Ecological Assessment of Cyclic Release Patterns (CRP) from … · 2013. 10. 21. · Ecological Assessment of Cyclic Release Patterns in the Mitta Mitta River, Victoria JOHNSTONE

JOHNSTONE CENTRE RESEARCH IN NATURAL RESOURCES & SOCIETY

Environmental Consulting Report No. 27

Ecological Assessment of Cyclic Release Patterns (CRP) from Dartmouth Dam to the Mitta Mitta River, Victoria.

MDBC

Lachlan Sutherland

Darren Ryder Robyn Watts

___________ August 2002

Ecological Assessment of Cyclic Release Patterns in the Mitta Mitta River, Victoria

JOHNSTONE CENTRE - CHARLES STURT UNIVERSITY 2

EXECUTIVE SUMMARY Background • The Johnstone Centre Environmental Consulting team were contracted to

undertake an ecological assessment of the CRP from Dartmouth Dam to the Mitta

Mitta River between December 2001 and February 2002.

• Four sites in the Mitta Mitta River and one reference site in the unregulated

tributary of Snowy Creek were sampled during this study. Extensive cobble banks

were present at all sites. Two cobble habitats were sampled during the study:

permanently inundated cobble that was inundated throughout the project and

newly inundated cobble that was inundated only during periods of high flow.

• Nine sampling events took place during the project, one on the final day of the

first variable flow release, three during each of the second and third variable flow

releases and two during the subsequent low and constant flow period.

• The following indicators were assessed:

Water Quality Dissolved organic carbon (DOC)

Particulate organic matter (POM)

Total suspended solids (TSS)

Water column chlorophyll-a (Chl-a)

Temperature, conductivity, dissolved oxygen, pH

River Productivity Biofilm Composition

Benthic production/respiration

Water column production

Bacterial activity of the water column

Macroinvertebrates Benthic invertebrates in cobble and littoral habitats

Effects of CRP • The cyclic release pattern from Dartmouth Dam to the Mitta Mitta River led to

substantial changes in the water quality and biotic parameters measured in this

study. In contrast, over the same period of time there was generally no change in

these parameters measured at the reference site in Snowy Creek.



• The water quality in the Mitta Mitta River during the CRP differed from that

during the 37 days of constant low flows that followed the releases. There was

lower conductivity, pH, temperature, and higher POM, TSS and Chl-a in the Mitta

Mitta River during the CRP than in the constant low flow period.

• There was scouring of biofilms during each peak flow of the CRP in the Mitta

Mitta River resulting in slight decreases in the biomass of biofilms, increased

activity of some dominant classes of enzymes in the water column, changed

composition of biofilm algal species and rapid changes in net productivity at all

four sites in the Mitta Mitta River.

• The response of macroinvertebrates to the CRP was more pronounced at site one

(the most upstream site) than at the other sites. There was an increased number of

families and increased SIGNAL scores observed only at site one, however there

were significant changes in the community composition of macroinvertebrates at

three of the four sites in the Mitta Mitta River.

Effects of 37 days of constant low flows following the CRP • Many water quality and biotic parameters displayed substantial changes during the

37 days of low and constant flows that followed the CRP in the Mitta Mitta River.

In contrast, over the same period there was no change in most of the parameters

measured at the reference site in Snowy Creek.

• Biofilm biomass increased during the constant flow period in the Mitta Mitta

River. This coincided with very low activity of enzymes in the water column,

altered composition of biofilm algal species to dominance of fewer, late

successional taxon and decreased net productivity.

• The response of macroinvertebrates during the constant flows differed between

sites. At site one there was no change in the number of families but the SIGNAL

score increased, suggesting that the ecological effects of the CRP at this site were

still being realised. In contrast, there was an increased abundance of tolerant

families and decreased abundance of more sensitive families at sites two and three

by the end of the constant flow period.



ACKNOWLEDGMENTS Many people have been involved in this project, mainly in the field and laboratory

components. Martin Asmus, Daniel Francis, Bruce Mullins, Dr Darren Ryder, Angus

Sutherland and Lachlan Sutherland all took part in sampling of the Mitta Mitta River

and Snowy Creek over the study period. Numerous people including, Nigel Anthony,

Michelle Burton, Toby Edmunds, Dr Darren Ryder, Claire Sims, Angus Sutherland,

Lachlan Sutherland and Dr Robyn Watts assisted in processing the samples that were

collected during the project. Special thanks must also go to Dr Adrienne Burns and

Andrea Wilson for identifying micro flora and fauna samples, respectively. Special

thanks to Lyn and Ted at the Mitta Mitta Caravan Park, and Steven Lord of Bowler

Station who allowed access through his property.

CONTENTS EXECUTIVE SUMMARY..................................................................................................... 2

ACKNOWLEDGMENTS....................................................................................................... 4

1.0 INTRODUCTION .......................................................................................................... 7

1.1 TERMS OF REFERENCE.................................................................................................. 7 1.2 MANAGEMENT EXPECTATIONS - INTENT OF THE PROGRAM........................................ 7 1.3 BACKGROUND PROJECT INFORMATION........................................................................ 8

1.3.1 Cyclic Release Pattern......................................................................................... 8 1.3.2 Recommended Environmental Indicators ............................................................ 9

2.0 PROJECT DESIGN ..................................................................................................... 10

2.1 PROJECT OBJECTIVES ................................................................................................. 10 2.2 ENVIRONMENTAL INDICATORS .................................................................................. 10

2.2.1 Additional Parameters....................................................................................... 12 2.3 STUDY AREA .............................................................................................................. 13

2.3.1 Hydrographic Data of the Mitta Mitta River and Snowy Creek ........................ 14 2.3.2 Sites Locations and Descriptions....................................................................... 15

2.4 PROJECT DESIGN ........................................................................................................ 17 2.4.1 Temporal Comparisons ..................................................................................... 18 2.4.2 Longitudinal Comparisons ................................................................................ 18 2.4.3 Reference Stream............................................................................................... 18 2.4.4 Relevance of Reference Site............................................................................... 19

2.5 SAMPLING REGIME ..................................................................................................... 20 2.5.1 Project Limitations ............................................................................................ 20

2.6 PREDICTIONS .............................................................................................................. 21 2.6.1 Water Quality..................................................................................................... 21 2.6.2 Enzyme activity .................................................................................................. 21 2.6.3 Biofilm composition ........................................................................................... 22 2.6.4 Benthic Metabolism ........................................................................................... 22 2.6.5 Macroinvertebrates............................................................................................ 23

3.0 HYDROGRAPHIC DATA DURING STUDY PERIOD .......................................... 24

4.0 WATER QUALITY...................................................................................................... 26

4.1 INTRODUCTION ........................................................................................................... 26 4.2 METHODS ................................................................................................................... 27

4.2.1 Field Methods .................................................................................................... 27 4.2.2 Laboratory Methods .......................................................................................... 28 4.2.3 Data Manipulation and Analysis ....................................................................... 29

4.3 RESULTS ..................................................................................................................... 29 4.3.1 POM, DOC, TSS and Chl-a ............................................................................... 29 Yeokal Multiprobes ......................................................................................................... 35 4.3.3 Water Column Nutrient...................................................................................... 36 4.3.4 DOC and POM Loading.................................................................................... 36

4.4 DISCUSSION ................................................................................................................ 38 4.4.1 Water Quality Parameters ................................................................................. 38 4.4.2 DOC and POM Loading.................................................................................... 41

4.5 SUMMARY OF FINDINGS ............................................................................................. 41



5.0 WATER COLUMN EXTRACELLULAR ENZYME ACTIVITY.......................... 43


5.2.1 Field methods..................................................................................................... 44 5.2.2 Laboratory Methods .......................................................................................... 44 5.2.3 Data Manipulation and Analysis ....................................................................... 45

5.3 RESULTS ..................................................................................................................... 45 5.4 DISCUSSION ................................................................................................................ 49 5.5 SUMMARY OF FINDINGS.............................................................................................. 51

6.0 BIOFILM STRUCTURE AND FUNCTION ............................................................. 52


6.2.1 Biofilm Structural Components ......................................................................... 54 6.2.2 Biofilm Taxonomy.............................................................................................. 55 6.2.3 Biofilm metabolism ............................................................................................ 55 6.2.4 Data Manipulation and Analysis ....................................................................... 57

6.3 RESULTS ..................................................................................................................... 58 6.3.1 Biofilm total, organic and algal biomass........................................................... 58 6.3.2 Biofilm Algal Species Composition.................................................................... 69 6.3.3 Biofilm metabolism ............................................................................................ 76

6.4 DISCUSSION. ............................................................................................................... 78 6.5 SUMMARY OF FINDINGS ............................................................................................. 82

7.0 MACROINVERTEBRATES....................................................................................... 84


7.2.1 Field methods-Cobble habitats.......................................................................... 85 7.2.2 Field methods-Littoral habitats ......................................................................... 86 7.2.3 Laboratory methods........................................................................................... 86 7.2.4 Data Manipulation and Analyses ...................................................................... 86

7.3 RESULTS ..................................................................................................................... 87 7.3.1 Overview of macroinvertebrate data ................................................................. 87

7.4 DISCUSSION .............................................................................................................. 103 7.5 SUMMARY OF FINDINGS............................................................................................ 105

8.0 SUMMARY & RECOMMENDATIONS................................................................. 107

8.1 EFFECTS OF VARIABLE FLOW RELEASES ON THE ECOLOGICAL CONDITION OF THE MITTA MITTA RIVER......................................................................................................... 107 8.2 EFFECTS OF 37 DAYS CONSTANT AND LOW FLOWS ON THE ECOLOGICAL CONDITION OF THE MITTA MITTA RIVER.................................................................................................. 109 8.3 RECOMMENDATIONS FOR FUTURE MONITORING PROGRAMS ................................... 111

9.0 REFERENCES ........................................................................................................... 114

APPENDIX 1 - PROJECT BREIF .................................................................................... 120

APPENDIX 2 - JOHNSTONE CENTRE PROPOSAL ................................................... 130



1.0 INTRODUCTION

1.1 Terms of Reference

The Johnstone Centre – Environmental Consulting (JC) team were contracted by the

Murray Darling Basin Commission (MDBC) to undertake an ecological assessment of

a cyclic release pattern (CRP) in the Mitta Mitta River, Victoria between December

2001 and February 2002. The Terms of Reference offered to JC were in the form of

four documents (Appendix 1): requirements for tenders (Tony McCleod, November

2001), summary of the proposed variable release pattern (David Dole, August 2001;

John Riddiford, November 2001) and the ecosystem components to be assessed

(Terry Hillman, November 2001).

The JC proposal (Appendix 2), with the inclusion of various amendments (Robyn

Watts, November 2001) was used as the Terms of Reference for the project.

1.2 Management Expectations - Intent of the Program Flow conditions within the Mitta Mitta River are highly regulated by Dartmouth Dam.

The timing and duration of releases from Dartmouth are dependent upon the status of

the other major water storages in the River Murray system, particularly Hume

Reservoir. Dartmouth plays an important role as a relatively secure ‘drought reserve

storage’.

In wet years, little or no transfers are required from Dartmouth Dam unless a spill

event occurs. However, in dry years, it can be necessary to transfer large volumes

from Dartmouth as reserves are depleted in downstream water storages. The risk that

transfers from Dartmouth may result in a spill from Hume during wet climatic

conditions, transfers are often delayed until later in the season. An exception to this

(which occurred in the 2001/2002 season) occurs when Dartmouth nears capacity

(≥80%) and ‘harmony transfers’ are required. These are made when the probability of

Dartmouth spilling exceeds Hume, and that for at which time transfers are made to

equalise the probability of spill from each storage.



River Murray Water’s (RMW) management of harmony transfers attempts to

minimise floodplain inundation and, maintain relatively constant discharge levels. The

River Murray Expert Panel for Environmental Flows (RMEPEF) highlighted constant

flow conditions as having detrimental impacts upon the instream and floodplain

environments of the Murray River Catchment.

Following the outcomes of discussions by the RMEPEF, RMW proposed the

introduction of a CRP to their harmony transfers from Dartmouth Dam to the Mitta

Mitta River in the 2001/02 season. The CRP comprises of repeated flow releases that

attempt to mimic minor natural flood events and are consistent with the

recommendations of the Expert Panel (Table 1.1).

The intent of the CRP was to introduce flow variability to their transfers from

Dartmouth Dam to Hume Weir, for the ecological benefit of the Mitta Mitta River. As

part of the introduction of the CRP the MDBC sought assistance in assessing the

ecological effects of this modification to management practice and intended that the

work would aim to:

• Identify components of the ecosystem which might be expected to respond to a

change from constant to variable flow patterns on the proposed scale;

• Provide measurements indicative of that response;

• Form the basis for preliminary assessment and advice for management of variable

releases, and;

• Provide data and insights, which could support and help direct more rigorous

studies in the future.

1.3 Background Project Information

1.3.1 Cyclic Release Pattern

River Murray Water presented the following CRP proposal to the Mitta Mitta Water

Services Committee on the 14/11/2001. The JC project was designed to assess the

ecosystem response to the proposed pattern (Table 1.1). The proposed CRP comprised



three identical variable flow releases. Detailed summary of the proposed CRP is

contained in Appendix 1 (David Dole, August 2001).

Table 1.1: Summary of the variable flow releases from Dartmouth Dam during the cyclic

release pattern (CRP) for the Mitta Mitta River between 19/11/2001 and 31/12/2001.

Prepared by River Murray Water, 14/11/2001.

Cyclic Release Pattern Number of variable flow releases 3 Duration of each release 14 days Flow Rise 2 days Flow Recession 12 days First pulse proposed to commence Week commencing 19/11/01 Colemans Gauge Average flow required 4000 ML/day Maximum Flow 4800 ML/dayMinimum Flow 3200 ML/day Average Water Level (Colemans gauge) 2.08 mWater Level Variation 0.25 m total Tallandoon Gauge * Average flow 5000 ML/day Maximum Flow 5800 ML/day Minimum Flow 4200 ML/day Average Water Level (Tallandoon gauge) 2.43 m Water Level Variation 0.25 m total

1.3.2 Recommended Environmental Indicators

A suite of environmental indicators were identified for assessment by Dr Terry

Hillman, 14/11/2001 (Appendix 1). The assessment of these parameters was intended

to provide an indication of ecosystem response to the CRP. The environmental

indicators recommended for assessment included water quality parameters, river

productivity parameters (eg. biofilm composition, benthic production / respiration,

water column production, enzyme activity), invertebrates (benthic

macroinvertebrates) and the activity of fish larvae.



2.0 PROJECT DESIGN

2.1 Project Objectives The main objective of the project was to record the response of selected

environmental indicators to the CRP from Dartmouth Dam to the Mitta Mitta River,

Victoria. The study was conducted between December 2001 and February 2002 and

comprised field and laboratory experiments for four sites on the Mitta Mitta River and

one site on Snowy Creek. Environmental indicators assessed in this project covered

pelagic and benthic cobble bench habitats. The response of environmental indicators

was used to make an assessment of ecological effects of the modifications to harmony

transfer management practice and provide recommendations for future manipulation

of transfers for environmental benefit.

The specific project objectives stated in a Draft Study Brief (Hillman, 14/11/2001)

provided in the tender documents were that the JC research team would:

• Identify components of the ecosystem which might be expected to respond to a

change from constant to variable flow patterns on the proposed scale;

• Provide measurements indicative of that response;

• Form the basis for preliminary assessment and advice for management of variable

releases, and;

• Provide data and insights, which could support and help direct more rigorous

studies in the future.

2.2 Environmental Indicators Assessing the ecological 'health' of river systems is an important issue for ensuring

both the long term ecological condition of the river system and the sustainability of

development dependant on the water resource. Indicators form the basis of most

empirical systems for assessing the status of the environment (Fairweather 1999).

Environmental indicators are measures of physical, chemical or biological responses

to environmental change.



In selecting environmental indicators for the assessment of ecological change, there is

a need to identify the stressors on the system and identify which components and

processes are likely to be affected (Cairns et al. 1993). The selection of environmental

indicators must therefore depend in part on the projected outcomes of the proposed

management regime. Fairweather (1999) postulates three approaches to selecting

specific indicators; (1) a haphazard selection from divergent perspectives (2) a single

perspective based on previous data, and (3) a synthetic approach that integrates

distinct perspectives.

