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Technical Service Center
Environmental Applications and Research Group — Publications

Suitability of Hygrohypnum ochraceum as an Indicator of Inorganic Pollutants in Streams and Rivers of North America: Laboratory Studies

Technical Memorandum No. 8220-98-15
by
Andrew M. Montaño
1998

Abstract
Introduction
Literature Review
Methods and Materials
Results
Discussion
References


Abstract

Aquatic bryophyte tissues were exposed in the laboratory to cadmium, zinc and selenium to assess their 10-day bioaccumulation potential and to determine physiological stress-response effects under laboratory conditions. Moss tufts of Hygrohypnum ochraceum (Turn. ex Wils.) Loeske, Moosfl. Harz. were collected from Nate Creek Ditch, Colorado USA. Mosses were acclimated to laboratory conditions and were then exposed to Cd, Zn and Se in independently run experiments at specified concentrations for the bioaccumulation portion of this study. Metal treatments for each experiment were performed using a static-renewal method. Physiological stress-response measurements were performed at the end of each bioaccumulation experiment by measurement of chlorophyll-to-phaeophytin ratios (OD665/OD665a). All experimental metals were accumulated in H. ochraceum tissues. Average daily uptake was 69.8% for Cd, 72.2% for Zn, and 10.6% for Se of their initial concentrations. Physiological stress-response measurements showed no significant differences between control- and metal-treated samples. H. ochraceum appears to successfully accumulate metals under certain environmental conditions. These environmental factors also seem to affect determination of physiological stress-response measurements.

Introduction

In the western United States, the impact of inorganic pollutants on water quality has become a major concern on water supplies. Inorganic contamination of natural waters can be attributed to both natural and anthropogenic sources. Connell and Miller (1984) described naturally occurring heavy-metal inputs caused by the chemical weathering of igneous and metamorphic rocks and soils into drainage basins as the most important source of background levels entering surface waters. Mineralized zones within these drainage basins contribute to spatial and/or temporal variance of metal concentrations. Other natural contributors of heavy metals include plant and animal detrital runoff and atmospheric deposition. Anthropogenic sources include domestic effluents and urban storm-water runoff, industrial wastes and discharges, agricultural runoff, and mining operations. Historical mining activities and the formation of acid mine drainage have been significant contributors to water-quality problems in the West (ie. the Upper Arkansas River in Colorado, USA). These activities can release metals such as arsenic, cadmium, cobalt, copper, iron, lead, manganese, nickel, silver, and zinc into natural waters.

Bioindicators are an essential component of environmental quality assessment techniques. Bioindicators can provide an integrated record of intermittent contamination and measure contaminant levels made available for bioaccumulation (López et al., 1994). Criteria for selecting biological indicators include ecological relevance, adequate sensitivity to contaminants, adaptability to both short- and long-term exposures, ability to culture organisms easily, and current and/or historical acceptance (USEPA, 1994; Phillips, 1980). Aquatic bryophytes (mosses) may serve as an ideal biological indicator for the following reasons:

  1. Mosses are ubiquitous in nature (primary producers that provide habitat to various aquatic organisms);
  2. Mosses exhibit a high tolerance to contaminants, allowing for bioaccumulation over long-term exposure;
  3. Mosses possess neither roots nor a vascular system, allowing for potentially easy culturing; and
  4. Once dried, mosses can be stored for long periods since they bind inorganic contaminants within their cell walls without reduction in concentration (Nelson and Campbell, 1995; López and Carballeira, 1993, 1990; Gonçalves et. al., 1992).

The purposes of this study were 1) to review pertinent literature dealing with background information, culture techniques, bioaccumulation and physiological stress in mosses; and 2) to determine the aquatic bryophyte Hygrohypnum ochraceum’s (Turn. ex Wils.) Loeske, Moosfl. Harz. ability to accumulate trace metals under laboratory conditions; and 3) to evaluate whether exposure to these trace metals contributes to stress responses as exhibited through the degradation of chlorophyll to phaeophytin.

