediment intact using a coring device. Each plug was then individually placed in a 270-mi glass jar. Upon arrival in Victoria, each plug was culled of dead ramets and debris and placed individually in a glass aquarium containing 2.75 1 synthetic seawater prepared from Instant Ocean ®. The sediment surface of each plug was approximately 120 mm below the surface of the seawater. The environmental conditions were measured in place on the day of collection, and the herbicide dosing was done on the following day. Following a trial, all aquaria and jars were washed in a dishwasher and then rinsed in methanol to remove any traces of atrazine that might remain. The dates that each trial began and its duration, and the collection and laboratory conditions are summarized in Table I. The trial conditions were chosen

27 TABLE I Summary of the field and laboratory conditions for the three trials Cropping interaction

Light interaction

Salinity interaction

Bay conditions Date Water temperature ( ° C) Salinity (ppt)

22 Oct. 1983 26 15

1 Sept. 1984 32 40

18 Nov. 1983 24 26

Trial conditions Duration {days) Water temperature (°C) Light reading (/~Em -2 s -1) Salinity (ppt)

22 26 108 35

22 27 115, 140, 173 35

23 25 108 15, 25, 35

to reflect as closely as possible an average set of conditions found in Redfish Bay throughout the year. The three levels of each environmental condition imposed in the interaction trials were also chosen to reflect conditions that can Occur.

The light in the laboratory was provided by twenty 40-W Sylvania Daylight F40/D fluorescent light tubes positioned 22 cm above the top of the aquaria. The photoperiod was 14 h light and 10 h dark ( McMillan, 1978). There was a 10% variation in light level according to the placement of the aquaria on the table; those on the outside edges received less light than those in the center. For the Light Interaction Trial, the light bank was lowered to 2.5 cm above the top of the aquaria. This produced a maximum reading of 172/zEm -2 s -1 at shoot level, with a 5% variation due to aquaria location. To reduce the amount of light transmitted for the two reduced light-level groups, aluminum and fiberglass screens were placed between the lights and aquaria for the first and second series of six light tubes, respectively (Drysdale and Barbour, 1975 ). The varying salinity levels were achieved by mixing Instant Ocean with tap water. Tap water has been used by other researchers ( McMillan and Moseley, 1967; McMillan, 1980a). Every 2-3 days, tap water was added to each aquarium to replace that lost due to evaporation. Evaporative water loss raised the salinities 1-2 ppt over the 2-3-day period. The water temperature in the laboratory was not controlled, but it remained fairly constant within and among trials. The water temperature was generally 1 °C cooler than average in the morning, when the lights had just come on, and 1 oC warmer than average when the lights went out at night. The 20 aquaria on the two outside rows were generally 0.5-1.0°C cooler than the four inner rOWS.

The aquaria were dosed with 30 ppm atrazine by adding 120 #l of Atrazine 4L to each herbicide-treated aquarium with a Hamilton No. 1725 syringe

28 equipped with a repeating dispenser. Thirty p p m is near the maximum amount of atrazine soluble in water, and was used to ensure the presence of toxic effects in order to assess any interactions with the three environmental variables. After the toxicant was added, the aquarium was gently stirred for about 15 s with a glass rod. All non-herbicide-treated aquaria were stirred for 15 s in a manner similar to the herbicide-treated aquaria. Atrazine residues were run on pooled water samples at the termination of the Cropping and Salinity Interaction Trials. From each herbicide-treated aquarium 100 ml samples were collected and pooled by environmental treatment. The samples were extracted with ethyl acetate and analyzed on a Tracor Model 222 gas chromatograph equipped with a nitrogen-phosphorous detector. The analyses were done by the Cooperative Extension Service, University of Georgia College of Agriculture. The detection limit was 0.5 ppb. Treatments were randomly assigned to the aquaria prior to the beginning of each trial. Beause of the method of light shading for the Light Interaction Trial, a randomized block design was used, with the herbicide treatments being randomly assigned within each light-level block. Every 2-3 days, each aquarium was checked and the number of live and dead ramets were recorded. A ramet was considered dead when all of its leaves had turned brown, fallen off, and it showed no regrowth. In the Cropping Interaction Trial, the leaf length of one ramet per aquarium was measured from the ground level to the tip of the longest leaf using a millimeter ruler. The same ramet and leaf were measured in each aquarium throughout the trial. Since there was a variable number of ramets present in each plug, the number of live ramets present on each day was expressed as a ratio. The ratio was the number of live ramets present on each day divided by the original number of ramets in the plug. A value of > 1 meant an increase in the total number of ramets, and a value of < 1 meant a decrease in the number of ramets. At termination, all ramets (except the center one which was reserved for chlorophyll analysis, data not presented here ) were clipped at ground level and dried to a constant weight to determine the above-ground biomass {Dawes et al., 1979; Dennison and Alberte, 1982). The dry weight per ramet was calculated by dividing the total above-ground biomass by the number of live ramets present at termination. Means were calculated for all parameters, and the data were analyzed using two-way analysis of variance {Sokal and Rohlf, 1969). Mean separations were done using Tukey multiple comparison method when the sample sizes were equal, and Bonferroni multiple comparison method when the sample sizes were unequal (Neter and Wasserman, 1974). Standard errors were compared using Bartlett's test. A significance level of P = 0.05 was used for all tests unless otherwise noted.