The project team identified the loss of flow variability to be the major stressor on the

pelagic environment of the Mitta Mitta River. Based on previous research conducted

within the Murrumbidgee River Catchment (Watts et al. 2000) the team identified that

benthic biofilm composition and production, enzyme activity and the structure of

benthic macroinvertebrate assemblages would be the instream components most likely

affected by the lack of flow variability. These components have also been

demontrated to respond rapidly to changes in flow conditions. The selection of

environmental indicators was based on previous data and research. The indicators

selected were:

• Extracellular enzyme activity of water column bacteria, included five

methylumbelliferyl (MUF) labelled carbon substrates that were used to estimate

bacterial activity in water samples (1) 4-MUF-butyrate (fatty acid esterase –

FAE); (2) α-D-glucosidase (carbohydrate), (3) β-D-glucosidase (carbohydrate);

(4) β-D-xylosidase (long chain carbohydrate, eg. woody substrates), and (5)

Leucine-7-amino-4-methyl-coumarin (aminopeptidase). These enzymes are

involved in the degradation of polysaccharides, carbohydrates and proteins

derived from a range of authochthonous and allocthonous organic matter (Chrost

1991);

• Biofilm structure and function, includes assessment of biofilm composition and

metabolism. Selected because changes to the species composition and metabolic

rate of algal biofilms can impact on their ecosystem function, by either reducing

or increasing oxygen production dependant on species present and controlling

food resources for primary consumers;



• Macroinvertebrate composition, included assessments of benthic and littoral

habitats. Macroinvertebrates are used in biological monitoring programs

worldwide as many taxa respond to changes in environmental conditions,

particularly to changes in flow condition.

2.2.1 Additional Parameters

A number of additional water quality parameters were also selected for assessment

during the CRP. These parameters included particulate organic matter (POM),

dissolved organic matter (DOC), total suspended solids (TSS), water column Chl-a

and water column nutrients (Total Phosphorus, NH3-N, PO-4). These parameters were

to be measured at all experimental sites. Two Yeokal multiprobes were also used to

collect water column data throughout the study at the top and the bottom of the study

reach in the Mitta Mitta River.



2.3 Study Area

The study area covers a 60km reach of the regulated Mitta Mitta River, from

downstream of Lake Banimboola downstream to Tallandoon (Figure 2.1).

Figure 2.1: Location of the experimental sites (1 – 5) used during the project on Mitta Mitta

River and Snowy Creek. Includes flow gauging stations.

The Mitta Mitta River in the study area consists of armoured cobble benches and

sandy depositional zones and flows through two morphological zones: restricted

upland stream of moderate gradient and a meandering floodplain river channel of

reduced gradient (Blyth et al. 1984).



The river between Lake Banimboola and the confluence of Snowy Creek flows

through upland and foothills habitat, with steep-sided valleys dominated by dry

sclerophyll woodland. Downstream of the township of Mitta Mitta the river flows out

into a wide floodplain that has been extensively cleared for agriculture, and is

dominated by dairy and beef cattle enterprises (Koehn et al. 1995). In this lower

section the riparian zone is mostly cleared with occasional stands of River Red Gum

(Eucalyptus camaldulensis) and Willows (Salix spp.).

2.3.1 Hydrographic Data of the Mitta Mitta River and Snowy Creek

Flow conditions within the Mitta Mitta River are highly regulated by Dartmouth Dam.

The timing and duration of releases from Dartmouth are dependent upon the status of

the other major water storages in the River Murray system, particularly Hume

Reservoir.

Annual flow patterns within the Mitta Mitta are therefore highly variable and are

dependent on the status of water volumes in Dartmouth Dam and Hume Reservoir.

Average discharge (ML/day) during the period of December through February

gradually decreases at Colemans gauging station, but remains relatively constant at

the Tallandoon gauging station (Figure 2.2). Given that the flow in the Mitta Mitta

River is controlled by irregularly timed releases from Dartmouth Dam, high

variability in discharge exists between years (Figure 2.2).

Snowy Creek, used as a reference stream for this project, is an unregulated upland

tributary of the Mitta Mitta River (Section 2.4.3). The flow pattern of Snowy Creek

reflects natural rainfall and snow melt events within the creek’s catchment, and is

characterised by low flows in late summer and early autumn and high flows in late

winter and early spring, and has low variability between years.



Figure 2.2: Average daily flow (+SD, n=10) (megalitres per day) for the study period

(November to February) over the past ten seasons. Data recorded on the Mitta Mitta River at

Colemans and Tallandoon gauging stations and at Granite Flat on Snowy Creek.

2.3.2 Sites Locations and Descriptions

Five cobble benches, four on the Mitta Mitta River (site 1 – 4) and one on Snowy

Creek (site 5) were selected as experimental sites for the ecological assessment of

cyclic release patterns from Dartmouth Dam to the Mitta Mitta River (Figure 2.1).

Cobble benches were selected as experimental sites because these areas would

undergo considerable hydrological change during the CRP. Further, cobble benches

were common attributes along the study reach in the Mitta Mitta River and were also

abundant within Snowy Creek. A summary of site details and locations is presented in

Table 2.1.

0100020003000400050006000700080009000

10000

19-Oct 2-Nov 16-Nov 30-Nov 14-Dec 28-Dec 11-Jan 25-Jan 8-Feb 22-Feb 7-Mar

(ML/

day) Colemans

TallandoonGranite Flat

Table 2.1: Summary of site locations and details for the five sampling sites on the Mitta Mitta River and Snowy Creek

Site No.

Rationale for site selection Distance below Dartmouth Dam(river km)

AMG co-ordinates

Mean Stream Width (m) during low and high flows

Riverbed/ bench type

Habitats Available Surrounding Environment and Riparian Vegetation

1 First accessible cobble bench downstream of Lake Banimboola used to assess ecological response at the top of study area.

26 534206, 5956959 Low 20 High 50

Cobble and coarse gravel

- Permanently and newly inundated cobble banks -Littoral zone

Cleared grazing land. Bottlebrush, tea-tree and willow scattered along banks.

2 Downstream of the Snowy Creek and Mitta Mitta River junction, used to assess potential impacts from inflows of major tributary.

28 533114, 5956322 Low 20 High 30

Cobble, coarse gravel and sand


Open eucalypt woodland. Eucalypt, Wattle, Tea-tree and Willow along banks.

3 In the mid-section of the study area used to gauge longitudinal responses to variable flows.

45 529971, 5964641 Low 30 High 50



Cleared grazing land. Willow, poplars and scattered tea-tree along banks.

4 Used to assess ecological response to variable flows at the end of the study area.

60 518232, 5967211 Low 30 High 60



Cleared grazing land. Willows and scattered eucalypts growing along banks.

5 Snowy Creek downstream of Granite Flat gauging station. To be used as a reference site.

- 536704, 5953663 15 Cobble and coarse gravel

-Cobble bench in permanently inundated areas -Littoral zone

Open eucalypt woodland. Dense Tea-tree, Wattle and Eucalypt along banks.



2.4 Project Design The project was designed to assess the response of environmental indicators to

disturbance as a result of CRP by dividing the cobble bench at each experimental site

into two habitats: permanently inundated and newly inundated. Permanently

inundated - areas of the cobble bench that were inundated throughout the project (eg.

main Mitta Mitta River channel, background of Plate 2.1). Newly inundated - areas of

the cobble bench that were only inundated during periods of high flows (Plate 2.2).

Areas of cobble that are permanently inundated can be subject to disturbances from

factors such as scouring and reduced light availability during periods of high flow.

These disturbances can trigger altered successional pathways in biological

communities and create new habitat for colonising organisms. The design aimed to

assess the response of selected environmental indicators under permanent inundation

to variation in flow during the CRP and constant flow period that followed the

variable releases from Dartmouth Dam.

Plate 2.1: Mitta Mitta River at Site 1 showing exposed cobble bench during an 800 ML/day

flow, and channel in background 19/2/2002.

The inundation of instream and floodplain surfaces by increasing river height creates

a range of new habitats and opportunities for colonisation by instream organisms. The

experimental design aimed to assess the response of selected environmental indicators

in newly inundated habitats during the CRP.



Plate 2.2: Mitta Mitta River at Site 1 showing a newly inundated cobble bench during a 4500 ML/day flow, 18/12/2001.

2.4.1 Temporal Comparisons

The proposal intended to simulate three individual flood peaks (variable flow

releases) during the CRP (Table 1.1), followed by a period of constant flow

conditions. The project design aimed to record the response of environmental

indicators during each variable flow release, and then to compare each of the

consecutive flow releases to one another. Further, the project aimed to compare the

responses of environmental indicators during the CRP with those observed during the

constant flow period.

2.4.2 Longitudinal Comparisons

The proposal included the comparison of loads (kg/day) of POM and DOM between

site 1 and site 4. These comparisons allow the project to infer cumulative downstream

affects of the CRP in the Mitta Mitta River.

2.4.3 Reference Stream

Snowy Creek, the main tributary of the Mitta Mitta River below Dartmouth Dam was

selected as a reference site for the project. This creek system has an average annual

discharge of 577 ML/day (Figure 2.2, Plate 2.3). The creek is unregulated and its



catchment has undergone minimal anthropogenic disturbance. Selection criteria were

quite pragmatic for the project given time constraints, and included a system with

unregulated flow, moderate annual discharge with a close proximity to the Mitta Mitta

River. Snowy Creek met all selection criteria and was selected during a

reconnaissance trip to the study area.

Plate 2.3: Site 5 located on Snowy Creek, during a 900 ML/day flow, 2/12/2002

Environmental indicators (except for benthic metabolism) were assessed within

Snowy Creek during the study period at the same time as the assessment of sites on

the Mitta Mitta River. Given the flow conditions within Snowy Creek environmental

indicators were only assessed within permanently inundated sections of cobble bench.

2.4.4 Relevance of Reference Site

The inclusion of a reference site provided the opportunity to compare the response of

environmental indicators to variable flow conditions in the Mitta Mitta River to the

response of indicators within a reference system with unregulated flow conditions.

The reference site was used as an ecological target, to provide a relatively undisturbed

system that could be used to gauge the ecological response of variable flow conditions

in the Mitta Mitta River.



2.5 Sampling Regime

The sampling regime used during the study period was constructed to assess three

stages at day 2, 7-8 and 14-19 of each variable flow release: peak flow (~4800

ML/day), mid flow (~4000 ML/day) and base flow (~3200 ML/day)(Figure 3.1).

Table 2.2 provides a matrix showing sampling dates for each environmental indicator

and water quality parameters, and the habitats sampled. The newly inundated habitat

was only sampled on peak flow and mid flow. A total of nine sampling events took

place during the project, one on the final day of the first variable flow release, three

during the second and third variable flow release and two during the subsequent

constant flow period, which commenced on the 4th of January 2002.

Table 2.2: Sampling regime used during the ecological assessment of the cyclic release pattern from Dartmouth Dam to the Mitta Mitta River, Victoria. Sampling commenced one day prior to the second variable flow release on the 2/12/2001 and continued over 71 days until the 11/02/2002. Perm denotes permanently inundated habitat; New denotes Newly inundated habitat.

Date Day Cumulative

Days Sample date

Water Quality

Water Column Enzyme activity andmetabolism

Benthic biofilm composition and metabolism

Macroinvertebrates

Perm New Perm New 2/12/01 14 0 1 4/12/01 2 2 2 10/12/01 8 8 3 16/12/01 14 14 4 18/12/01 2 16 5 23/12/01 7 21 6 4/01/02 19 33 7 21/01/02 17 50 8 11/02/02 38 71 9

2.5.1 Project Limitations

A major limitation of the project was the absence of before data describing the

ecological condition of the Mitta Mitta River prior to the commencement of the CRP.

The need for before data in restoration ecology is set out clearly in Underwood (1996)

and again in Chapman and Underwood (2000). RMW commenced the first variable



flow release on the 19/11/2001, and JC did not receive formal acceptance of its

proposal until the 26/11/2001. The lack of before data has placed major constraints

upon the statistical analyses and the strength of the conclusions and inferences that

could be drawn from the data recorded during the second and third variable flow

releases. Further, the subsequent lack of data from the first variable flow release

limited the project’s ability to infer the benefits of multiple flood pulses and the

introduction of variable releases following constant flow conditions.

The proposed flow regime set out in Table 1.1 was altered without notice during the

CRP (see Table 3.1). The alterations occurred during the peak of the third variable

flow release and the constant flow period. These anomalies limited comparisons that

could be made between peaks of each variable flow release and introduced variability

during the proposed constant flow period. The availability of one reference site and no

control site was seen as a limitation given that Chapman and Underwood (2000) state

that a restoration activity requires having a control and a reference site. This issue was

unavoidable given the logistics of sampling in a control stream, possibly within

another catchment and the short time frame for project preparation.

2.6 Predictions

2.6.1 Water Quality

1. The concentration of DOC, POC and suspended solids will increase during the

CRP compared to constant flows as a result of increased riverbank and floodplain

inundation and in channel resuspension. We predict there will be increased

loading of carbon and POM with distance downstream.

Water quality parameters listed in the tender will be used to aid in the interpretation of

biofilm composition and productivity and macroinvertebrate data.

2.6.2 Enzyme activity

1. Peak flows will increase the overall activity of water column bacteria, specifically

increase the activity of carbohydrase enzymes.



2. The CRP will increase the overall enzyme activity in the water column

(specifically increase the activity fatty acids and proteins) due to increased

riverbank and floodplain inundation and in channel resuspension. We predict there

will be increased overall enzyme activity with distance downstream.

2.6.3 Biofilm composition

Based on the results of Watts et al. (2001) we predict that if there are forty days of

constant low flows after the third variable flow release, the biofilms will reach a stable

state for biomass, composition and productivity. These data will be compared to the

data collected during the CRP.

1. Algal and total biomass from cobble substrata will decrease following peak flows

during the CRP compared to the biomass prior to the peak due to scouring from

increased velocity.

2. Peak flows will change the community composition of algal biofilms and promote

early successional algal taxa on cobble substrata due to scouring from increased

water velocity.

2.6.4 Benthic Metabolism

1. Peak flows will increase carbon respiration of biofilms on cobble substrata from

deep habitats due to scouring from increased water velocity and light deprivation

from increased water depth.

2. Newly inundated cobble with established biofilm communities will have increased

carbon production relative to those newly inundated cobbles that do not have an

established biofilm community.



2.6.5 Macroinvertebrates

The samples collected during the constant flow period following the third variable

flow release will be compared to the data collected during the CRP.

1. Variable flow releases will increase algal diversity on cobble substrata and will

result in a higher diversity of macroinvertebrates in cobble habitats.

2. Variable flow releases will increase algal diversity on cobble substrata and

increase the relative abundance of primary consumers on cobble habitats.



3.0 HYDROGRAPHIC DATA DURING STUDY PERIOD Table 1.1 provides a summary of the proposed CRP from Dartmouth Dam to the Mitta

Mitta River. The CRP was to consist of three variable flow releases, commencing at

4000 ML/day, rising over two days to a 4800 ML/day flow peak, then receding over

twelve days to a minimum flow of 3200 ML/day (flows taken at Colemans gauge).

The three variable flow releases were then to be followed by a period of constant

flows of approximately 800 ML/day. This proposed pattern was not implemented as

substantial changes occurred. Table 3.1 provides a summary of the hydrographic data

recorded during the CRP, and is displayed in Figure 3.1. The major anomalies that

occurred were the extended peak of the third variable flow release which lasted four

days instead of the proposed one day, and a spike of 2500 ML/day that occurred

during the constant flow period.

Table 3.1. Summary of hydrographic data obtained from Colemans and Tallandoon gauging

stations during the CRP.

Variable flow release

number

1 2 3 Constant Flow Period

Duration (days) 14 14 18 45

Flow Rise (days) 2 2 2

Flow Recession (days) 12 12 14

Flow Peak (days) 1 1 4

Commencing Date 19.11.01 03.12.01 17.12.01 04.01.02

Colemans Gauge

Maximum (ML/day) 4754 4684 4824 2490

Minimum (ML/day) 3200 3200 1518 590

Average 879

Tallandoon Gauge

Maximum (ML/day) 5933 5387 5691 2540

Minimum (ML/day) 3859 3778 2186 1100

Average 1266



Figure 3.1: Hydrograph for the Mitta Mitta River recorded at Tallandoon and Colemans gauging stations, and Snowy Creek recorded at Granite Flat gauging station for the period 1/11/2001 to 19/2/2002. See Figure 1.3 for locations of each gauging station. Hydrograph shows the position of the three variable flow peaks and 12 day recession periods. VR denotes variable flow release.