Literature Review

An intensive literature review was conducted to prepare for this study. This portion is divided into the categories of literature that help define this study.

Bryophytes
The phylum Bryophyta includes liverworts and mosses. Bryophytes can be characterized as being mostly terrestrial. They all develop from simple embryos and are composed of parenchymous cell tissues except during one phase of the moss life cycle as a filamentous stage. Bryophytes exhibit an alternation of generations with their sporophyte generation dependent upon the gametophyte generation. Lastly, their rhizoids serve to anchor and to absorb water and nutrients from their substrate.

The phylum Bryophyta is divided into 3 classes: Hepaticae, Anthocerotae, and Musci. The aquatic moss Hygrohypnum ochraceum is a member of the class Musci and is included under the family Amblystegiaceae. The family Amblystegiaceae includes mosses that are found in predominantly aquatic or damp environments. The genus Hygrohypnum consists of semi-aquatic pleurocarpous mosses. Pleurocarpous mosses have female sex organs (archegonia) and capsules (spore bearers) which are borne on short, lateral branches and not at the tips (or apical tissues) (Jamieson, 1976). Jamieson (1976) continues to describe Hygrohypnum’s habitat requirements as:

"Hygrohypnum normally occurs on rocks or stones between the high and low water levels; or, in or beside small, cold, swiftly running mountain streams. Like other plants of similar environments various real and alleged species of Hygrohypnum have responded to the fluctuating water levels by assuming a myriad of forms which have blurred species limits."

Common morphological trends that can be noted for the genus Hygrohypnum are:

  1. Broad to narrow leaves;
  2. Straight to straight and/or falcate leave shape;
  3. Mostly undifferentiated to variously differentiated alar cells;
  4. Stems exhibit a small cortex of thick-walled cells, a medulla that has larger, thinner-walled cells, and a central strand distinguished by a more variable pattern of differentiation; and,
  5. Autoecious plants to plants of more variable sexuality (Jamieson, 1976).

More specifically, Smith (1978) and Crum and Anderson (1981) identified Hygrohypnum ochraceum as being a dioecious medium-sized to robust plant with shoots that are soft, procumbent, and usually 1-8 cm in length. H. ochraceum’s stems are irregularly branched with branches that are parallel to the stems. It’s leaves are varied and can be described as loosely erect and when moist can be slightly undulate, concave, ovate-oblong to ovate-lanceolate. When dry the leaves are usually glossy, usually falcato-secund, and often twisted. The leaf apex is rounded to obtuse or sub-acute. The leaf margins are erect or inflexed above and tend to be denticulate towards the apex. Figure 1 depicts various traits of H. ochraceum. The H. ochraceum used in this study was identified by Norton Miller with the New York State Museum.

Studies with Bryophytes
The majority of studies performed with aquatic bryophytes (mosses) have focused on the physiological adaptations that allow them to survive in a variety of severe habitats (Glime and Vitt, 1984; Proctor, 1984). A variety of photoperiod, temperature, flow and water level studies dominate the literature. Li and Glime (1991) discuss the effects of photoperiod in two Sphagnum species and how both increases in growth and biomass can be attributed to continual illumination. Glime and her co-authors have also vigorously studied the effects of temperature and/or flow and water levels on various species of aquatic mosses (Glime and Carr, 1974; Glime and Acton, 1979; Glime, 1980; Glime, 1982; Glime, 1987a, b, c; Glime and Raeymaekers, 1987; and Li et al., 1992). Other investigators have also observed the effects of temperature on growth patterns with a variety of mosses (Sanford, 1979; Furness and Grime, 1982; Jenkins and Proctor, 1985; and Kelly and Whitton, 1987).