29 1.2

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Fig. I.Average ratioof totalnumber of liveramets on each observationdate to originalnumber of rametspresentforthe Cropping InteractionTrial.Solidlinesrepresentnon-herbicide-treated aquariaand dottedlinesrepresentherbicide-treatedaquaria.The averagestandarderrorsforthe noncropped,60- and 40-mm groupsin the non-herbicide-and herbicide-treatedaquariaare 0.05, 0.03,0.05,0.08,0.04and 0.04,respectively. RESULTS In the Cropping Interaction Trial, the average number of ramets per aquarium (11.8) was not significantly different a m o n g treatment groups. The standard errors were small and not significantly different a m o n g dates for each treatment group individually, therefore, only the average S.E. for each group is given in Fig. 1. Beginning on 30 October, the average ratio was significantly higher in the non-herbicide-treated group than in the herbicide-treated group, and this difference continued until termination of the trial 2 weeks later (Fig. 1 ). Beginning on 8 N o v e m b e r and continuing until termination, the non-herbicide-treated group had significantly higher ratios than their herbicide-treated counterparts at the non-cropped and 40-ram cropped levels,but there was no significant difference at the 60-ram cropped level.Trimming the ramets seemed to stimulate the production of new ramets. This was seen in the non-herbicidetreated group as well as in the herbicide-treated group. Because of this stimulatory effect,on 30 October and i and 6 November, the 40-ram cropped group (which combined the non-herbicide-treated with the herbicide-treated aquaria) as a whole had a higher ratio than the non-cropped group. After 8 days, however, the herbicide effects began to dominate, and the two cropped groups also began to show a decline in ramet numbers. B y the end of the trial,the herbicide-treated groups had fewer ramets present than at the beginning of the trial and the non-herbicide-treated groups had more ramets present than at the beginning. Overall, the average above-ground biomass was significantly greater in the

3O TABLE II Total above-ground biomass (mean ___1 s.e.) in the Cropping Interaction Trial Cropping level (mm) None 60 40 Overall

Sample size 10 10 10 30

Dry weight (rag)

Significance'

Without herbicide

With herbicide

50.8 _+8.92 47.5_+4.1 39.2 ___6.3 45.8 _+3.9

25.1 + 5.62 25.6_+ 2.1 16.3 _+3.2 22.3 __+2.3

* NS * *

'NS =not significant; • =significant at P=0.05. 2There were no significant differences among the three cropping level groups. n o n - h e r b i c i d e - t r e a t e d group ( T a b l e I I ) . T h e overall r e d u c t i o n in t h e biomass of the h e r b i c i d e - t r e a t e d group was 51%, a n d averaged 51, 46, a n d 57% less in the t h r e e stress groups, respectively. On a p e r r a m e t basis, the dry weight per r a m e t was significantly g r e a t e r in t h e n o n - h e r b i c i d e - t r e a t e d group t h a n in t h e h e r b i c i d e - t r e a t e d group ( T a b l e I I I ) , b u t it was n o t significant at t h e t h r e e c r o p p i n g levels individually. T h e stress f a c t o r was also significant. W h e n the n o n - a n d 60- a n d 4 0 - m m c r o p p e d groups were c o m p a r e d , c o m b i n i n g t h e n o n herbicide a n d herbicide groups w i t h i n each, t h e average dry weight p e r r a m e t was 3.04 + 0.29, 2.96 _+0.18 a n d 2.25 _+0.21 mg, a n d was significantly h i g h e r in the n o n - c r o p p e d group t h a n in t h e 4 0 - m m c r o p p e d group. T h e 6 0 - m m c r o p p e d group was i n t e r m e d i a t e a n d was n o d i f f e r e n t f r o m t h e o t h e r groups. T h i s docu m e n t s the obvious, since the 6 0 - m m a n d 4 0 - m m groups were cut c o n s i d e r a b l y s h o r t e r t h a n t h e n o n - c r o p p e d group a n d s h o u l d h a v e less biomass. A t r a z i n e affected t h e g r o w t h o f c r o p p e d r a m e t s (Fig. 2). B e g i n n i n g on 25 O c t o b e r a n d c o n t i n u i n g u n t i l the e n d o f t h e trial, t h e n o n - h e r b i c i d e - t r e a t e d group h a d significantly longer r a m e t s t h a n t h e h e r b i c i d e - t r e a t e d group. W h e n the 60- a n d 4 0 - m m groups were e x a m i n e d individually, t h e n o n - h e r b i c i d e TABLE III Dry weightper ramet (mean _+s.e.) in the CroppingInteraction Trial