0

1000

2000

3000

4000

5000

6000

7000

1/11/0

1

8/11/0

115

/11/01

22/11

/0129

/11/01

6/12/0

113

/12/01

20/12

/0127

/12/01

3/01/0

210

/01/02

17/01

/0224

/01/02

31/01

/02

7/02/0

214

/02/02

ML/

day

Colemans gaugeTallandoon gaugeGranite Flat gauge1st flow peak

2nd flow peak 3rd flow peak

VR1 VR2 VR3Constant Flow Period



4.0 WATER QUALITY

4.1 Introduction As streams flood and subsequently inundate floodplain, fluxes of terrestrial organic

and inorganic materials occur between the floodplain and aquatic systems. Current

reviews show that these fluxes play significant roles in aquatic food webs (e.g.

Findlay & Sinsabaugh 1999; Robertson et al. 1999). Floodplain inundation also drives

changes in physico-chemical parameters by altering thermal, optical and chemical

properties within instream ecosystems.

A major component of floodplain to instream fluxes is dissolved organic matter

(DOM). DOM constitutes a large proportion of the organic carbon in all aquatic

ecosystems and is often a significant carbon resource for heterotrophic

microorganisms (predominantly bacteria)(Findlay & Sinsabaugh 1999). DOM plays a

significant role in aquatic food webs (Findlay et al. 1986), mediates the availability of

dissolved nutrients and metals (e.g. Carlson et al. 1993), and modifies the optical

properties of water bodies (Robertson et al. 1999).

Dissolved organic carbon (DOC) is the carbon component of DOM and in Australian

river systems is predominantly derived from the decomposition of leaf litter

(Robertson et al. 1999). Large quantities of DOC are leached from floodplain litter

during inundation, and drive instream productivity as floodwaters drain back into the

river channel.

Litter and soil borne organic nutrients are another component of the DOM flux that

occurs between floodplain and aquatic ecosystems during inundation (Baker et al.

2001; Mettler et al. 2001). Mineralised nitrogen (N) and phosphorus (P) from these

sources are generally the limiting factors for instream primary production. However,

recent research has shown that the quality of floodplain litter mediates the quantity of

nutrient mineralisation during inundation (Mettler et al. 2001).

Another important fraction of the material entrained by floodwaters is particulate

organic carbon (POM). POM is derived from a number of sources including coarse



particulate organic matter (CPOM), large woody debris (LWD), riparian soil particles,

flocculated DOC, scoured biofilms and autochthonous production (Ward 1986). POM

is a major food source for fine particle feeders (e.g. collector gatherers and filterers,

sensu Cummins & Klug 1979), these feeders being the dominant functional group in

many stream and river systems (Robertson et al. 1999).

The flux of floodplain carbon and nutrient sources to instream ecosystems during

inundation is also responsible for increases in water column and instream

productivity. Water column Chl-a and benthic biofilms are important food resources

for all functional feeding groups, and their availability controls the structure of

macroinvertebrate assemblages in stream and river systems (Matonickin et al. 2001).

Given the importance of DOC, POM, nutrient and water column Chl-a to instream

productivity, the project aimed to assess the response of these factors to the CRP from

Dartmouth Dam in the Mitta Mitta River, Victoria from December 2001 to February

2002. Further, physico-chemical parameters were also measured throughout the study

period as supporting data for assessments made upon environmental indicators. The

aim of this section is to examine the response of these parameters. The following

prediction can be made:

1. Concentrations of DOC, POM, organic and inorganic nutrients, total

suspended solids (TSS) and Chl-a will increase during periods of high flows

and decrease as flood waters recede.

4.2 Methods

4.2.1 Field Methods

Three replicate samples were taken from flowing surface waters at each study site on

each sampling event for determination of DOC, POM, TSS and water column Chl-a,

total phosphorus (TP), ammonia-nitrogen (NH3-N) and soluble reactive phosphorus

(PO-4) concentration. Two Yeokal multiprobes were placed near the water surface at

the top and bottom study site in the Mitta Mitta River. These probes recorded a

number of water column parameters every two hours.



4.2.2 Laboratory Methods

Particulate organic matter

Water column POM was measured using filtered (pre-weighed 70µm glass fibre

filters, GFF) water samples. The dry and ash weights of the filters plus retained

material were then used to estimate POM concentration’s per meter3.

Dissolved organic carbon

Water column DOC was measured using filtered (0.45µm) water samples and

analysed for the concentration of carbon (mg/L) using a Total Organic Carbon

Analyser.

Total suspended solids

TSS was calculated using the dry and ash weights obtained during the POM analysis.

TSS was calculated as the total dry weight of filtrate and estimated as g/m3.

Chlorophyll-a

Water column Chl-a was measured using filtered (70µm GFF) water samples. The

Chl-a was fixed by placing filters and retained material into 10ml vials containing a

solution of 150mg of magnesium carbonate and 8mL of 90% aqueous methanol.

Following refrigeration vials were centrifuged and the resulting supernatant was

analysed for light absorbance at 750 and 666λm using a light spectrophotometer. One

hundred microlitres (100µL) of 4 percent hydrochloric acid was then added to the

supernatant and the absorbance of light was measured again at 750 and 666λm for the

determination of phaeophytin concentrations.

Nutrient (TP, NH3-N, PO-4)

Total phosphorus (TP) concentration was measured using an Inductively Coupled

Plasma Atomic Emission Spectrometer (ICP). Ammonia – Nitrogen (NH3-N) and

Phosphate (PO-4) concentrations were measured using a Segmented Flow Auto

Analyser (ALCHEM) at the Environmental and Analytical Laboratory at Charles

Sturt University (NATA accredited).



4.2.3 Data Manipulation and Analysis

The water quality parameters (except data from the Yeokal multiprobes) have been

summarised by the calculation of mean and standard deviation on a per site basis. The

comparison of parameters at each site utilised visual interpretation of differences. The

absence of replication in the data from the Yeokal multiprobes meant that

interpretation was also visually compared but without calculated mean and standard

deviation.

Concentrations of DOC and POM at sites 1 and 4 were multiplied by discharge

(ML/day) at Colemans and Tallandoon gauging stations respectively to attain a daily

load (kg/day) of each parameter at each site and on each sampling date. An organic

matter budget for the study reach was calculated for DOC and POM concentrations

for each sampling date by subtracting the loads at site 4 from site 1.

4.3 Results

4.3.1 POM, DOC, TSS and Chl-a

The concentration of POM at each site in the Mitta Mitta River increased during the

peak of the second variable flow release (date 2) (Figure 4.1). These changes were

most pronounced at sites 3 and 4 with increases of 22 and 24 g/m3, respectively. POM

concentration in the reference stream varied little during this period.

The peak of the third variable flow release (date 5) resulted in a substantial decrease

in POM at sites 1, 2 and 4, but an increase at site 3. POM increased at all sites during

the recession of the third variable flow release, and then gradually decreased to less

than 8 g/m3 at the end of the constant flow period. POM at the reference site followed

a similar trend gradually decreasing over time to its lowest concentration on date 9.

The contribution of POM from the reference stream to the Mitta Mitta River does not

appear to be substantial given the minor difference in concentrations between sites

one and two.



Figure 4.1: Water column concentrations (g/m3) of particulate organic matter (POM) at sites 1 to 4 on the Mitta Mitta River and site 5 on Snowy Creek (reference site) on nine sample dates from December 2001 to February 2002 (mean ± SD, n=3).

Site 1

0.00

10.00

20.00

30.00

40.00

2/12/0

1

9/12/0

1

16/12

/01

23/12

/01

30/12

/01

6/01/0

2

13/01

/02

20/01

/02

27/01

/02

3/02/0

2

10/02

/02g/

m3

Site 2

0.00

10.00

20.00

30.00

40.00

2/12/0

1

9/12/0

1

16/12

/01

23/12

/01

30/12

/01

6/01/0

2

13/01

/02

20/01

/02

27/01

/02

3/02/0

2

10/02

/02

g/m

3

Site 3

0.00

10.00

20.00

30.00

40.00

2/12/0

1

9/12/0

1

16/12

/01

23/12

/01

30/12

/01

6/01/0

2

13/01

/02

20/01

/02

27/01

/02

3/02/0

2

10/02

/02

g/m

3

Site 4

0.00

10.00

20.00

30.00

40.00

2/12/0

1

9/12/0

1

16/12

/01

23/12

/01

30/12

/01

6/01/0

2

13/01

/02

20/01

/02

27/01

/02

3/02/0

2

10/02

/02

g/m

3

Site 5

0.00

10.00

20.00

30.00

40.00

2/12/0

19/1

2/01

16/12

/0123

/12/01

30/12

/016/0

1/02

13/01

/0220

/01/02

27/01

/023/0

2/02

10/02

/02

g/m

3



The concentration of DOC was generally low at all sites on the Mitta Mitta River and

in the reference stream throughout the study period (Figure 4.2). There were no

substantial changes in DOC concentration at the reference site during the study period

although an increase in DOC concentration at the recession of the third variable flow

release was consistent with increases found at all sites on the Mitta Mitta River during

the same period. DOC decreased slightly at sites 1, 2 and 4 following the flow peaks

of the second (date 2) and third (date 5) variable flow release. All Mitta Mitta sites

showed substantial increases in DOC during the recession period of the third variable

flow release (dates 6 and 7), which then gradually decreased during the constant flow

period.

TSS increased substantially at all sites on the Mitta Mitta River during the peak flow

of the second variable flow release (date 2) and a minimal increase in TSS in the

reference stream was also evident at this time. TSS concentrations varied little at sites

1 and 2 on the Mitta Mitta River as well as in the reference stream following date 2

and until the end of the constant flow period (Figure 4.3). TSS at sites 3 and 4 in the

Mitta Mitta decreased rapidly during the recession of the second variable flow release

(dates 3 and 4), and increased again during the peak flow of the third variable flow

release. TSS concentrations were maintained above 30 g/m3 at sites 3 and 4 on the

Mitta Mitta until midway through the constant flow period and then decreased

gradually to the end of the study.

Chl-a concentrations increased substantially at all sites on the Mitta Mitta River

during the peak of the second variable flow release (date 2). Only at sites 2, 3 and 4

recorded increased Chl-a concentrations during the peak of the third variable flow

release (Figure 4.4). The concentration of Chl-a decreased rapidly at all Mitta Mitta

River sites during the recession period of both variable flow releases. The reference

stream did not follow a similar pattern in Chl-a concentration to sites on the Mitta

Mitta, varying little at approximately 300µg/m3 throughout the study period.



Figure 4.2: Water column concentrations (mg/L) of dissolved organic carbon (DOC) at sites 1 to 4 on the Mitta Mitta River and site 5 on Snowy Creek (reference site) on the nine sample dates from December 2001 to February 2002 (mean ± SD, n=3).

Site 1

0.00

10.00

20.00

30.00

2/12/0

1

9/12/0

1

16/12

/01

23/12

/01

30/12

/01

6/01/0

2

13/01

/02

20/01

/02

27/01

/02

3/02/0

2

10/02

/02

mg/

L

Site 2

0.00

10.00

20.00

30.00

2/12/0

1

9/12/0

1

16/12

/01

23/12

/01

30/12

/01

6/01/0

2

13/01

/02

20/01

/02

27/01

/02

3/02/0

2

10/02

/02

mg/

L

Site 3

0.00

10.00

20.00

30.00

2/12/0

1

9/12/0

1

16/12

/01

23/12

/01

30/12

/01

6/01/0

2

13/01

/02

20/01

/02

27/01

/02

3/02/0

2

10/02

/02

mg/

L

Site 4

0.00

10.00

20.00

30.00

2/12/0

1

9/12/0

1

16/12

/01

23/12

/01

30/12

/01

6/01/0

2

13/01

/02

20/01

/02

27/01

/02

3/02/0

2

10/02

/02

mg/

L

Site 5

0.00

10.00

20.00

30.00

2/12/0

19/1

2/01

16/12

/0123

/12/01

30/12

/016/0

1/02

13/01

/0220

/01/02

27/01

/023/0

2/02

10/02

/02

mg/

L



Figure 4.3: Water column concentrations (g/m3) of total suspended solids (TSS) at sites 1 to 4 on the Mitta Mitta River and site 5 on Snowy Creek (reference site) on the nine sample dates from December 2001 to February 2002 (mean ± SD, n=3).

Site 1

0.0010.0020.0030.0040.0050.0060.0070.00

2/12/0

1

9/12/0

1

16/12

/01

23/12

/01

30/12

/01

6/01/0

2

13/01

/02

20/01

/02

27/01

/02

3/02/0

2

10/02

/02

g/m

3

Site 2

0.0010.0020.0030.0040.0050.0060.0070.00

2/12/0

1

9/12/0

1

16/12

/01

23/12

/01

30/12

/01

6/01/0

2

13/01

/02

20/01

/02

27/01

/02

3/02/0

2

10/02

/02

g/m

3

Site 3

0.0010.0020.0030.0040.0050.0060.0070.00

2/12/0

1

9/12/0

1

16/12

/01

23/12

/01

30/12

/01

6/01/0

2

13/01

/02

20/01

/02

27/01

/02

3/02/0

2

10/02

/02

g/m

3

Site 4

0.0010.0020.0030.0040.0050.0060.0070.00

2/12/0

1

9/12/0

1

16/12

/01

23/12

/01

30/12

/01

6/01/0

2

13/01

/02

20/01

/02

27/01

/02

3/02/0

2

10/02

/02

g/m

3

Site 5

0.0010.0020.0030.0040.0050.0060.0070.00

2/12/0

19/1

2/01

16/12

/0123

/12/01

30/12

/016/0

1/02

13/01

/0220

/01/02

27/01

/023/0

2/02

10/02

/02

g/m

3



Figure 4.4: Water column concentrations (ug/m3) of chlorophyll-a (Chl-a) at sites 1 to 4 on the Mitta Mitta River and site 5 on Snowy Creek (reference site) on the nine sample dates from December 2001 to February 2002 (mean ± SD, n=3).

Site 1

0

300

600

900

1200

2/12/0

1

9/12/0

1

16/12

/01

23/12

/01

30/12

/01

6/01/0

2

13/01

/02

20/01

/02

27/01

/02

3/02/0

2

10/02

/02

ug/m

3

Site 2

0

300

600

900

1200

2/12/0

1

9/12/0

1

16/12

/01

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/01

30/12

/01

6/01/0

2

13/01

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/02

27/01

/02

3/02/0

2

10/02

/02

ug/m

3

Site 3

0

300

600

900

1200

2/12/0

1

9/12/0

1

16/12

/01

23/12

/01

30/12

/01

6/01/0

2

13/01

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/02

27/01

/02

3/02/0

2

10/02

/02

ug/m

3

Site 4

0

300

600

900

1200

2/12/0

1

9/12/0

1

16/12

/01

23/12

/01

30/12

/01

6/01/0

2

13/01

/02

20/01

/02

27/01

/02

3/02/0

2

10/02

/02

ug/m

3

Site 5

0

300

600

900

1200

2/12/0

19/1

2/01

16/12

/0123

/12/01

30/12

/016/0

1/02

13/01

/0220

/01/02

27/01

/023/0

2/02

10/02

/02

ug/m

3



4.3.2 Yeokal Multiprobes

Figure 4.5: Yeokal multiprobe data collected from site 1 and site 4 from 16th of December 2001 to 12th of February 2002. Multiprobes recorded temperature in degrees Celius, Conductivity (µS/cm), dissolved oxygen (DO) concentration (mg/L) and pH.

S i t e 4

0

1 0

2 0

3 0

4 0

5 0

6 0

10:15

:00 17

/12/01

10:15

:00 24

/12/01

1 10:1

5:00 3

1/12/0

1

2 10:1

5:00 7

/01/02

17:53

:00 15

/01/02

17:53

:00 22

/01/02

17:53

:00 29

/01/02

17:53

:00 5/

02/02

17:53

:00 12

/02/02

oC, u

S/c

m, m

g/L

0

1

2

3

4

5

6

7

8

9

1 0

pH

T e m p o C

C o n d u S / c m

D O m g / L

p H

S i t e 1

0

1 0

2 0

3 0

4 0

5 0

6 0

10:15

:00 17

/12/01

10:15

:00 24

/12/01

1 10:1

5:00 3

1/12/0

1

2 10:1

5:00 7

/01/02

17:53

:00 15

/01/02

17:53

:00 22

/01/02

17:53

:00 29

/01/02

17:53

:00 5/

02/02

17:53

:00 12

/02/02

oC, u

S/c

m, m

g/L

0

1

2

3

4

5

6

7

8

9

1 0

pH

T e m p o C

C o n d u S / c m

D O m g / L

p H



The pH, conductivity (µS/cm) and temperature (oC) were lower and dissolved oxygen

concentration (DO) was higher at site 1 during the CRP compared to the constant flow

period (Figure 4.5). The pH increased from 7.5 to 8.5 over the course of the study,

while conductivity and temperature recorded a minimal increase of approximately

7µS/cm and 3oC, respectively. Temperature and DO displayed strong diurnal

fluctuations.