Culture Methods
Mosses have been thought of as excellent study material because of their ease of culturing in laboratory conditions during the vegetative phase (or gametophyte generation) of their life cycle (Belkengren, 1962). However, it is difficult to carry mosses through their complete life cycle under sterile conditions. Belkengren (1962) successfully experimented with different aseptic growth mediums to culture Amblystegium riparium and help induce sexual reproduction (sporophyte generation) under laboratory conditions. Methods have since been developed to help in the axenic culturing of bryophytes as well as comparisons of macronutrient media for successful culturing (Basile, 1972; Basile, 1975; Basile et al., 1985; Sargent, 1988; Basile and Basile, 1988; Li and Glime, 1990; Li et al., 1993; and Smith, 1993). Lastly, other growth observations have been made in situ through phosphorous fertilization of the Kuparuk River, Alaska, and its effects on bryophyte distribution (Bowden et al., 1994; Finlay and Bowden, 1994).

Bioaccumulation
Hawkes (1962) said that river pollution "is essentially a biological phenomenon" despite its chemical and physical influences. Hawkes went on to describe the lack of biological surveys seen in his time since emphasis was mostly placed upon biochemical, chemical, and physical standards for determining river pollution. In the early 1970's studies began including plants in investigations of water pollution. Dietz (1972) used several different species of aquatic plants to determine concentrations of macronutrients and trace elements in the Ruhr river. Skaar et al. (1973) used moss to help determine the accumulation of lead in vegetation growing near busy roads. However, a milestone in the use of plants for determining effects of water pollution was set by Benson-Evans and Williams (1976) in their paper describing the use of transplanted aquatic bryophytes for bioassessment of river pollution. With the potential of aquatic bryophytes discovered for use in bioassessments, a variety of studies began to surface with respect to heavy metal monitoring (Burton and Peterson, 1979; Thomas, 1979; Hasseloff and Winkler, 1980; Say et al., 1981; Harding and Whitton, 1981; Whitton et al., 1981; Whitton et al., 1982; Say and Whitton, 1983; Wehr et al., 1983; Mouvet, 1984; Brown, 1984; Kelly et al., 1987; Empain, 1988; Kelly and Whitton, 1989; Arts, 1990; Hawker, 1990; Jackson et al., 1991; Sérgio et al., 1992; Nimmo et al., 1992a, b; Jackson et al., 1993; López and Carballeira, 1993; and Nelson and Campbell, 1995; Carter and Porter, 1997). Each one of these studies tend to agree that the use of aquatic bryophytes in integrated bioassessments can be beneficial, however, not all of these studies were strictly concerned with the use of mosses.

Physiological Stress and Pigment Responses
A variety of studies have been performed to determine chlorophyll’s degradation to phaeophytin in several plants and its spectrophotometric determination (Orr and Grady, 1957; Odum et al., 1958; Vernon, 1960; Wetzel, 1963; Lorenzen, 1967; Moss, 1967a, b; Hendry et al., 1987; and APHA, 1989). Peñuelas (1984a, b) used these known spectrophotometric methods to determine pigment responses in six species of aquatic mosses to water pollution. Patidar et al. (1986) used chlorophyll ratios to determine responses in three species of Riccia to habitat. Lastly, López and Carballeira (1989) led the next wave of studies with aquatic mosses by using their pigment responses to water pollution and their usefulness in determination of water quality (López et al., 1994; López et al. 1997).

Methods and Materials

This study is divided into three major sections: culture, bioaccumulation and depuration, and stress responses. Mosses were collected from Nate Creek Ditch, CO USA, and cultured in the laboratory in Conviron® environmental growth chambers (Model E15) at 15° C ± 2° C with an 18:6 hr. light:dark cycle (light intensity ~130 uE/ sm2). Hygrohypnum ochraceum was subjected to a static-renewal culture method where filtered dechlorinated water was replaced on a weekly basis. The mosses were kept in polyethylene containers with only enough culture water to keep them moist. Upon successful culturing, the mosses were then used for the experimental phase of this study.