Cropping level (ram) None 60 40 Overall

Sample size 10 10 10 30

Dry weight/ramet (mg)

Significance 1

Without herbicide

With herbicide

3.23 _+0.392 3.05 +0.13 2.76 + 0.33 3.02 _ 0.17

2.84 + 0.432 2.86__.0.34 1.74__+0.16 2.48_ 0.21

'NS--not significant; * =significant at P=0.05. 2There were no significant differences among the three cropping level groups.

NS NS NS •

31 6O m m

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80 70

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50 .40 I

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Fig. 2. Average cumulative height for mmet~ cropped to 60- end 40-ram above the soil surface J_n the Cropping Interaction Trial. Solid lines represent non-herbicide-treated aquaria and dotted lines represent herbicide-treated aquaria. T h e average standard errors for the 60- and 40-ram groups in the non-herbicide- and herbicide-treated aquaria are 3.1, 3.1, 2.3 and 2.5, respectively.

treated group for both the 60- and 40-ram groups had significantlylonger rarnets beginning 1 week later on 1 November, and this difference continued until termination. By 6 November, the shoot lengths for the 60- and 40-ram groups not exposed to atrazine were no longer statisticallydifferent. In the herbicidetreated group, however, the 40-rnm group did not statisticallycatch up in length to the 60-rnm group until 13 November. Thus, herbicide-treated groups grew slower and did not achieve the length of their respective non-herbicide-treated group. The results of the Salinity and Light Interaction Trials were similar to those of the Cropping Interaction Trial (Figs. 3 and 4). There were significantly fewer rarnets surviving in the herbicide-treated group than in the non-herbicide-treated group; the above-ground biornass and dry weight per rarnet were also significantly reduced by atrazine. The three salinity and three light levels did not alter the toxicity of atrazine. At the end of the Cropping and Salinity Interaction Trials, the atrazine level in the pooled samples averaged 17.7 + 1.55 and 16.4 _+ 1.82 pprn. This was 59 and 55% of the amount originally added. DISCUSSION

Varying environmental conditions did not alterthe toxicity of atrazine under experimental conditions. Within each trial,H. wrightiiresponded similarly to atrazine whether the light level was 173, 140 or 115/zEm-2s -I, the salinity level was 35, 25 or 15 ppt, or whether the shoots were not cropped or cropped to 60 or 40 mrn above the soil surface. There were no significant differences

32

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Fig. 3. Average ratioof totalnumber of liveramets on each observation date to originalnumber of ramets presentforthe Light InteractionTrial.Solidlinesrepresentnon-herbicide-treatedaquaria and dotted linesrepresentherbicide-treatedaquaria.The average standard errorsfor the 173, 140 and 115/~Em - 2s- tgroups in the non-herbicide-and herbicide-treatedaquariaare 0.03,0.03,0.01, 0.03, 0.03 and 0.02, respectively.

1.3

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Fig. 4. Average ratioof totalnumber of liveramets on each observation date to originalnumber of tamers present for the SalinityInteractionTrial. Solid linesrepresentnon-herbicide-treated aquaria and dotted linesrepresentherbicide-treatedaquaria.The average standard errorsfor the 35, 25 and 15 plotgroups in the non-herbicide-and herbicide-treatedaquaria are 0.04,0.03,0.08, 0.03,0.04 and 0.02,respectively. within the herbicide-treated group or the non-herbicide-treated groups at the end of each trial for the parameters measured. Reduction in the a m o u n t of light reaching the H. wrightiim e a d o w s should not alter the toxicity of atrazine because of epiphytic shading or increased turbidity due to dredging. Likewise, salinity changes caused by hurricanes, altered freshwater inflow patterns, cropping d a m a g e from boat propellers or grazing activities should not m a k e