Conductivity and pH varied little at site 4 in the period following the CRP.

Temperature was slightly lower during the variable flow releases when compared to

the constant flow period. DO concentration also decreased at site 4 following the

CRP, from approximately 11 mg/L to around 7 mg/L at the end of the constant flow

period.

4.3.3 Water Column Nutrient

Water column nutrient concentrations were exceptionally low at all sites on the Mitta

Mitta River and Snowy Creek for the duration of the study period (Table 4.1). The

majority of samples collected were below the detectable limits for the each of the

three nutrients analysed (PO-4, NH3-N and TP). Those samples that were above the

detectable limits displayed very low concentrations

4.3.4 DOC and POM Loading

Water column loads of POM were consistently higher at site 4 when compared to site

1. This indicates organic matter inputs from floodplain inundation, scouring of

substrata or runoff from influent streams occur along the length of the Mitta Mitta

River study area (Figure 4.6a).

The peak flow of the second variable flow release led to a rise in POM at both sites

peaking at 146.03 ± 11.54 kg/day and 65.75 ± 3.54 at sites 4 and 1 respectively

(Figure 4.6a). The peak flow of the third variable flow release led to a substantial

decrease in POM at both sites, which then increased to date 8, the midpoint of the low

constant flow period.



Table 4.1: Summary of Nutrient analyses upon PO-4, NH3-N and TP for sites 1 to 4 on the

Mitta Mitta River, and site 5 on the reference stream Snowy Creek for nine sampling dates from December 2001 to February 2002 (mean ± SD, n=3). <0.02 denotes undetectable limits for PO-

4 and TP. <0.05 denotes undetectable limits for NH3-N. Highlighted values represent samples above detectable limits.

PO-

4 Site Date 1 2 3 4 5 02/12/01 <0.02 <0.02 <0.02 <0.02 <0.02 04/12/01 <0.02 <0.02 <0.02 <0.02 <0.02 10/12/01 <0.02 <0.02 <0.02 <0.02 <0.02 16/12/01 <0.02 <0.02 <0.02 <0.02 0.02 ± 0.00 18/12/01 <0.02 <0.02 0.03 ± 0.01 <0.02 0.07 ± 0.05 23/12/01 <0.02 <0.02 <0.02 <0.02 <0.02 04/01/02 <0.02 <0.02 <0.02 <0.02 <0.02 21/01/02 <0.02 <0.02 <0.02 <0.02 <0.02 06/02/02 <0.02 0.02 ± 0.00 <0.02 <0.02 <0.02 NH3-N Site Date 1 2 3 4 5 02/12/01 <0.05 <0.05 <0.05 <0.05 <0.05 04/12/01 <0.05 <0.05 <0.05 <0.05 <0.05 10/12/01 <0.05 <0.05 <0.05 <0.05 <0.05 16/12/01 <0.05 <0.05 <0.05 <0.05 <0.05 18/12/01 <0.05 <0.05 <0.05 <0.05 <0.05 23/12/01 <0.05 <0.05 <0.05 <0.05 <0.05 04/01/02 <0.05 <0.05 <0.05 <0.05 <0.05 21/01/02 <0.05 <0.05 <0.05 <0.05 <0.05 06/02/02 <0.05 <0.05 <0.05 <0.05 <0.05 TP Site Date 1 2 3 4 5 02/12/01 <0.02 <0.02 <0.02 <0.02 <0.02 04/12/01 <0.02 <0.02 <0.02 <0.02 <0.02 10/12/01 <0.02 <0.02 <0.02 <0.02 <0.02 16/12/01 <0.02 <0.02 <0.02 0.02 ± 0.00 0.02 ± 0.00 18/12/01 <0.02 <0.02 0.05 ± 0.04 <0.02 0.09 ± 0.07 23/12/01 <0.02 <0.02 <0.02 <0.02 <0.02 04/01/02 <0.02 <0.02 <0.02 <0.02 0.04 ± 0.2 21/01/02 <0.02 <0.02 <0.02 <0.02 <0.02 06/02/02 <0.02 0.02 ± 0.00 <0.02 <0.02 <0.02 Loads of DOC displayed only minimal differences between sites 1 and 4 (Figure

4.6b). DOC loads were consistently higher at site 4 except on date 3 following the

peak of the second variable flow release. DOC loads were lowest under low flow

conditions and during this time displayed similar concentrations to POM. DOC

concentrations decreased on the flow peak of the second variable flow release at both

sites, but increased at site 1 and decreased at site 4 by date 5 the peak of the third

variable flow release. Maximum DOC loads at both sites occurred on date 6, the

midpoint of the third variable flow release recession of 38.17 ± 6.58 kg/day at site 1

and 65.75 ± 3.54 at site 4.



Net gain of POM in the water column increased during the second variable flow

release but decreased rapidly with the onset of the third variable flow release peak

(Figure 4.6c). Net DOC decreased during each peak flow event of the CRP suggesting

there is a dilution of DOC in river water by water from Dartmouth Dam. DOC was

negative on date 3 following the second variable flow release peak showing there was

a net loss of DOC in the system under those flow conditions. Under constant flow

conditions both DOC and POM consistently recorded net gains.

4.4 Discussion

4.4.1 Water Quality Parameters

The effects of the CRP in the Mitta Mitta River on the movement of DOC, POM, TSS

and water column Chl-a were examined. It was predicted there would be an overall

increase in concentrations of these four parameters during the peak discharges of the

CRP in the Mitta Mitta River compared to the reference stream.

The results of this study have shown a general trend of a short-term increase for all

parameters (except DOC) during the second variable flow release peak that was not

evident in any parameter measured in the reference stream during the same period.

Water column Chl-a, TSS and DOC concentrations also showed similar increases

during the peak of the third variable flow release and again this was not evident in the

reference stream.

The increases in water column Chl-a during each variable flow release peak is

suspected to be the result of scouring activity caused by increased flow velocity. The

scouring process can be a positive influence to the ecological health of stream

environments by resetting benthic biofilm successions, a process which increases the

algal species diversity and habitat available for consumers.



Figure 4.6: Mean (± SD) loadings of (a) POM and (b) DOC per day at sites 1 and 4 on the Mitta Mitta River, and (c) a reach scale budget of POM and DOC between sites 1 and 4 on the Mitta Mitta River.

0

20

40

60

80

100

120

140

160

180

2/12/0

1

9/12/0

1

16/12

/01

23/12

/01

30/12

/01

6/01/0

2

13/01

/02

20/01

/02

27/01

/02

3/02/0

2

10/02

/02

kg P

OM

/day

Site 1 Site 4

0

10

20

30

40

50

60

2/12/0

1

9/12/0

1

16/12

/01

23/12

/01

30/12

/01

6/01/0

2

13/01

/02

20/01

/02

27/01

/02

3/02/0

2

10/02

/02

kg D

OC

/day

Site 1 Site 4

-20

0

20

40

60

80

100

120

2/12/0

1

9/12/0

1

16/12

/01

23/12

/01

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/01

6/01/0

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13/01

/02

20/01

/02

27/01

/02

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10/02

/02

kg P

OM

and

DO

C/d

ay

POM DOC



The first variable flow release which was not measured in this study may have

resulted in the entrainment and flux of larger quantities of organic matter than were

observed during the second and third variable flow releases. The primary inundation

caused by the first peak flow may have caused a major breakdown and movement of

floodplain and instream sources of POM and DOC, resulting in a loss of quality and

quantity of these sources in the river system.

There were no obvious cumulative effects of successive variable flow releases on

concentrations of the four water quality parameters measured downstream of site 2.

These results suggest that continuous input of organic matter does not occur below the

Mitta Mitta and Snowy Creek confluence. The floodplain downstream of the township

of Mitta Mitta has been cleared for agricultural development, resulting in a narrow

riparian corridor dominated by Salix spp, an introduced deciduous tree with a defined

seasonal pulse of litter fall. Subsequently, the quantity and quality of floodplain

organic matter has been substantially altered and thus flow peaks in the study area

may not result in relatively large and consistent accumulations of organic matter

entrained into the water column.

Nutrient concentrations in the Mitta Mitta River and Snowy Creek were extremely

low throughout the study period, indicating a regional trend of low nutrient levels in

flowing surface waters. Further, the results from the Mitta Mitta River also

demonstrate that the water released from Dartmouth Dam is also very low in the

concentrations of the analysed nutrients. Trace nutrient levels in flowing surface water

within the region and from Dartmouth Dam coupled with a lack of quality floodplain

litter may result in minimal transport and or availability of organic and inorganic

nutrient within the Mitta Mitta River.

The CRP did not result in any distinct changes in the physicochemical parameters

measured at reaches located at the top and bottom of the study area. However, the

onset of constant flow conditions within the Mitta Mitta River resulted in an increase

in water column conductivity, pH and temperature and a decrease in DO

concentration. It is suspected that the reductions in DO concentrations may be a result

of a high benthic oxygen demand generated by the senescence of benthic biofilm

communities. This is evidenced by the presence of layers of dead algae within the



river following prolonged low flows. Increases in temperature are probably the result

of changes in season from November to February, decreased discharge levels and

flow rates as well as decreased water depth, allowing greater exposure to solar

warming. Increases in conductivity and pH during the constant flow conditions may

be due to increased contribution from ground water sources to the main river channel

relatively to water from Dartmouth Dam.

4.4.2 DOC and POM Loading

The loads of POM and DOC were relatively low, but consistent with loads recorded

from similar headwater stream systems (Webster & Meyer 1997). Water column loads

of POM were consistently higher at site 4 than at site 1, indicating that organic matter

inputs from floodplain inundation, scouring of substrata or runoff from influent

streams occur along the entire Mitta Mitta River study area. This process appears to

occur under the range of flow conditions experienced during this study as the organic

matter budget showed net POM to be consistently positive. The constant flow period

was characterised by reduced loadings of POM at both sites indicating minimal

scouring or input from influent streams occurred under these flow conditions.

Loads of DOC displayed only minimal differences between sites 1 and 4. These

trends indicate that either the input of DOC is not continuous along the study area or,

that it is rapidly utilised by bacteria and does not accumulate in the water column.

Similarly, net DOC decreased on each variable flow release peak indicating there is a

dilution of DOC in river water by water from Dartmouth Dam.

4.5 Summary of Findings • The release of peak flows of 4800 ML/day during the second and third variable

flow release resulted in short-term increases in the concentration of water column

organic matter in the form of DOC, POM, TSS and Chl-a.

• Periods of constant flow conditions decreased the concentrations of water column

organic matter.



• Increased concentrations of instream Chl-a following the peak flows of each

variable flow release may be the result of scouring caused by increased flow

velocity on benthic biofilms.

• The lack of data from the first variable flow release makes interpretation of the

concentrations of each parameter very difficult as potential organic matter sources

may have been degraded and or removed during the first variable flow release.

• There were extremely small concentrations of water column nutrients within all

sites on the Mitta Mitta River and Snowy Creek.

• Constant flow conditions resulted in increases in water column temperature, pH

and conductivity and decreased DO concentration.



5.0 WATER COLUMN EXTRACELLULAR ENZYME ACTIVITY

5.1 Introduction

Heterotrophic microorganisms (predominantly bacteria) form a key level in aquatic

ecosystems, being supported by DOM. Bacterial communities in aquatic habitats can be

derived from either aquatic or terrestrial habitats, although terrestrial bacteria do not

survive for extended periods in aquatic habitats (Veal et al. 1998). Bacterial communities

can occur in all aquatic habitats but are abundant in the water column and surface

sediments. Bacteria are responsible for the biogeochemical cycling of all elements in the

biosphere (Hart et al. 1996). They are generally considered as key organisms responsible

for the decomposition of organic material and the regeneration of nutrients. The

composition and productivity of aquatic bacterial communities are the consequence of

multiple interactions between hydrological, chemical and biotic factors. Bacteria have been

extensively used as indicators of pollution such as contamination by heavy metals. More

recently there has been a focus on developing microbial indicators as measures of

ecosystem function, where the aim is to measure the activity of key microbial processes.

Carbon compounds in aquatic environments are utilised by bacteria through the release of

extracellular enzymes. A technique has been developed which uses the activity of bacterial

extracellular enzymes to link bacterial productivity to the concentrations and classes of

available organic matter. (Sinsabaugh et al. 1997). Organic matter in aquatic systems

occurs as carbohydrates, proteins, fatty acids and other compounds. Each class of organic

matter requires a specific extracellular enzyme to be excreted from bacteria in order for the

organic matter to be utilised. Bacteria can shift their composition of extracellular enzymes

in response to changes in the classes of available organic matter. This technique has

successfully been used to identify the response of microbial metabolism, composition and

utilisation of organic matter to disturbance in a wide variety of aquatic habitats (Chappell

& Goulder 95; Boschker & Cappenberg 1998; Findlay et al. 2001) and in Australian river

systems by Burns & Ryder (2001a). By monitoring these shifts in enzyme activity it is

possible to examine the classes and quantity of organic matter available to these microbial

communities.



The aim of this section is to examine the response of water column bacterial activity to a

CRP from Dartmouth Dam to the Mitta Mitta River, Victoria from December 2001 to

February 2002. The following prediction can be made:

1. There will be an overall increase in rates of water column bacterial enzyme

activity during the peak discharges of the CRP from Dartmouth Dam to the Mitta

Mitta River.

5.2 Methods

5.2.1 Field methods

Five replicate water samples were taken from flowing surface waters at each study site on

each sampling event for determination of extracellular enzyme activity. Samples were

collected and stored in 30ml polycarbonate vials and frozen immediately after collection.

5.2.2 Laboratory Methods

The following methylumbelliferyl (MUF) labelled carbon substrates were used to estimate

bacterial activity in water samples (1) 4-MUF-butyrate (fatty acid esterase – FAE); (2) α-

D-glucosidase (carbohydrate), (3) β-D-glucosidase (carbohydrate); (4) β-D-xylosidase

(long chain carbohydrate, eg. woody substrates), and (5) Leucine-7-amino-4-methyl-

coumarin (aminopeptidase). These enzymes are involved in the degradation of

polysaccharides, carbohydrates and proteins derived from a range of autochthonous and

allocthonous organic matter (Chrost 1991).

Stock solutions (1mM) of each MUF-linked substrate were made and diluted to known

concentrations that assume substrate saturation (50-100µM) for each water sample. Water

samples were defrosted and activities were assayed by mixing 750 µL of each substrate

with 750 µL of sample water in 3ml disposable fluorometric cuvettes and conducted at

20oC. Four replicate samples of river water from each site for each of the sampling dates

(Table 2.2) were analysed using this technique. The α- β- glucosidases and xylosidase

were assayed at pH 8 in 5mM bicarbonate buffer, and esterase and aminopeptidase in pH 7



5mM phosphate buffer. Fluorescence was read repeatedly over a 1 to12 hour period at

365nm excitation and 450nm emission on a Shimadzu Fluorometer.


Enzyme activities are expressed as rates of accumulation of MUF equivalents (µmol/L/h),

by using the emission coefficient calculated by regression from MUF standards in

bicarbonate buffer and phosphate buffer. Individual samples were corrected for substrate

degradation using a boiled replicate water sample. All data were log10 transformed to

remove dependencies between mean and variance.

Statistical analysis employed single factor ANOVA planned comparisons of date 1 (end of

first variable flow release) to date 2 (peak flow of second variable flow release) and date 4

(end of second variable flow release) to date 5 (peak flow of third variable flow release) for

each of the five sites using Statistica V4 (Statistica 1995). This design is based on the rapid

and short term response of the enzyme activities to changes in discharge and tests the

response of each enzyme at each site to the predicted increases in DOM during the flood

peaks.

5.3 Results

Activities of all enzymes were generally highest during the peak discharges of the second

and third variable flow releases, although trends often differed between the two releases

and between sites for each enzyme. Very low activities were recorded from all sites on the

Mitta Mitta River throughout the period of low and constant flows (dates 7 to 9). Enzyme

activities at the reference site on Snowy Creek were consistently low, with no significant

difference in activities found between the sampling dates examined (Table 3.1).

The two carbohydrase enzymes, alpha and beta glucosidase displayed significant increases

in activity at all sites during the third variable flow release, with activities increasing with

distance downstream (Table 5.1, Figures 5.1, 5.2). Activities ranged from 1.56 ± 0.09

µM/L/h at site 1 to 2.64 ± 0.46 µM/L/h at site 4 for alpha glucosidase and 0.54 ± 0.02

µM/L/h at site 2 to 1.33 ± 0.34 µM/L/h at site 4 for beta glucosidase. However the second

variable flow release did not result in such a consistent response across sites. Significant



increases in alpha and beta glucosidase enzyme activity from dates 1 to 2 were only found

at site 1.