Trace metal bioaccumulation experiments with Cd, Zn, and Se treatments lasted 10 days. Each of the trace metal experiments was run independently of the other at separate time intervals. Trace metals were obtained from an outside source at specified concentrations. Concentrations (in solution) were 5.0 ug/L Cd (from CdCl2 x 2.5H2O), 500 ug/L Zn (from ZnCl), and 5.0 ug/L Se (from SeO2). Each 10-day experiment used 46 testing containers: 30 each contained 5.0 grams of moss apical tissues per 1000 mL of metal treatment; 10 each contained 10.0 grams of moss apical tissues per 1000 mL of filtered-dechlorinated water (daily controls); 3 each contained 5.0 grams of moss apical tissues per 1000 mL of filtered-dechlorinated water (10-day controls); and 3 each contained 5.0 grams of moss apical tissues per 1000 mL of metal treatment for use in growth experiments (or depuration). Each day of the experiments, three replicates were randomly drawn along with one daily control replicate. The solutions in which the mosses were contained were saved for concentration analysis; and a fraction (0.1 gram) of the moss was removed for use in the physiological stress-response experiment. The remainder of the moss was then dried for 48 hours in a 60°C drying oven. This routine continued until the experiment was concluded.

Dried samples were analyzed for trace-metal concentration through gaseous hydride atomic absorption (GHAA) or by inductively coupled plasma spectroscopy (ICP). Certified reference samples (National Institute of Standards and Technology, SRM 8031) were submitted for analytical precision and accuracy of metal analysis in bryophyte tissue. The corresponding water samples were also analyzed by GHAA and/or ICP for metal content.

Chlorophyll and phaeophytin analyses were conducted as described by López et al. (1997) using a Perkin-Elmer Lambda 3B UV/VIS spectrophotometer. Physiological stress-responses were estimated by chlorophyll-to-phaeophytin ratios (López et al., 1997, 1994; López and Carballeira, 1989; and Peñuelas, 1984a, b). This ratio, OD665/OD665a, was used by Peñuelas (1984) to describe ecophysiological classes for the aquatic moss Fontinalis antipyretica. This ratio was then used to examine whether "stress" occurred during the 10-day experiments when H. ochraceum was exposed to the different metal concentrations.

Growth measurements were conducted by placing string identifiers on apical tissues for each metal treatment. Growth was then measured at the end of the experiment in both the metal-treated replicates and the 10-day control replicates.

Data obtained from the bioaccumulation experiments and the stress-response experiments were tested for normality and homogeneity of variance and transformed (log10) when necessary. Analysis of variance (ANOVA) was used to determine whether significant differences existed between the daily metal concentrations in moss for each metal-treatment experiment followed by the multiple comparison Tukey’s test (P < 0.05) if significance was indicated by ANOVA.

Results

Precision and accuracy were tested by submittal of certified reference bryophyte material in conjunction with sample bryophyte material. The procedures of Colinet et al. (1982) were followed to determine whether sufficient precision was achieved for the experimental metal-treated samples. Results suggest precision for Zn and Se were acceptable, but Cd had a slightly higher range than that recommended. Accuracy for the Zn samples was 90 percent. The accuracy for Se samples, however, was only 73 percent. Therefore, the analytical values for Se may be slightly lower. Conversely, the accuracy for Cd samples was 130 percent; which suggests that the reported values for Cd are probably somewhat higher than actual values. Comparisons were made between metal concentrations in the moss tissue to that in the treated water in a pair-wise fashion and are presented here despite the interpretive limitations of the precision and accuracy procedures.

Culture

Initially, mosses were cultured in acid-washed jars in aerated filtered-dechlorinated water using a static-renewal method where the water was replaced on a daily basis. Mosses were monitored for new growth and general appearance. However, use of this particular method seemed to induce algal growth and the appearance of the moss was not ideal. Instead of showing bright-green new growth, the moss exhibited a slight brownish-green color and little-to-no new growth was noted.

The culture method used in this study which was suggested by D.W. Jamieson (pers. comm.) appeared to be more successful. Dr. Jamieson suggested culturing Hygrohypnum ochraceum in the laboratory by keeping the moss maintained at 15°C in growth chambers with a 18:6 light:dark cycle (light intensity ~130 uE/ sm2). Plastic containers were used with just enough filtered-dechlorinated water to keep the moss moist. This method seemed to keep the mosses viable and little growth was noted. However, since the genus Hygrohypnum is best suited for cold, swift flowing waters, the use of this type of growth method was not ideal and may have caused some general stress to the moss.