33 atrazine any more or less toxic than it would be under normal unstressed conditions. The effects of the varied environmental conditions are similar to those reported by others. In the Light Interaction Trial, the three light levels are less than the natural irradiance levels produced by sunlight (Pulich, 1980a), but are within the range used elsewhere (Correll et al., 1978; McMillan, 1980b; Correll and Wu, 1982; Jones and Winchell, 1984). The 175/~Em-2s - 1 group produced more shoots than those grown at the two lower levels, but the differences were not great nor were they significant. In a field experiment, Dennison and Alberte (1982) found that an artificially induced 35% increase or a 55% decrease in the amount of light had no effect on ramet size, leaf production rates or chlorophyll content in Zostera marina L. growing in a shallow location. Halodule wrightii is a euryhaline species and seems to grow well in salinities between 9 and 60 ppt (McMahan, 1968; Phillips, 1980b). This was true in the Salinity Interaction Trial as well. There were no patterns in the Salinity Interaction Trial due to the three salinity levels in the non-herbicide- or herbicidetreated group. In the non-herbicide group, the 15 ppt group produced the most ramets followed by the 35 and 25 ppt groups respectively, however, the aboveground biomass was highest in the 25 ppt group and least in the 15 ppt group. In the herbicide-treated group, no patterns relating to salinity were obvious. The 35 ppt group showed the largest decline in numbers followed by the 15 and 25 ppt groups. Halodule wrightii grows by basal elongation from the meristemic region (Virnstein, 1982 ). Halodule wrightii ramets reacted to cropping by producing new ramets. The most severely cropped plants had the greatest increase in ramet numbers; the non-cropped plants had the lowest increase. In the herbicide-treated group, the non-cropped plants began an immediate decline in numbers, while the two cropped groups showed an initial increase in numbers as a result of the cropping stimulus, but then began to decline after about 1.5 weeks. In all three trials, atrazine adversely affected H. wrightii. In the herbicidetreated groups, the number of live ramets declined throughout the trial. There were significantly fewer ramets per aquarium in the herbicide-treated group than in the non-herbicide-treated group by the end of each trial. The aboveground biomass was reduced by 51, 37 and 51% in the three trials, respectively, in the herbicide-treated groups, and the weight per ramet was significantly reduced in most cases. It was not true in all instances because new ramets were smaller and weighed less than old established ramets, therefore, the average dry weight per ramet was reduced in proportion to the number of new ramets present in the aquarium. Zieman (1975) also found this inverse relationship in his work with Thalassia testudinum Banks ex KSnig. Halodule wrightii seems to be less sensitive to atrazine than is VaUisneria americana Michx or Z. marina. Correll and Wu (1982) found 100% mortality

34 in V. americana within 22 and 30 days at 1.06 and 0.12 ppm atrazine, respectively. There was 50% mortality at the end of 47 days at 0.012 ppm. Delistraty and Hershner (1984) found 100% mortality in Z. marina at 1.0 ppm atrazine after 21 days. In four replications, they estimated the concentration that could cause 50% mortality ranged from 0.1 to 0.5 ppm and averaged 0.35 ppm. In this study, even at 30 ppm atrazine, mortality never averaged more than about 30% after 21 days. Finally, atrazine reduced the amount of regrowth in shoots cropped to 40 and 60 mm when compared to the growth rate of cropped shoots not exposed to atrazine. Atrazine was found to inhibit the growth rate in other species as well. Atrazine at 0.32 ppm significantly reduced the leaf length of V. americana by an average of 36% over a 6-week period (Forney and Davis, 1981). Correll and Wu (1982) found that 0.012 ppm atrazine significantly decreased the rate of leaf growth by 75% in V. americana by the end of a 47-day period. The growth rate of Z. marina leaves seemed to be reduced at 1.0 ppm atrazine, but it was not statistically tested nor were any variation statistics given (Delistraty and Hershner, 1984). Again, H. wrightii seems to be less sensitive to atrazine than V. americana or Z. marina. The amount of atrazine present at the end of the trial was intermediate to levels found by others. Kemp et al. (1985) had 84-89% of the original atrazine remaining after 28 days, while Jones et al. {1982 ) had between 18 and 40% left after 20 days. I do not know why H. wrightii seems to be less sensitive to atrazine than other species. One explanation could be that H. wrightii is a pioneering species (Phillips, 1980a; Williams and McRoy, 1982). It is able to recover more quickly from propeller damage than is T. testudinum (Virnstein, 1982) and is able to better withstand being covered with dredge material than other marine angiosperms (Phillips, 1980a). It also has a broader tolerance for temperature (McMillan, 1984) and salinity changes, thus H. wrightii may also be less affected by chemical perturbations in its environment t h a n are the other less resilient species. ACKNOWLEDGEMENTS I benefited from the ideas, time and equipment shared freely by the following individuals whom I wish to thank: James Andreason, Steven Barnes, George Branson, Christine Bunck, Parshall Bush, Leonard Cartwright, Brian Chapman, Thomas Custer, Elwood Hill, Joe Loter, Douglas Maley, Calvin McMillan, Andrew and Kathryn Mitchell, Warren Pulich, Jr., John Tunnell, Jr., Donald White and the U.S. Fish and Wildlife Service. I also wish to thank the editor and two anonymous reviewers for helpful comments on the manuscript. This research was done in partial fulfillment of a M.S. Degree at Corpus Christi State University.

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36

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