Table 5.1: Probability value and significance levels of planned comparison one-way ANOVAs comparing enzyme activities of river water from dates 1 to 2, and 4 to 5 at four sites in the Mitta Mitta River (sites 1 to 4) and in Snowy Creek (site 5). Post hoc tests indicating direction of trend are shown in brackets. * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Release 2 (Dates 1 to 2)

Site 1 Mitta Mitta

Site 2 Mitta Mitta

Site 3 Mitta Mitta

Site 4 Mitta Mitta

Site 5 Snowy Creek

Alpha glucosidase 0.001 *** (1<2) 0.263 0.814 0.381 0.613

Beta glucosidase 0.001 *** (1<2) 0.039 * (1>2) 0.016 * (1>2) 0.026 * (1>2) 0.061

Butyrate 0.087 0.001 *** (1<2) 0.714 0.072 0.803

Leucine 0.138 0.515 0.113 0.048 * (1<2) 0.347

Xyloside 0.903 0.888 0.790 0.693 0.746

Release 3 Dates (4 to 5)

Site 1 Mitta Mitta

Site 2 Mitta Mitta

Site 3 Mitta Mitta

Site 4 Mitta Mitta

Site 5 Snowy Creek

Alpha glucosidase 0.001 *** (4<5) 0.001 *** (4<5) 0.001 *** (4<5) 0.001 *** (4<5) 0.464

Beta glucosidase 0.008 ** (4<5) 0.021 * (4<5) 0.001 *** (4<5) 0.001 *** (4<5) 0.084

Butyrate 0.212 0.004 ** (4>5) 0.010 ** (4>5) 0.613 0.771

Leucine 0.106 0.041 * (4>5) 0.184 0.001 *** (4<5) 0.355

Xyloside 0.093 0.084 0.006 ** (4<5) 0.002 ** (4<5) 0.887

Differences in the responses of the other enzymes were not as definitive as the

carbohydrase enzymes due to small variations in their response to altered flow regimes and

large standard deviations. Although not significantly different at all sites, the butyrate

(fatty acid esterase) and leucine (protein) substrates displayed trends of higher activity

during the second Release and lower activity during the third Release (except site 4)

(Figures 5.3, 5.4). This indicates they may have potential for detecting change in microbial

production with increased replication. Xyloside activities were very low compared to the

other substrates indicating low concentrations of long chain carbohydrates (derived from

woody debris) in the water column (Figure 5.5).



Figure 5.1: Alpha glucosidase activities in µM substrate/L/h at sites 1 to 4 on the Mitta Mitta River and site 5 on Snowy Creek (reference site) on the nine sample dates from December 2001 to February 2002 (mean ± SD, n=4).

Figure 5.2: Beta glucosidase activities in µM substrate/L/h at sites 1 to 4 on the Mitta Mitta River and site 5 on Snowy Creek (reference site) on the nine sample dates from December 2001 to February 2002 (mean ± SD, n=4).

Beta Glucosidase

0

0.5

1

1.5

2

2.5

3

3.5

4

2/12/2

001

9/12/2

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16/12

/2001

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/2001

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/2001

6/01/2

002

13/01

/2002

20/01

/2002

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/2002

3/02/2

002

10/02

/2002

uM s

ubst

rate

/L/h

site1 site2 site3

site4 site5

Alpha Glucoside

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

2/12/2

001

7/12/2

001

12/12

/2001

17/12

/2001

22/12

/2001

27/12

/2001

1/01/2

002

6/01/2

002

11/01

/2002

16/01

/2002

21/01

/2002

26/01

/2002

31/01

/2002

5/02/2

002

10/02

/2002

uM s

ubst

rate

/L/h

site1 site2 site3

site4 site5



Figure 5.3: Butyrate activities in µM substrate/L/h at sites 1 to 4 on the Mitta Mitta River and site 5 on Snowy Creek (reference site) on the nine sample dates from December 2001 to February 2002 (mean ± SD, n=4).

Figure 5.4: Leucine activities in µM substrate/L/h at sites 1 to 4 on the Mitta Mitta River and site 5 on Snowy Creek (reference site) on the nine sample dates from December 2001 to February 2002 (mean ± SD, n=4).

Butyrate

0

0.5

1

1.5

2

2.5

3

3.5

4

2/12/2

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/2001

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/2001

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002

13/01

/2002

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/2002

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10/02

/2002

uM s

ubst

rate

/L/h

site1 site2 site3

site4 site5

Leucine

0

0.5

1

1.5

2

2.5

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3.5

4

2/12/2

001

9/12/2

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16/12

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23/12

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/2001

6/01/2

002

13/01

/2002

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27/01

/2002

3/02/2

002

10/02

/2002

uM s

ubst

rate

/L/h

site1 site2 site3

site4 site5



Figure 5.5: Xylosidase activities in µM substrate/L/h at sites 1 to 4 on the Mitta Mitta River and site 5 on Snowy Creek (reference site) on the nine sample dates from December 2001 to February 2002 (mean ± SD, n=4).

5.4 Discussion The effects of the CRP in the Mitta Mitta River on the sources and utilisation of DOC in

river water by bacterial communities were examined. It was hypothesised that there would

be an overall increase in rates of water column bacterial enzyme activity during peak

discharges of the CRP compared to the reference stream. The results of this study have

shown a general trend of a very short-term, but substantial increase in activity associated

with the peak flows for most carbon substrates that was not evident in any substrate in the

reference river. The release of peak flows of 4800 ML/day on dates 2 and 5 have increased

the rates of microbial productivity for a diverse range of water column bacteria, which in

turn can be expected to increase the biomass of microbial communities available to

successive trophic levels.

Few studies have explored the response of microbial communities to short time scales such

as peak flow events with the majority of studies using the extracellular enzyme technique

identifying seasonal patterns or sources of carbon (Chappell & Goulder 1995; Boschker &

Xyloside

0

0.5

1

1.5

2

2.5

3

3.5

4

2/12/2

001

9/12/2

001

16/12

/2001

23/12

/2001

30/12

/2001

6/01/2

002

13/01

/2002

20/01

/2002

27/01

/2002

3/02/2

002

10/02

/2002

uM s

ubst

rate

/L/h

site1 site2 site3

site4 site5



Cappenberg 1998; Foreman et al 1998). Short term peaks of enzyme activity similar to

those seen in the Mitta Mitta River during flow peaks were noted by Burns and Ryder

(2001b) who provide an example of rapid, short term (21 days) changes in enzyme activity

associated with the inundation of river sediments. However, rates of peak enzyme activities

in the Mitta Mitta River are at the lower end of those found in other river systems (eg.

Sinsabaugh et al 1997; Findlay et al 1998; Burns & Ryder 2001b) possibly due to limiting

concentrations of inorganic nutrients (Findlay & Sinsabaugh 1999) demonstrated in

Section 4.

The low rates of activity for xylosidase compared to the other enzymes is expected due to

the relatively low abundance of woody debris in the Mitta Mitta River. However, a number

of issues such as the removal and transport leaf litter and woody debris during the first

Release (not studied as part of this contract), timing of natural litterfall and replenishment

of coarse organic debris may all influence the activity rates of xyloside. Therefore, this

enzyme should not be discounted from any future studies in this or similar river systems.

For example, the pulse of leaf litter entering the stream during leaf fall from willows and

poplars that line the banks of the Mitta Mitta River may an important seasonal source of

carbon for microbial production that could be quantified using xylosidase substrates.

Similarly, the high level of variability seen in the butyrate and leucine substrates should

not discount them from future monitoring studies, although it highlights the need for

increased sample replication.

Activities of alpha and beta glucosidase displayed the most consistent response to increases

in flow discharge. These enzymes are responsible for the degradation of carbohydrase

substrates such as those found in cell walls of aquatic plants and green algae. The Mitta

Mitta River contains extensive benthic algal communities that contain large populations of

filamentous green algae (particularly Stigeoclonium and Oedogonium – Section 6). An

increase in water column Chl-a (Section 4) and a decrease in biofilm Chl-a (Section 6)

during peak flows in the Mitta Mitta River suggest that increases in alpha and beta

glucosidase activity may the result the scouring of benthic algae during peak flows. This is

supported by trends of increased protease and fatty acid esterase activity during flood

peaks, enzymes associated with benthic algal communities containing cyanobacteria. An

increase in the activity of carbohydrase enzymes with distance downstream during the third



Release suggests these carbon resources originate from along the entire study reach

indicating the physical disturbance of biofilms along the entire study length.

Findlay and Sinsabaugh (1999) demonstrated increased microbial activity following cell

breakage by macroinvertebrate consumers. We hypothesise that the peak flows of 4800

ML/day are sufficient to result in the physical abrasion and damage of algal cells along the

entire study reach leading to an increase in microbial productivity. This hypothesis is

supported by substantial decreases in biofilm net productivity on flood days possibly due

to the increase in heterotrophic microbial activity (Section 6).

5.5 Summary of findings • The release of peak flows of 4800 ML/day on dates 2 and 5 resulted in short-term

increases (days) in the rates of microbial productivity along the entire study reach for a

diverse range of water column bacteria. This in turn can be expected to increase the

biomass of microbial communities available to successive trophic levels.

• Rates of peak enzyme activities in the Mitta Mitta River were at the lower end of rates

found in other river systems possibly due to limiting concentrations of inorganic

nutrients.

• The lack of data from the first variable flow release makes interpretation of some

enzyme activities, particularly xylosidase (leaf/woody debris) very difficult, as

potential carbon sources may have been affected by the first variable flow release.

• Activities of alpha and beta glucosidase displayed the most consistent response to

flooding. These enzymes are responsible for the degradation of carbohydrase substrates

such as those found in cell walls of aquatic plants and green algae.

• We hypothesise that consistent increases in enzyme activity during peak flows are the

result of the physical abrasion and damage to biofilm algal cells, particularly

filamentous green algae, leading to an increase in microbial productivity.



6.0 BIOFILM STRUCTURE AND FUNCTION

6.1 Introduction Submerged surfaces in lakes and rivers are colonised by assemblages of algae, fungi and

bacteria in a mucilaginous matrix of algal and bacterial exudates and detritus (Wetzel

1983). These are the biofilms (or periphyton) that cover rocks, wood, sediment particles

and other surfaces in aquatic systems. Biofilms form a key level in aquatic ecosystems, and

are a major instream source of carbon in river systems, often making a greater contribution

to biomass and metabolic activity than phytoplankton and macrophytes (Bott 1983). These

organisms are central to important nutrient and biogeochemical processes, and as such may

respond to disturbance before effects on higher organisms are detected. This is because the

higher organisms depend on processes mediated by algal and microbial communities.

Consequently, they form the base of food webs supporting zooplankton, grazers such as

crustaceans, insects, molluscs and some fish (Lock et al. 1984; Rounick & Winterbourn

1986; Stevenson 1996).

Biofilms are assemblages that contain both autotrophic and heterotrophic microorganisms.

The composition and productivity of biofilms are the consequence of multiple interactions

between hydrological, chemical and biotic factors. Processes that control resources

ultimately affect biomass accumulation, with disturbances leading to losses (Biggs 1996).

Succession of biofilms is driven by differential species performance in dispersal, survival

and reproduction; through factors such as resource availability, ecophysiology, life history

and disturbance (Pickett & McDonnell 1989). Nutrients, light and available substrata form

the basic resources for biofilms. These can be modified by external factors, which

ultimately regulate local resource availability. Physical disturbances, such as flow and

changes in water level, act as resource modulators for biofilms through changes to nutrient

and light availability, and by clearing substrata through scouring and abrasion. Principally,

flow regulates accrual of biofilm biomass in river systems through scour, substratum loss

and by increasing light attenuation through higher loads of suspended material. The

scouring of biofilms can lead to changes in the community composition of algal species

within the biofilm by favouring species that can tolerate high velocities (such as diatoms).

Changes to the species composition and biomass of algal biofilms can impact on their



ecosystem function, by either reducing or increasing oxygen production dependant on

species present.

The short generation time, sessile nature, responsiveness to environmental condition and

the availability of sound, quantitative methodologies make biofilms ideally suited as

indicators of disturbance in aquatic systems. Information can be collected, processed and

analysed at time scales relevant to both scientific and management interests. Measures of

the structural characteristics of biofilms can be obtained rapidly and at low cost through

measurements such as biomass or taxonomic composition. The determination of biofilm

function can be achieved through measuring changes in system state using techniques such

as biofilm metabolism and extracellular enzyme activity. Functional measures can be used

to integrate diverse communities into a few attributes, allowing easier comparison among

different systems and within systems over time (Pratt & Cairns 1996). The combination of

structural and functional information from biofilms at population, community and

ecosystem levels offers even greater potential for ecologically meaningful analysis (Hart et

al. 1996; Veal et al. 1998).

The aim of this section is to examine the response of structural and functional attributes of

biofilms attached to cobble substrata to variable flow releases in the Mitta Mitta River,

Victoria from December 2001 to February 2002. The following predictions can be made:

1. Decrease in the total and algal biomass, and an increase in the organic biomass of permanently inundated biofilms in the Mitta Mitta River resulting from peak flow releases.

2. Change in the relative proportion of algal species of permanently inundated biofilms in the Mitta Mitta River resulting from peak flow releases.

3. Decrease in the net primary productivity of permanently inundated biofilms in the Mitta Mitta River from peak flow releases.

4. Variable release patterns associated with the CRP will result in the creation of new cobble habitat, and these habitats will have biofilms that are structurally and functionally distinct from permanently inundated cobbles in the Mitta Mitta River.



6.2 Methods

6.2.1 Biofilm Structural Components

Total, organic and algal biomass

Biofilms were collected from cobble that remained permanently inundated throughout the

study on each sampling date and from all sites. Biofilms were also collected from newly

inundated cobble at sites 1 to 4 on dates 2, 3, 5 and 6. On each sampling occasion, 5

cobbles (ranging between 12 and 25cm diameter) were randomly selected from each

relevant habitat at each site, placed in labelled sealed plastic bags and stored on ice in the

dark. Markers were placed at each site to delineate the permanently inundated zone and

cobbles taken from 0.5 to 1.0m from this edge. Newly inundated cobble were taken from

within 0.5m of the water/land boundary on relevant days. Replication and cobble size were

based on the results of Watts et al. (2001).

In the laboratory, the biofilm was scrubbed from each cobble within 48 hours of field

collection into 100 to 500ml of distilled water (dependant on cobble size) using a soft

toothbrush. The volume of water was recorded for individual cobbles. The slurry

containing the scrubbed biofilm was thoroughly homogenised using a household blender

and sub-samples removed for determination of Chl-a (30ml filtered through a GFF 0.75µm

filter) and taxonomic composition (10ml stored in Lugol’s solution). The remaining

solution was placed in an evaporation dish for analysis of organic and inorganic biomass.

Each biomass sample was dried at 80°C for 48 hours, weighed, combusted for 4h at 550°C,

then reweighed. All samples were weighed to four decimal places and converted to dry

weight (DW) and ash free dry weight (AFDW). Chl-a was determined following Tett et al.

(1975). Samples were placed in 10mL methanol containing 150mg MgCO3 to prevent

premature acidification, extracted for 18h at 4°C, transferred to a 70°C water bath and

boiled for 2 minutes. Samples were centrifuged at 4500rpm for 5 minutes and optical

densities at 750 and 666nm were measured pre- and post-acidification (2N HCl) using a

Shimadzu UV/Visible Spectrophotometer.

Each cobble was measured for colonisable rock surface area (CRSA) by covering the

exposed surface area of the rock (excluding the buried surface) with aluminium foil (after



Doeg & Lake 1981). CRSA measurements were used to standardise biofilm dry weight

(DW) and ash free dry weight (AFDW) to g/m2 and Chl-a to mg/m2. Percent organic

matter was calculated as the proportion of AFDW to DW and converted to a percentage to

standardise across sites and dates.

6.2.2 Biofilm Taxonomy

Taxonomic composition of the algae was estimated by calculating the biovolume of the

first 750 cells counted by light microscopy at 400x magnification of two replicate biofilm

samples from sites 1 and 4 for all dates and habitats (newly and permanently inundated).

Replication was limited due to the high costs associated with algal taxonomic

identification. The cell dimensions and approximated geometric shape of each taxon were

recorded and used to calculate the biovolumes using the biovol program (Kirschtel 1999).