Bioaccumulation

Cadmium
The average amount of Cd in moss tissues before the experimental addition of cadmium to the water was 0.57 ppm (baseline). After the addition of cadmium, moss tissue concentrations ranged from 3.4 ppm (day 1) to 36.0 ppm (day 10) exhibiting an increasing trend over time (Figure 2a). As seen in Figure 2a, uptake of cadmium exhibited a significant difference on a daily basis up to day 6 and no significant differences noted from day 6 to day 10. On average, the daily accumulation of Cd in moss tissues was 69.83 percent (S.D. 46.06) of cadmium from solution. It does not appear that an asymptote was reached during this experimental period. Lastly, the moss in control treatments showed no great increase over the 10-day experimental period relative to treated moss. The range of Cd values in the control mosses was 0.70 ppm to 0.85 ppm.

The average amount of Cd found naturally (baseline) in the filtered-dechlorinated water used for this experiment was below the detection limit of 0.0001 ppm. The control waters also had undetectable levels of Cd in each daily test. The amount of Cd found in the experimental water after the daily accumulation tests were performed ranged from 0.00013 ppm (day 2) to 0.00028 ppm (day 10) as shown in Figure 3a (see also Appendix 1). The daily average of Cd found in solution was 4.57 percent (S.D. 1.58) of the initial concentration suggesting rapid uptake by the moss.

Zinc
The baseline values of Zn found naturally in the moss tissues employed for this study were 57.33 ppm. Concentrations ranged from 381 ppm (day 1) to 3320 ppm (day 10) after exposure to Zn under experimental conditions (Figure 2b). There were significant differences in concentrations of Zn between day 0 to day 6 as shown by Tukey’s test results. The average daily accumulation of Zn into moss tissues was 72.17 percent (S.D. 27.22) from solution. As observed in the Cd series, it does not appear that an asymptote was reached during the Zn experimental period. Relative to the treated moss, the moss in control treatments does not appear to have increased over time and ranged in concentration from 66 ppm to 115 ppm. This slight increase may be related to accumulation of Zn found naturally in the filtered-dechlorinated water.

Baseline Zn concentrations in the filtered-dechlorinated water were 0.01286 ppm. The control waters ranged from 0.00417 ppm to 0.00863 ppm for Zn concentration during the course of the experiment. During the 10-day experiment for Zn accumulation, the daily amount found in the treatment solutions ranged from 0.01976 ppm to 0.11620 ppm (Figure 3b, see also Appendix 1). The daily average of Zn found in solution was 12.41 percent (S.D. 4.16) of the initial concentration suggesting uptake by the moss.

Selenium
Moss tissues had baseline selenium concentrations of 0.46 ppm. After the addition of Se to the experimentally treated water, the moss tissues ranged in Se concentrations from 4.6 ppm (day 1) to 13.2 ppm (day 10) increasing over time as seen in Figure 2c. Significant differences between daily concentrations were not as pronounced as in the Cd and Zn experiments. Day 0 and day 1 differed from one another and both of these days differed from days 9 and 10 as seen through Tukey’s test results. The average daily accumulation of Se into moss tissues was 10.63 percent (S.D. 15.41) from solution. The moss in control treatments also increased over time with ranges from 3.7 ppm (day 1) to 7.8 ppm (day 10). This increase in the control moss treatment can be attributed to the naturally-occurring amounts of Se found in the filtered-dechlorinated water.

Baseline Se concentrations in the filtered-dechlorinated water were 0.00315 ppm. The control waters, however, ranged from 0.00200 ppm (the detection limit) to 0.00433 ppm for Se concentrations occurring naturally in the filtered-dechlorinated water. The daily amount found in the treatment solutions after the 10-day experiments ranged from 0.00666 ppm to 0.00774 ppm (Figure 3c, see also Appendix 1). The daily average of Se remaining in solution was 93.49 percent (S.D. 11.15) of the initial concentration suggesting little uptake by the mosses.