Biovolume provides a more accurate estimate of relative abundance than cell number as it

standardises results by cell size and removes complications associated with species such as

filamentous Cyanobacteria which do not have individual cells.

Taxonomy was confirmed at 1000x magnification for some specimens. Biovolume of each

taxon was converted to a relative percentage of the total biovolume. Relative biovolume

gives a good assessment of broad taxonomic shifts in biomass. The algae were grouped by

division into Chlorophyta (green algae), Cyanophyta (cyanobacteria) and Bacillariophyta

(diatoms). The algal cells were mainly identified to genus. Species were named where

sufficient detail was available from microscopy, and keys were available for the genus.

6.2.3 Biofilm metabolism

Biofilm metabolism was measured at sites 1 and 4 from permanently inundated cobble on

dates 1 to 9 and from newly inundated cobble on dates 2, 3, 5 and 6. Rates of

photosynthesis and dark respiration of cobble biofilm and of the water column were

measured by monitoring changes in oxygen concentrations within closed chambers using

separate light and dark incubations. Eight dome shaped chambers (4 dark, 4 light)

constructed from clear or opaque perspex with a 50 cm inner diameter were used. The

chambers were mounted in a mobile trailer that could be located immediately adjacent to



the river channel (Figure 6.1). For each incubation, a high volume pump mounted on the

trailer filled the trailer body and chambers with river water. Once filled, one or two large

cobble (20-30cm diameter) were placed in each chamber and the chambers sealed to the

base of the trailer to isolate them from the water in the trailer. Water was recirculated

within each chamber using a Whale 911 recirculating pump (max 30L/hour) with inlet and

outlet valves located at the top and bottom of the chamber respectively (Figure 6.2). The

water in the trailer was continuously replaced with new river water to reduce changes in

water temperature during the incubations.

Plate 6.1: Chambers mounted in the mobile trailer used to measure biofilm metabolism.

A port with a rubber o-ring in each chamber fitted the probe from an Orion 835A oxygen

logger, which recorded oxygen concentrations at 10 minute intervals. Each DO probe was

calibrated immediately before each incubation. Incubations were for 3 hours during peak

irradiance between 10am – 4pm on each sample date. Water column metabolism was

assessed by measuring change in DO concentrations over time inside smaller (4L) sealed

light and dark perspex chambers with no cobble inside.

At the end of each incubation the cobble in each chamber was removed and sealed in a

plastic bag for determination of total, organic and algal biomass and CRSA as outlined

above. Logged DO data were downloaded into the Orion Paraly SW105 software package.



Net primary production (NPP), Gross Primary Productivity (GPP) and Respiration (R)

were determined by calculating the rate of change in DO concentration within each

chamber over the incubation period using the regression function of Statistica (Statistica

1995). Only R2 values above 0.6 were used in subsequent calculations. All NPP,

GPP and R data were standardised to mgO2/m2/hour.

Plate 6.2: One transparent metabolism chamber mounted in the mobile trailer showing location of oxygen probe and recirculating pump.


Statistical analyses for biofilm DW, AFDW, organic percent and Chl-a employed single

factor ANOVAs for selected sample dates based on a priori planned comparisons of dates

and habitats. Biofilm DW, AFDW and Chl-a data were square root transformed and

organic percent data were arcsine transformed to remove dependencies between means and

variance. The following planned comparisons were undertaken:

Date 1 (end of first variable flow release) to date 4 (end of second variable flow release)

and date 1 (end of first variable flow release) to date 7 (end of third variable flow release 3)

for each of the five sites to examine the individual and cumulative effects of 2 and 3

variable flow releases.

Newly inundated cobble to permanently inundated cobble at sites 1 to 4 for each of dates 2

(flow peak of the second variable flow release), date 3 (day 8 of the second variable flow

Oxygen probe

Recirculating pump



release), date 5 (flow peak of the third variable flow release) and date 6 (day 8 of third

variable flow release). Date 7 (end of third variable flow release and start of constant flow

period) to date 9 (end of constant flow period) to examine the effect of extended periods of

constant flows.

The low number of replicates examined for taxonomic composition of algal species

associated with biofilms limited the statistical analysis. Data are presented as relative

percentage of the total biovolume for Chlorophyta (green algae), Cyanophyta

(cyanobacteria) and Bacillariophyta (diatoms) for permanently and newly inundated cobble

habitats from sites 1 and 4.

No statistical analyses were conducted on metabolism data. To maximise the sites and

habitats sampled, separate light and dark incubations were used to estimate GPP and R.

This meant that GPP and R were not calculated for the same cobble, and therefore one

NPP estimate was derived from the mean value of GPP and R for a particular treatment.

6.3 Results

6.3.1 Biofilm total, organic and algal biomass

The dry weight and ash free dry weight of permanently inundated biofilms was highly

variable at all sites on the Mitta Mitta River throughout the CRP and constant flow periods

and were generally less than 100 g/m2 (Figure 6.1) and 10 g/m2 respectively (Figure 6.2).

Biofilms at site 4 were the exception with a dry weight ranging from approximately 100 to

225 g/m2, and an ash weight ranging from approximately 5 to 15 g/m2 during the CRP.

Biofilm dry and ash weight in Snowy Creek were less than those in the Mitta Mitta River,

consistently below 20 g/m2 and 5 g/m2 respectively. Dry and ash weights decreased

following 2 and 3 variable flow releases at all sites. Dry and ash weights increased at all

sites during the period of constant flows following the end of the CRP.

The organic matter percent of biofilms ranged between 5 and 15% at all sites on the Mitta

Mitta River during the CRP (Figure 6.3). An increase in biofilm organic matter percent as

a result of 2 or 3 variable flow releases was only evident at site 1, with little difference

between dates at the other sites. Biofilm organic percent at sites 1 and 2 was more variable

during the period of constant flows following the end of the CRP. Biofilm organic matter



percent was highest and most variable at Snowy Creek ranging from around 6 to 29%

throughout the study period.

Similar to the other biomass estimates, the concentrations of Chl-a in permanently

inundated biofilms in the Mitta Mitta River were highly variable, ranging from around 180

mg/m2 to over 1700 mg/m2 throughout the study (Figure 6.4). A trend of decreasing

biofilm Chl-a following 2 or 3 variable flow releases was consistently evident at sites 1, 2

and 4, however, the Chl-a concentration of biofilms increased at site 3 throughout the

CRP. An increase of biofilm Chl-a was seen at all sites during the period of constant flows

following the end of the CRP, with sites 2 and 3 showing a 5 fold increase in this period.

In general, biofilm attributes in the newly inundated cobble habitat were different to those

in the permanently inundated cobbles. Biofilm dry weight in newly inundated cobble was

generally higher that in the permanently inundated cobble (Figures 6.1). The ash free dry

weight of biofilms was generally similar in both habitats throughout the study. The organic

matter percent of biofilms in the permanently inundated habitat was similar to that in the

newly inundated habitat in sites 2 and 3, but generally higher in the newly inundated

cobble at sites 1 and 4. Biofilm Chl-a concentrations were consistently lower in the newly

inundated cobble habitat throughout the study.



Figure 6.1: Biofilm dry weight in permanently inundated and newly inundated cobble habitats at sites 1 to 4 on the Mitta Mitta River and site 5 on Snowy Creek (reference site) on the nine sample dates from December 2001 to February 2002 (mean ± SD, n=5).

Site 1

0

50

100

150

200

250

2/12/2001 3/12/2001 4/12/2001 5/12/2001 6/12/2001 7/12/2001 8/12/2001 9/12/2001 10/12/2001 11/12/2001 12/12/2001 13/12/2001 14/12/2001 15/12/2001 16/12/2001 17/12/2001 18/12/2001 19/12/2001 20/12/2001 21/12/2001 22/12/2001 23/12/2001 24/12/2001 25/12/2001 26/12/2001 27/12/2001 28/12/2001 29/12/2001 30/12/2001 31/12/2001 1/01/2002 2/01/2002 3/01/2002 4/01/2002 5/01/2002 6/01/2002 7/01/2002 8/01/2002 9/01/2002 10/01/2002 11/01/2002 12/01/2002 13/01/2002 14/01/2002 15/01/2002 16/01/2002 17/01/2002 18/01/2002 19/01/2002 20/01/2002 21/01/2002 22/01/2002 23/01/2002 24/01/2002 25/01/2002 26/01/2002 27/01/2002 28/01/2002 29/01/2002 30/01/2002 31/01/2002 1/02/2002 2/02/2002 3/02/2002 4/02/2002 5/02/2002 6/02/2002 7/02/2002 8/02/2002 9/02/2002 10/02/2002 11/02/2002

g/m

2Newly inundatedPermanently inundated

Site 2

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g/m

2

Site 3

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2/12/2

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/2002

g/m

2

Site 4

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100

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2/12/2

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Site 5

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g/m

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Figure 6.2: Biofilm ash free dry weight in permanently inundated and newly inundated cobble habitats at sites 1 to 4 on the Mitta Mitta River and site 5 on Snowy Creek (reference site) on the nine sample dates from December 2001 to February 2002 (mean ± SD, n=5).

Site 1

0

5

10

15

20

25

2/12/2001 3/12/2001 4/12/2001 5/12/2001 6/12/2001 7/12/2001 8/12/2001 9/12/2001 10/12/2001 11/12/2001 12/12/2001 13/12/2001 14/12/2001 15/12/2001 16/12/2001 17/12/2001 18/12/2001 19/12/2001 20/12/2001 21/12/2001 22/12/2001 23/12/2001 24/12/2001 25/12/2001 26/12/2001 27/12/2001 28/12/2001 29/12/2001 30/12/2001 31/12/2001 1/01/2002 2/01/2002 3/01/2002 4/01/2002 5/01/2002 6/01/2002 7/01/2002 8/01/2002 9/01/2002 10/01/2002 11/01/2002 12/01/2002 13/01/2002 14/01/2002 15/01/2002 16/01/2002 17/01/2002 18/01/2002 19/01/2002 20/01/2002 21/01/2002 22/01/2002 23/01/2002 24/01/2002 25/01/2002 26/01/2002 27/01/2002 28/01/2002 29/01/2002 30/01/2002 31/01/2002 1/02/2002 2/02/2002 3/02/2002 4/02/2002 5/02/2002 6/02/2002 7/02/2002 8/02/2002 9/02/2002 10/02/2002 11/02/2002

g/m

2Newly inundatedPermanently inundated

Site 2

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Site 3

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Site 4

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g/m

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Figure 6.3: Biofilm organic matter percent in permanently inundated and newly inundated cobble habitats at sites 1 to 4 on the Mitta Mitta River and site 5 on Snowy Creek (reference site) on the nine sample dates from December 2001 to February 2002 (mean ± SD, n=5).

Site 1

0

5

10

15

20

25

30

35

2/12/2001 3/12/2001 4/12/2001 5/12/2001 6/12/2001 7/12/2001 8/12/2001 9/12/2001 10/12/2001 11/12/2001 12/12/2001 13/12/2001 14/12/2001 15/12/2001 16/12/2001 17/12/2001 18/12/2001 19/12/2001 20/12/2001 21/12/2001 22/12/2001 23/12/2001 24/12/2001 25/12/2001 26/12/2001 27/12/2001 28/12/2001 29/12/2001 30/12/2001 31/12/2001 1/01/2002 2/01/2002 3/01/2002 4/01/2002 5/01/2002 6/01/2002 7/01/2002 8/01/2002 9/01/2002 10/01/2002 11/01/2002 12/01/2002 13/01/2002 14/01/2002 15/01/2002 16/01/2002 17/01/2002 18/01/2002 19/01/2002 20/01/2002 21/01/2002 22/01/2002 23/01/2002 24/01/2002 25/01/2002 26/01/2002 27/01/2002 28/01/2002 29/01/2002 30/01/2002 31/01/2002 1/02/2002 2/02/2002 3/02/2002 4/02/2002 5/02/2002 6/02/2002 7/02/2002 8/02/2002 9/02/2002 10/02/2002 11/02/2002

Org

anic

Mat

ter %

Newly inundatedPermanently inundated

Site 2

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2/12/2

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anic

Mat

ter %

Site 3

0

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10

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2/12/2

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/2002

2/02/2

002

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/2002

Org

anic

Mat

ter %

Site 4

0

5

10

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2/12/2

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001

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/2001

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Comparison of the effect of two variable flow releases and three variable flow releases on biofilm biomass in permanently inundated cobble habitat.

Significant reductions in the dry weight and ash free dry weight of permanently inundated

biofilm were evident at all sites on the Mitta Mitta River of biofilm biomass by the end of

the third variable flow release (date 7) compared the end of the first variable flow release

(date 1). This occurred over a period of time when there was no significant change in dry

and ash weights at the reference site (site 5 Snowy Creek) (Table 6.1, Figures 6.1, 6.2). A

comparison of only 2 variable flow releases (dates 1 and 4) shows evidence of loss of

biomass but not consistently across all sites (Table 6.1). A significant decrease in biofilm

dry weight was found at sites 1, 2 and 4, with ash weights significantly different between

dates 1 and 4 only at sites 2 and 3 in the Mitta Mitta River. There was also a significant

difference between dates 1 and 4 at the reference site (site 5 Snowy Creek) for biofilm ash

free dry weight.

The organic matter percent of permanently inundated biofilms did not display any

consistent trends between sites or sample dates. A significant increase in organic percent

was only evident at site 1 at the end of both the second and third variable flow release

(dates 4 and 7) (Table 6.1, Figure 6.3). This trend was also apparent at Snowy Creek (site

5).

A significant reduction in biofilm Chl-a concentration on permanently inundated cobble

was found only at site 2 at the end of the third variable flow release (Table 6.1, Figure 6.4).

Conversely, the Chl-a concentration of biofilms at site 3 increased significantly following

the end of both the second and third variable flow releases.



Figure 6.4: Biofilm chlorophyll-a (Chl-a) concentrations in permanently inundated and newly inundated cobble habitats at sites 1 to 4 on the Mitta Mitta River and site 5 on Snowy Creek (reference site) on the nine sample dates from December 2001 to February 2002 (mean ± SD, n=5).

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Table 6.1: Probability value and significance levels of one-way ANOVAs comparing biofilm dry weight, ash free dry weight, organic matter percent and chlorophyll-a (Chl-a) in permanently inundated cobble habitats between sample dates 1 and 4 and between dates 1 and 7 at four sites in the Mitta Mitta River (sites 1 to 4) and in Snowy Creek (site 5). Direction of trend is shown in brackets. ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Dry weight Ash free dry weight

Date1 to 4 Date 1 to 7 Date1 to 4 Date 1 to 7

Site 1 0.001*** (1>4) 0.001*** (1>7) 0.212 0.001*** (1>7)

Site 2 0.001*** (1>4) 0.001*** (1>7) 0.001*** (1>4) 0.001*** (1>7)

Site 3 0.135 0.001*** (1>7) 0.007** (1>4) 0.001*** (1>7)

Site 4 0.047* (1>4) 0.001*** (1>7) 0.459 0.001*** (1>7)

Site 5 (ref) 0.097 0.052 0.001*** (1>4) 0.627

Organic matter % Chlorophyll-a

Date1 to 4 Date 1 to 7 Date1 to 4 Date 1 to 7

Site 1 0.008 ** (1>4) 0.001*** (1<7) 0.132 0.089

Site 2 0.153 0.225 0.421 0.011* (1>7)

Site 3 0.018* (1<4) 0.772 0.017* (1<4) 0.001*** (1<7)

Site 4 0.077 0.201 0.265 0.651

Site 5 (ref) 0.001*** (1>4) 0.008** (1>7) 0.110 0.009** (1<7)

Sample dates 2 and 3 in variable flow release 2, and dates 5 and 6 in variable flow release

3 show that many of the biofilm biomass attributes are highly variable within an individual

variable flow release. For example, there are clear reductions in the Chl-a concentrations

of biofilms at sites 1 and 2 during the flow peaks of the variable flow releases (dates 2 and

5) (Figure 6.4), indicating that algae are being scoured from cobble during peak flows but

the algal biomass is recovering quickly to pre flow peak levels.



Comparisons of biofilm biomass in permanently inundated and newly inundated cobble

habitat.

Newly inundated cobble had significantly lower biofilm dry weight at all sites only on date

2 (the peak of the second variable flow release) (Table 6.2). The dry weight of newly

inundated biofilms at site 1 continued to increase over time resulting in no significant

difference between the habitats by the last sample date (date 6)(Figure 6.1). Biofilm dry

weight at sites 2 and 3 was highly variable in both habitats resulting in similar weights on

dates 3 and 5. An increase in weight in the permanently inundated habitat and a

corresponding decrease in weight in newly inundated habitats resulted in a significantly

higher biomass in the permanently inundated habitats between dates 5 and 6. Values for

biofilm dry weight in the newly inundated habitat at site 4 were similar to the other sites,

however, significant differences between permanently inundated and newly inundated

habitats were found on all dates as a result much higher weights in the permanently

inundated habitat.

Similar to biofilm dry weight, ash free dry weights were only significantly lower in the

newly inundated habitat at all sites on the first sample date (flow peak of the second

variable flow release) (Table 6.2, Figure 6.2) The ash weight of newly inundated biofilms

at site 1 increased over time with little change in the ash weight of the permanently

inundated biofilms resulting in no significant difference between the habitats by the last

sample date (date 6). Biofilm ash weight at sites 2 and 3 were relatively stable throughout

the study period. Significant differences between the two habitats at these sites, particularly

date 6 resulted from increased in ash weights in the permanently inundated habitat.

Biofilm organic matter percent was similar between the newly and permanently inundated

habitats at sites 1 to 3 on most dates (Figure 6.3). Increased biofilm organic matter percent

in newly inundated habitats on day 8 of each variable flow release (dates 3 and 6) at site 1

resulted in significant differences between habitats on these dates (Table 6.2). Similarly,

increased biofilm organic matter percent in permanently inundated habitats on the flow

peak of each variable flow release (dates 2 and 5) at site 3 resulted in significant

differences between habitats on these dates. The most notable result was the significantly

higher biofilm organic matter percent in the newly inundated cobble at site 4 on all dates.

Biofilm Chl-a concentration was significantly lower on all dates and at all sites (except site

3, date 3) in newly inundated cobble during the CRP (Table 6.2, Figure 6.4). This suggests



that newly inundated cobble were not inundated long enough (with individual or

cumulative inundations) to accumulate the algal biomass found in the permanently

inundated habitats during the CRP.

Table 6.2: Probability value and significance levels of one-way ANOVA comparing biofilm dry weight, ash free dry weight, organic matter percent and chlorophyll-a (Chl-a) between permanently inundated (P) and newly inundated (N) cobble habitats at four sites in the Mitta Mitta River on four sample dates. Direction of trend is shown in brackets. ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Date 2 Date3 Date 5 Date 6

Dry Weight

Site 1 0.001*** (N<P) 0.001*** (N<P) 0.054 0.052

Site 2 0.001*** (N<P) 0.491 0.113 0.001*** (N<P)

Site 3 0.001*** (N<P) 0.057 0.071 0.013* (N<P)

Site 4 0.001*** (N<P) 0.001*** (N<P) 0.001*** (N<P) 0.001*** (N<P)

AFDW

Site 1 0.009** (N<P) 0.055 0.071 0.771

Site 2 0.001*** (N<P) 0.630 0.059 0.001*** (N<P)

Site 3 0.001*** (N<P) 0.001*** (N<P) 0.227 0.013* (N<P)

Site 4 0.048* (N<P) 0.573 0.021* (N<P) 0.045* (N<P)

Organic %

Site 1 0.143 0.010** (N>P) 0.498 0.014* (N>P)

Site 2 0.235 0.441 0.460 0.692

Site 3 0.041* (N<P) 0.096 0.011* (N<P) 0.336

Site 4 0.001*** (N>P) 0.001*** (N>P) 0.001*** (N>P) 0.001*** (N>P)

Chlorophyll a

Site 1 0.001*** (N<P) 0.009** (N<P) 0.001*** (N<P) 0.021* (N<P)

Site 2 0.001*** (N<P) 0.001*** (N<P) 0.001*** (N<P) 0.001*** (N<P)

Site 3 0.001*** (N<P) 0.059 0.001*** (N<P) 0.001*** (N<P)

Site 4 0.001*** (N<P) 0.004** (N<P) 0.001*** (N<P) 0.001*** (N<P)



Effect of 37 days of constant and low flows on biofilm biomass in permanently inundated cobble

The dry weight and ash free dry weight of permanently inundated biofilms were

significantly higher at all sites on the Mitta Mitta River at the end of the constant flows

(date 9) compared to the start of constant flow period (date 7) (Table 6.3). This trend was

most evident at sites 2 and 3 which showed up to a 5 fold increase in weights between

dates 7 and 9 (Figures 6.1, 6.2).

Biofilm organic matter percent on permanently inundated cobble was not significantly

different between dates 7 and 9 (Table 6.1). Date 8, midway into the period of constant

flows shows that biofilm organic matter percent is not stable throughout this period (Figure

6.2). Biofilms at all sites display a trend of reduced organic percent to day 8 and an

increase to date 9 to values similar to those on date 7. Site 5, the reference stream that

received relatively constant flows during this period, displayed the opposite trend, with

organic matter percent decreasing significantly during the same period.

The Chl-a concentration of permanently inundated biofilms increased significantly from

dates 7 to 9 only in sites 2 and 3, (Table 6.3) displaying a 5 fold increase during this

period. Similar to organic percent, biofilms at sites 1 and 4 displayed a trend of reduced

organic percent to day 8 followed by an increase to date 9 to values similar to those on date

7.

Table 6.3: Probability value and significance levels of one-way ANOVAs comparing biofilm dry weight, ash free dry weight, organic matter percent and chlorophyll-a (Chl-a) between the end of the cyclic flow release pattern (date 7) to the end of the 37 days constant flows (date 9) in permanently inundated cobble habitats at four sites in the Mitta Mitta River and in Snowy Creek. ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

DW AFDW Organic matter % Chlorophyll a

Date7 to 9 Date7 to 9 Date7 to 9 Date7 to 9

Site 1 0.043* (7<9) 0.022* (7<9) 0.061 0.261

Site 2 0.002** (7<9) 0.001*** (7<9) 0.051 0.001*** (7<9)

Site 3 0.001*** (7<9) 0.001*** (7<9) 0.092 0.001*** (7<9)

Site 4 0.007** (7<9) 0.009** (7<9) 0.088 0.519

Site 5 (ref) 0.117 0.338 0.003** (7>9) 0.068



6.3.2 Biofilm Algal Species Composition

The species richness of benthic algae within biofilms attached to cobble in the Mitta Mitta

River did not vary greatly throughout the study with no trends apparent over time or

between sites. Species richness ranged from 9 to 14 taxon for both habitats at each site

(Table 6.4). There was no obvious shift over time, or between permanently and newly

inundated habitats, in the broad divisions of algae recorded at each site with diatoms

consistently dominating the biofilm species richness. Thirty six species of algae were

found at sites 1 and 4 across the 9 sample dates, 5 species of Chlorophyta (green algae), 4

species of Cyanobacteria (blue-green algae) and 27 species of diatoms. Most species were

present at both sites 1 and 4. Spyrogyra (a filamentous green) was only recorded from site

4 and unicellular green algae were found only at site 1, and 4 species of diatoms were

restricted to only one site.

Table 6.4: Number of species recorded within each algal division in permanently (P) and newly (N) inundated habitats at sites 1 and 4 on the Mitta Mitta River for sample dates 1 to 9.

Site 1 Site 4

Sample date

Green Blue-green

Diatoms Total Green Blue-green

Diatoms Total

1 P 2 3 9 14 1 1 11 13 2 P 2 2 9 13 2 2 8 12 3 P 1 1 9 11 2 1 6 9 4 P 2 2 8 12 2 2 6 10 5 P 2 2 6 10 2 1 8 11 6 P 0 3 11 14 3 2 8 13 7 P 2 1 7 10 1 0 11 12 8 P 2 2 9 13 0 2 12 14 9 P 3 1 7 11 2 2 8 12 2 N 3 1 9 13 2 1 8 11 3 N 1 1 12 14 1 1 8 10 5 N 0 1 12 13 0 1 12 13 6 N 2 1 6 9 0 1 10 11

Comparison of the effect of two variable flow releases and three variable flow releases on biofilm species composition in permanently inundated cobble habitat. There were substantial changes in the relative biovolume of green, blue-green and diatom

algal divisions throughout the CRP at both sites (Figures 6.5, 6.6). The peak of the second



variable flow release (date 2) resulted in the removal of all blue green algae (dominated by

Planktothrix a filamentous blue-green algae, Table 6.5) at site 1 and an increase of 55% in

the relative biovolume of diatoms within the biofilm.

Table 6.5: The percentage contribution of individual algal taxon to total relative biovolume in permanently (P) and newly (N) inundated habitats at sites 1 and 4 on the Mitta Mitta River for sample dates 1 to 9.

Chlorophyta Cyanobacteria Diatoms Stigeoclonium Oedogonium Lyngbya Planktothrix Phormidium Fragillaria Achnanthidium Site 1 1 P 58.9 2.5 29.0 1.2 2 P 42.6 1.2 4.1 3 P 25.5 16.1 1.9 4 P 14.4 0.7 58.9 19.9 3.9 5 P 40.8 1.3 18.8 23.0 12.0 6 P 28.8 21.6 14.4 0.7 14.1 7 P 15.6 3.6 19.5 24.4 7.3 8 P 18.5 25.0 20.9 16.1 9 P 12.8 19.3 16.4 37.2 2.1 2 N 51.4 5.8 12.0 4.9 14.7 3 N 43.8 41.8 4.4 5 N 47.8 16.5 25.7 6 N 72.5 11.2 6.9 Site 4 1 P 97.2 1.7 2 P 67.5 13.0 1.9 3 P 84.5 4.7 3.2 4 P 18.5 17.3 27.2 5 P 89.3 2.4 3.6 3.1 6 P 16.8 3.5 25.4 7.3 22.1 7 P 42.2 18.2 8 P 39.4 48.9 1.5 0.8 9 P 3.9 38.9 56.3 2 N 22.0 38.1 6.2 3 N 53.7 9.3 11.1 5 N 36.1 7.4 20.8 6 N 85.2 5.2



Figure 6.5: Cumulative percentage of the relative biovolume of the three major algal divisions (Chlorophyta, Cyanobacteria and diatoms) recorded on permanently inundated cobble in the Mitta Mitta River at Site 1.

Figure 6.6: Cumulative percentage of the relative biovolume of the three major algal divisions (Chlorophyta, Cyanobacteria and diatoms) recorded on permanently inundated cobble in the Mitta Mitta River at Site 4.

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However, by the end of the second variable flow release the relative biovolume of green

algae was less than date 1, and the relative biovolume of blue-green algae had increased

from 31.7% to 78.8% and dominated by two filamentous species (Lyngbya and

Planktothrix). The third flow peak (date 5) again saw a reduction in the relative biovolume

of blue greens of approximately 37% and small increases in the other two divisions. At the

end of the third variable flow release (date 7) the biofilms were substantially different to

those at the end of the first variable flow release being dominated by diatoms, particularly

Fragillaria, a very large non colonial species.

The flow peak of the second variable flow release (date 2) did not result in an immediate

shift in algal divisions at site 4 (Figure 6.6), with only a slight decrease in the relative

biovolume of the filamentous branching green algae Stigeoclonium (Table 6.5). However,

by the end of the second variable flow release (date 4) the relative biovolume of green

algae had dropped by approximately 65% and the Stigeoclonium replaced completely by

Oedogonium, with diatom species dominating the biofilm. The third flow peak (date 5)

again saw an increase in the in the relative biovolume of greens (almost entirely

Stigeoclonium ) of approximately 70%. At the end of the third variable flow release (date

7) this species of filamentous algae was again substantially reduced in biovolume. Similar

to site 1, biofilms at site 4 were substantially different to those at the end of the first

variable flow release being dominated by diatoms, particularly Achnanthidium, an early

successional taxon.

Comparisons of biofilm species composition in permanently inundated and newly inundated cobble habitats.

The wetting of cobble banks as a result of the CRP in the Mitta Mitta River created biofilm

assemblages in newly inundated habitats that were consistently different at the division and

species levels to the biofilms on permanently inundated cobble (Figures 6.7, 6.8). The

relative biovolume of major algal divisions on newly inundated cobble at site 1 are

relatively similar on each date, and therefore differences between habitats are driven by

changes in the permanently inundated biofilms. Biofilms at site 1 in both habitats are most

similar on dates 3 and 5, but an absence of blue-green algae on date 2 and green algae on

date 6 in the permanently inundated habitat result in very different biofilm assemblages

between habitats on these dates. Green algae, specifically Stigeoclonium, dominate the



newly inundated biofilms on each date ranging from 43.8% to 73.2% of the total

biovolume. An increase in the relative biovolume of Achnanthidium, an early successional

taxon on dates coinciding with peak flows (dates 2 and 5) has resulted in an increase in the

overall relative biovolume of diatoms on these dates. Differences in the species

contributing to the relative biovolume of blue-green algae between habitats is only evident

on the last date as the permanently inundated biofilms have 3 species of blue-green algae

compared to only 1 species, Lyngbya (a filamentous species), present in the newly

inundated biofilms.

Unlike site 1, differences between habitats in the relative biovolume of the major algal

divisions at site 4 are driven by changes in both permanently and newly inundated biofilms

over time. Newly inundated biofilms have substantially higher biovolumes of diatoms,

dominated by Achnanthidium, than permanently inundated habitats on dates 2, 3 and 5. On

date 6 (day 8 of the third variable flow release), permanently inundated biofilms have a

substantially higher biovolume of Achnanthidium than newly inundated biofilms.

Permanently inundated biofilms consistently have a higher percentage of green algae

(Stigeoclonium) than newly inundated biofilms. On dates 5 and 6 newly inundated biofilms

have no green algae recorded, dominated by 70% diatoms on date 5 and 85% blue greens

(solely Lyngbya) on date 6.



Figure 6.7: Cumulative percentage of the relative biovolume of the three major algal divisions (Chlorophyta, Cyanobacteria and diatoms) recorded on permanently (P) and newly (N) inundated cobble in the Mitta Mitta River at Site 1.

Figure 6.8: Cumulative percentage of the relative biovolume of the three major algal divisions (Chlorophyta, Cyanobacteria and diatoms) recorded on permanently (P) and newly (N) inundated cobble in the Mitta Mitta River at Site 4.

Site 1

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Effect of 37 days of constant and low flows on biofilm species composition in permanently inundated cobble

The extended period of constant flows at the end of the CRP resulted in only a minor

change in algal divisions but larger changes in dominant taxa at site 1. The relative

biovolume of green algae increases by approximately 13% due to an increase in the

biovolume of Oedogonium, a late successional filamentous green algae. Diatom species

(particularly Achnanthidium and Fragillaria) dominate the biofilm biovolume on dates 7

and 9 at approximately 62% and 52% respectively. This dominance is reduced on date 8 by

an increase in the biovolume of blue-green algae, which constitute 46% of the total

biovolume on that date.

The shift in algal composition of biofilms is more dramatic at site 4 where the biofilm

shifts from one dominated by diatoms (57% mainly Achnanthidium and Fragillaria) and

green algae (43% entirely Oedogonium) on date 7 to one with 88% on date 8 and 96% blue

green algae on date 9.



6.3.3 Biofilm metabolism

Comparison of the effect of two variable flow releases and three variable flow releases on

biofilm net productivity in permanently inundated cobble habitat.

The net primary productivity of permanently inundated biofilms displayed enormous

variability during the CRP, ranging from 12.82 mgO2/m2/h at site 1 on date 1, to 3.69

mgO2/m2/h at site 1 on date 5 (Figure 6.9). The rates of net productivity of biofilms were

high and similar at both sites following the end of the first variable flow release. Net

primary productivity was reduced by up to one third at both sites to approximately 4

mgO2/m2/h during the peak of the second variable flow release. Response trajectories at

sites 1 and 4 differed after this point. Biofilm metabolism at site 1 increased rapidly to date

4 (end of the second variable flow release), returning to rates similar to those at the end of

the first variable flow release. Conversely, the biofilm metabolism at site 4 increased by

three-fold to date 3 and then fell to approximately 6 mgO2/m2/h by the end of the second

variable flow release. The second variable flow release resulted in less marked changes to

biofilm net productivity with rates on dates 5 and 7 relatively similar at approximately 6

mgO2/m2/h. Rates of biofilm net productivity were substantially lower at the end of the

CRP than at the end of the first variable flow release. Water column net productivity varied

little throughout the CRP consistently less than 2 mgO2/m2/h. Slight increases were noted

during the peaks of each variable flow release.

Comparisons of biofilm net productivity in permanently inundated and newly inundated

cobble habitat.