Physiological Stress and Growth

The D665/D665a index (Peñuelas, 1984a, b) was used to determine the degradation of chlorophyll to phaeophytin for H. ochraceum. In the Cd experiments, the chlorophyll-to-phaeophytin ratio decreased for both the control- and Cd-treated mosses. This decrease ranged from 1.59 to 1.06. The observed standard deviation for the control- and Cd-treated samples varied considerably during the experiment (Figure 4a). The Zn experiments showed a slight increase in the D665/D665a ratio for the Zn-treated samples. This increase ranged from 1.04 to 1.61. The control samples in the Zn experiments varied from 1.25 to 1.61 following a similar trend as those exposed to Zn. The observed standard deviations for this experiment varied considerably with the only difference seen between the control- and Zn-treated samples on day 1 (Figure 4b). Lastly, the Se experiments ranged from 1.51 to 1.62 for the Se-treated samples and from 1.52 to 1.14 for the control-treated samples. Variance between the control- and Se-treated samples showed no difference except for day 7 (Figure 4c). Overlapping standard deviations for all metals tested suggests no significant differences in the stress index between the control- and the metal-treated samples.

Growth measurements were made during each metal-treatment experiment and also during two of the 10-day control replicates. However, even though not much growth was noted in either the metal-treated experiments or in the 10-day control replicates, there were new branches of growth noted. (Appendix 1, Tables 4-8).

Discussion

Past studies have mostly dealt with growing various species of moss under sterile or axenic culture (Belkengren, 1962) along with various sorts of culture media (Basile, 1975; Basile and Basile, 1988; Basile et. al., 1988; and Sargent, 1988). Other studies have focused on temperature effects, photoperiod, and varying water levels for determining growth patterns (Glime and Carr, 1974; Glime and Acton, 1979; Sanford, 1979; Glime, 1980; Furness and Grime, 1982; Glime, 1982; Glime, 1987b-d; Glime and Raeymaekers, 1987; Li and Glime, 1991; and Li et. al., 1992). However, laboratory culture and growth of these aquatic bryophytes remains as the primary focus of most of these studies. Laboratory culturing of field-collected aquatic bryophytes can be difficult because of the presence of biological organisms as well as dirt and organic matter can be found in conjunction with the moss. If the moss is not washed completely these other components can serve as a problem, especially in axenic cultures. Sargent (1988) has recommended that field collected mosses should be cultured under conditions of cooler temperatures (10-15°C), high light intensity and low humidity for morphological growth to close to normal.

Culture techniques used in this study were probably not optimal, however, H. ochraceum was still viable for the experimentation series. The replication of natural environmental conditions in the Conviron® growth chambers appeared to cause physiological stress on the moss tissue. This stress may also have affected the mosses ability to effectively accumulate the experimental metals. A remedy to this would be to have employed the use of a flow-through system for culturing the moss. This would have simulated H. ochraceum’s affinity for growth in these types of systems as suggested by Jamieson (1976). On rare occasions, H. ochraceum can be found growing in submerged conditions. However, H. ochraceum is most commonly known for its growth on wet rocks alongside fast flowing waters or near the splash of waterfalls. (Crum and Anderson, 1981).

The static-renewal experiments as used in this study were not ideal and resulted in some problems in the study of bioaccumulation. The static-renewal replacement of metals did not provide a constant concentration of trace elements available in solution to the moss. Steady-state conditions are commonly assumed in bioaccumulation models. These sorts of models are often used to determine the uptake and retention of pollutants. Due to the lack of steady-state metal concentrations in this study, the bioaccumulation factor could not be calculated. This calculation would have provided a ratio for the uptake and depuration rate for each metal used; Cd, Zn and Se.

Increasing the ratio of metal solution to grams might ameliorate the problems associated with the water-metal concentration observed in this study. The mosses accumulated Cd and Zn in greater concentrations linearly over time (Figure 2); and, as seen in Figure 3, the daily amounts of Cd and Zn found in solution after each 24-hour period over the course of the experiment were very small. Had an increase in the metal:filtered-dechlorinated water ratio been performed, an asymptote may have been reached over the course of the experiment for these metals.

In these experiments, H. ochraceum demonstrated varying ability to bioaccumulate trace metals. Although Cd and Zn accumulated in or on the moss tissues over time, the uptake of selenium was not as obvious since both the control- and metal-treated samples varied in Se concentration over time (Figure 2). The variation observed in the Se-treated samples may be the result of physiological stress caused by the culturing methods. However, the variation observed in the control samples may have also been caused by naturally-occurring levels of selenium found in the filtered-dechlorinated water (Figure 3).

Aquatic bryophytes accumulate trace elements through the processes of adsorption to their external cell-wall surfaces as well as through cellular uptake. These processes aid in determining the fate of trace metals in the natural environment. Trace elements are known to adversely effect aquatic biota and impact ecosystems when mining wastes, sewage, and/or industrial discharges enter these aquatic systems. However, as noted by Carter and Porter (1997), trace elements within the water column and sediment beds can often be in concentrations smaller than detectable through conventional chemical methods. The main focus of this particular study was to determine how effective H. ochraceum would be at accumulating known concentrations of trace metals in the laboratory. This application is important because aquatic bryophytes can accumulate trace elements in their tissues that are fairly dilute within the water column and sediment beds in the natural environment (Carter and Porter, 1997).

The physiological stress-response index (OD665/OD665a) in mosses was extensively used in European experiments (López et al., 1997, 1994; López and Carballeira, 1989; and Peñuelas, 1984a, b). However, this index was applied in situ and not in a laboratory setting. The transition from natural setting to growth chamber, as seen in this experiment, probably caused more physiological stress than the in situ European experiments. Also, the static-renewal culture method employed in this study did not replicate the natural environment for these bryophytes (ie. a fast-running stream). Therefore, in laboratory bioaccumulation experiments and the culturing of bryophytes, the OD665/OD665a index used to determine physiological stress-responses to metal treatments cannot be used effectively.

Subjective observations such as color changes, vigor, and/or growth patterns of the mosses were noted during the bioaccumulation experiments. The Cd-treated bryophyte material exhibited little or no new growth on the apical tissues. In addition, the normally deep-green tissue color changed to a lighter yellow-green color after 10 days. However, in both the Zn- and Se-treated bioaccumulation experiments, the moss apical tissues seemed to change from deep-green to a bright-green color. Branched new growth was also observed in these zinc and selenium bioaccumulation experiments.

Growth measurements can be very important when working with aquatic bryophytes. When dealing with moss in field conditions, growth is dependent upon such variables as substrate type, water chemistry, exposure to light, temperature, and speed of flow (Glime, 1987d). However, under laboratory conditions, these variables may not always be available for replication. The growth measurements seen in these experiments were noted, but the data collected was not sufficient for use in statistical comparisons and was placed in Appendix 1 (Tables 4-8).

In conclusion, the results of these experiments show that H. ochraceum could be considered to be a satisfactory bioindicator of trace metal contamination in surface waters. Because of their natural hardiness, mosses can be readily used in bioindication experiments in the field. Also, these types of accumulation experiments could be used in conjunction with standard water quality and/or sediment quality testing methods to show integration of metals into tissues over time rather than instantaneous measurements. Because laboratory settings are different from those experienced in natural environments, great care should be taken to determine proper culturing techniques for the moss species to be used in experimentation when initiating laboratory studies. These culture techniques would be helpful in also determining the best exposure method(s) when subjecting H. ochraceum to trace metals (ie. Live streams vs. static-renewal methods). The correct culturing and exposure methods might also give rise to performing physiological stress-response experiments without worry of any additional stress to H. ochraceum. Further investigation is needed to determine under what laboratory conditions and under what experimental concentrations would be sufficient for H. ochraceum to reach an asymptote or a state of equilibrium when being exposed to experimental trace metals. Also, further study would be necessary to improve understanding of specific rates of bioaccumulation with respect to varying trace metals to determine if there is element-specific uptake in the laboratory setting.

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