The net productivity of newly inundated cobble was substantially lower than permanently

inundated cobble at both sites for the first three dates. Newly inundated cobble that had not

been inundated for approximately 14 days (since the previous flow peak) on dates 2 and 5

were net consumers of oxygen. Newly inundated biofilms at site 1 was strongly

heterotrophic with a net productivity of approximately minus 2.5 to 3 mgO2/m2/h. Newly

inundated biofilms at site 4 were less heterotrophic consuming approximately 0.7

mgO2/m2/h on date 2 and displaying low levels of positive net productivity during the third

flow peak. Biofilms on newly inundated cobble that had been inundated for 8 days on date



3 were net producers of oxygen but still has much lower rates of productivity than biofilms

on permanently inundated cobble. However, by date 6 (8 days after the third flow peak) net

productivity in the newly inundated cobble was slightly higher than from biofilms that

were permanently inundated. This suggests that cumulative wetting and/or drying periods

stimulate biofilm productivity.

Effect of 37 days of constant flows on biofilm net productivity in permanently inundated

cobble

The extended period of constant flows at the end of the CRP resulted in a rapid decline in

biofilm productivity at both sites. After 37 days constant flows, biofilm net productivity at

site 1 was only slightly higher than in the water column (approximately 2 mgO2/m2/h) and

negative at site 4.

Figure 6.9: Biofilm and water column net primary productivity (mgO2/m2/h) from newly inundated and permanently inundated habitats at sites 1 and 4 on the Mitta Mitta River.

-4

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newly inundated site 1

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permanently inundated site 1

permanently inundated site4

water column



6.4 Discussion.

This study examined the effects of the CRP from Dartmouth Dam to the Mitta Mitta River

on the biomass, species composition and productivity of biofilms attached to cobble

substrata. The implementation of this release pattern resulted in substantial changes to the

structure and function of biofilms through scouring and sloughing of biofilms and the

creation of newly inundated cobble areas through variable discharge volumes. However,

many of the changes associated with the CRP were short lived, with the period of constant

flows following the variable releases resulting in biofilms with a high biomass, a very low

net productivity and dominated by few species of blue-green algae.

The growth and prosperity of biofilms in river systems has been shown to be controlled by

complex interactions between hydrological, water quality and biotic variables including

factors such as flow disturbance, resource supply and grazer control (Biggs 1996).

Resources such as light, relatively low concentrations of organic and inorganic nutrients,

availability of cobble substrata and general water quality were similar across all sites in the

Mitta Mitta River. This suggests that responses of biofilm attributes during and after the

CRP are predominantly the result of changes in either flow variables such as velocity and

variability in water level heights or altered rates of biofilm herbivory (which may also be

flow mediated).

Biofilms were found to respond very rapidly to changes in flow regime. Structural and

functional responses in biofilms were evident immediately following each peak flow, as

well integrating responses to flow regime over longer time periods. The recovery of algal

communities following disturbance can vary from days to months depending on the

duration and frequency of the disturbance (Dodds et al. 1996; Benenati et al. 1998; Biggs

et al. 1999), which may explain the occurrence of significant results across the range of

temporal scales for different biofilm attributes. Biofilms also displayed structural and

functional responses to the wide variety of flow conditions experienced in the Mitta Mitta

River during the study. This finding is consistent with the results of Watts et al. (2001),

who found that there were highly predictable associations between biofilm attributes such

as biomass and net productivity and hydrological variables at both short (up to 10 days)

and long (annual) temporal scales.



The total, organic and algal biomass of biofilms and their rates of net productivity in the

Mitta Mitta River are within the range of those found in other regulated south-east

Australian upland streams (see review in Burns & Ryder 2001b). Similarly, the low levels

of these attributes recorded from the unregulated reference river, Snowy Creek, were

similar to those from reference rivers in the headwaters of the Murrumbidgee catchment

used by Watts et al (2001). The stark differences between the biofilms in Snowy Creek and

the Mitta Mitta River highlight the impact of flow regulation on instream biofilm

communities.

Comparison of the effect of two variable flow releases and three variable flow releases on

biofilm attributes in permanently inundated cobble habitat.

The sloughing or scouring of biofilms is an important process in regulating sediment

accumulation and resetting biofilm structure (Peterson 1996; Mosisch & Bunn 1997) and

function (Bunn et al. 1999). The release of three successive flow peaks as part of the CRP

resulted in the significant reduction of biofilm dry weight and organic biomass at all sites

in the Mitta Mitta River when compared to biofilms developed under prolonged and

constant low flow conditions. Results from two successive variable flow releases were not

as conclusive. This suggests that the magnitude and shape of the three successive variable

releases were sufficient to ‘reset’ permanently inundated benthic algal communities

through mechanisms such as scouring along the entire study reach. Determining the effects

of individual and/or successive variable flow releases is difficult as data were not obtained

for the period prior to or during the first release.

The relative biovolume and species composition of permanently inundated biofilms

changed rapidly, while species richness remained static throughout the CRP. Significant

losses of biomass as a result of three successive variable flow releases may be due to shifts

in algal communities from ones dominated by filamentous green and blue-green taxa on

sample date 1 to biofilms dominated by diatoms at the end of the flow peak of variable

flow releases at both sites. This suggests the cumulative effect of three releases is to favour

early successional algal species, particularly evident by the dominance of early

successional diaton species such as Fragillaria sp. and Achnanthidium sp. at sites 1 and 4.

The rapid changes in the dominance of different algal divisions associated with changes in



discharge magnitude and variability during the releases may favour a diversity of primary

consumers in the Mitta Mitta River.

Similar to changes in taxonomic structure, the net productivity of permanently inundated

biofilms varied substantially during the CRP, as the balance between autotrophy and

heterotrophy within a biofilm is often determined by physical disturbances (Peterson

1996). The most obvious trend is the reduction in net productivity following the peak flows

of each release. This reduction in net productivity occurs concurrently with a 250 fold

increases in heterotrophic activity in the water column (section 3). We hypothesise that the

peak flows of 4800 ML/day are sufficient to result in the physical abrasion and damage of

algal cells along the entire study reach leading to an increase in heterotrophic microbial

productivity. This depression in net productivity is short lived, with rates of oxygen

production rising rapidly by the end of the second variable flow release. Scouring events

have been shown to favour the exponential growth phase of filamentous algal biofilms

attached to cobble (Uehlinger et al. 1996; Biggs & Stokseth 1996) which can promote high

levels of biofilm productivity through rapid increases in biomass (Ryder in press). This is

evident particularly at site 1, where a reduction in filamentous algal species by

approximately 50% immediately following the flow peak returns to a biofilm comprising

over 90% filamentous green and blue green algae. Similarly, peaks and troughs of biofilm

productivity in permanently inundated biofilms follow trends in the relative biovolume of

Stigeoclonium, filamentous green algae.

Comparisons of biofilm attributes in permanently inundated and newly inundated cobble

habitat.

An increase in the diversity of biofilm structure and function in the Mitta Mitta River by

creating habitats of newly inundated cobble was evident during the CRP. The wetting and

drying of biofilms has been shown to significantly alter their structure and function (e.g.

Fisher et al. 1982; Jowett and Duncan 1990, Άcs and Kiss 1993, Benenati et al. 1998,

Burns and Walker 2000, Ryder in press). Newly inundated cobble was generally lower in

total and organic biomass and consistently lower in Chl-a concentrations. The taxonomic

composition was similar between newly and permanently inundated biofilms, however the

relative dominance of different taxa was different between habitats. However, there were

no consistent trends in biofilm structural or composition attributes over time to suggest that



there were cumulative ecological benefits of three over two peak variable flow releases for

newly inundated biofilms.

However, the net productivity of newly inundated biofilms showed a trend of increasing

productivity between day 1 and 8 of each variable flow release, and between 2 and 3

variable flow releases. Biofilm net productivity was negative on the first day of inundation

associated with the flow peaks of variable flow release 2 and 3. Rewetting of biofilms

following desiccation has been shown to enhance rates of respiration in many systems as it

can respond more rapidly to rewetting than production (Sabater and Romani 1996, Romani

and Sabater 1997). However by day 8 of each variable flow release the net productivity

had increased substantially indicating the rapid response of biofilm metabolism to

rewetting. By day 8 of the third variable flow release, net productivity in the newly

inundated biofilms were strongly autotrophic and similar to rates in the permanently

inundated biofilms even though newly inundated biofilms had significantly lower Chl-a

concentrations. Differential responses by algal taxa to survive desiccation may be

responsible for increased productivity in these biofilms, with Chlorophytes and

Cyanobacteria showing the greatest resistance to desiccation (Davis 1972). This is

consistent with Ryder (in press) who found biofilm net productivity was enhanced by

wetting and drying cycles that promoted desiccation tolerant algal taxa. The recovery of

biofilms following desiccation can vary from days to months depending on the duration,

frequency and the speed of the drying process (Dodds et al. 1996, Benenati et al. 1998,

Biggs et al. 1999). It may therefore be possible to enhance the productivity of biofilms

through regulating the rates of rises and falls in water levels in the Mitta Mitta River.

However the slow drying of biofilms is necessary for surviving desiccation (Davis 1972)

and increasing their recovery potential (Robson 2000). Rapid rises and falls in water levels

(over days) in the Mitta Mitta River may be detrimental to biofilm productivity by

preventing the establishment of desiccation tolerant algal taxa (Ryder in press).

Effect of 37 days of constant flows on biofilm attributes in permanently inundated cobble

Under conditions of low to moderate disturbance, accrual processes dominate biofilm

development, especially where resources are not limited (Biggs 1996). This is evident at all

sites in the Mitta Mitta River where significant increases in total and organic biomass

occur at all sites during the period of lower and constant flows. These changes were not



evident in the reference river. This period also led to substantial changes in the taxonomic

composition and relative biovolume of major algal divisions at site 4. The filamentous

green algae Stigeoclonium, which requires substantial water velocities to support its

branching form was completely replaced by Oedogonium, a late successional taxon that

thrives in low velocity habitats (Burns & Walker 2000). More dramatic was the change to a

biofilm with 2 species of blue-green algae comprising over 95% of the biovolume by the

last sample date at site 4. These changes in biofilm composition may be directly related to

the flow velocity being insufficient to disturb the ‘boundary layer’ of algal cells. The

disturbance of the outer layers of algal cells within biofilms allows the transfer of organic

and inorganic nutrients and matter in and out of the biofilm (Peterson 1996). The two

species of blue-green algae, which dominate site 4 are capable of internally fixing their

own nitrogen, and are therefore not as reliant on external supplies of nutrients as other

taxa, and may have led to their complete dominance of the biofilm assemblage.

The period of lower and constant flows at the end of the CRP also led to a rapid decrease

in net productivity to a point where biofilms at site 4 were net consumers of oxygen. The

change in flow conditions from high flows that promoted large assemblages of filamentous

algae such as Stigeoclonium, Lyngbya and Planktothrix to low velocity and constant flows

over a relatively rapid period resulted in the death of large proportions of these algae.

These algae formed dense mats of rotting organic matter that coated the cobble and

remained attached to cobble due to persistent low velocity conditions. This material

provides a large resource to benthic heterotrophic bacteria which increases their activity

and biomass and reduces net biofilm productivity. Once biofilm communities have reached

this state, major changes to their structure and function may only be achieved by increases

in flow magnitude or variability.

6.5 Summary of Findings

• The implementation of the CRP resulted in substantial changes to the structure and

function of biofilms through changes in water velocity and the creation of newly

inundated cobble areas through variable discharge volumes. However, many of these

changes associated with the CRP were short lived, with the period of constant flows



following the variable releases resulting in biofilms with a high biomass, a very low net

productivity and dominated by few species of algae.

• Following the release of three successive variable flow releases there was a reduction

in biofilm dry and organic weight and a shift towards biofilm communities dominated

by early successional algal species at all sites in the Mitta Mitta River. This suggests

that the magnitude and shape of the three successive variable releases were sufficient to

scour algae and sediment from the permanently inundated cobble and reset biofilm

development along the entire study reach.

• A substantial and rapid change in net productivity of biofilms was evident as a result of

the CRP, providing a diversity of productivity rates along the entire length of the study.

Flow peaks caused a reduction in net productivity from increased heterotrophic

microbial activity associated with scouring of algal cells. This depression in net

productivity is short lived, with rates of oxygen production rising rapidly by the end of

the second variable flow release.

• A diversity in the structure and function of biofilms in The Mitta Mitta River was

evident as a result of the CRP by creating habitats of newly inundated cobble. Newly

inundated cobble was generally lower in total and organic biomass and Chl-a

concentrations, and altered the relative dominance of algal species compared to

permanently inundated cobble. The net productivity of newly inundated biofilms

showed a trend of increasing productivity between day 1 and 8 of each variable flow

release, and between 2 and 3 releases suggesting cumulative effects of successive flows

as part of the CRP.

• All sites in the Mitta Mitta River displayed significant increases in total and organic

biomass occur during the period of lower and constant flows. These changes were not

evident in the reference river. This period also led to changes in the taxonomic

composition and relative biovolume of major algal divisions at site 4, and a rapid

decrease in net productivity to a point where biofilms at site 4 were net consumers of

oxygen. Once biofilm communities have reached this state, major changes to their

structure and function may only be achieved by increases in flow magnitude or

variability.



7.0 MACROINVERTEBRATES

7.1 Introduction

Freshwater macroinvertebrates are non-vertebrate animals (eg. insects, crustaceans, snails

and worms) that are visible to the naked eye and live at least part of their life within a body

of freshwater. Macroinvertebrates have been used in biological monitoring programs

worldwide as many taxa respond to changes in environmental conditions. In addition,

many species live for months or years, so they can integrate the impacts on the ecosystem

over an extended period of time, rather than just at the time of sampling.

Several macroinvertebrate indices have been developed to assess ecosystem health. Indices

such as the British River Invertebrate Prediction and Classification Scheme

(RIVPACS)(Wright 1995) and the Australian River Assessment System

(AusRivAS)(Simpson et al. 1997) compare the macroinvertebrate community structure at a

test site with that at reference sites. In these indices, poorer environmental conditions are

usually associated with a loss of taxa. Macroinvertebrate indices have also been developed

to monitor the ecological response to specific environmental stressors, such as pollution.

Examples of these types of biotic indices include the Biological Monitoring Working Party

Score (BMWP)(Chesters 1980, cited in Mason 1991) and the SIGNAL biotic index of

Chessman (1995). These indices are calculated from scores that have been assigned to

macroinvertebrate families, depending on their susceptibility to disturbance and pollution.

Higher levels of disturbance or pollution are generally associated with lower BMWP or

SIGNAL scores.

Macroinvertebrate indices have also been used to assess the impact of river regulation on

aquatic ecosystems. Many studies have shown there are differences in macroinvertebrate

species richness and community composition between regulated and unregulated river

reaches (eg. Brittain & Saltveit 1989; Richter et al. 1995). Other studies have observed a

reduction in the diversity of macroinvertebrates following construction of an impoundment

(eg. Armitage 1984; Doeg 1984; Boon 1988; Pardo et al. 1998).

Recently there has been a move towards reinstating elements of natural flow regimes in

regulated rivers as a means of river restoration. However, there is a considerable gap in



knowledge as to how biological indicators will respond to the changed flow conditions

(Watts & Ryder 2001). The highly predictable associations between macroinvertebrate

attributes and hydrological variables reported by Watts et al (2001) suggests that

macroinvertebrates can be used to provide a reliable assessment of the ecological impact of

changed flow management at relatively short temporal scales. Results from studies that

have compared macroinvertebrate indices between regulated and unregulated rivers (eg.

Brittain & Saltveit 1989; Richter et al. 1995) can be used to make predictions of the

changes expected following changed flow management.

The aim of the current project is to examine the response of macroinvertebrates to the CRP

in the Mitta Mitta River, Victoria. The ecological condition of the Mitta Mitta River is

expected to improve in response to the CRP. The following hypotheses will be examined

by this study:

1. There will be an increase in the diversity of macroinvertebrate families in the

Mitta Mitta River following the CRP relative to the diversity of macroinvertebrate families in the reference reach Snowy Creek.

2. There will be an increase in the abundance of macroinvertebrate individuals in

the Mitta Mitta River following the CRP relative to the abundance of macroinvertebrate individuals in the reference reach Snowy Creek.

3. There will be an increase in the SIGNAL scores in the Mitta Mitta River following

the CRP relative to any changes in the SIGNAL score in the reference reach Snowy Creek

7.2 Methods

7.2.1 Field methods-Cobble habitats

Macroinvertebrates in cobble habitats were sampled quantitatively using a surber sampler

comprising a rectangular quadrat (20cm by 20cm) to delineate the area of bed to be

sampled and a net (250 µm mesh) into which the disturbed benthic invertebrates are swept

by the current. The sampler was placed on the benthos facing upstream and the substrate

within the quadrat was thoroughly disturbed. Following sampling, net contents were

emptied into a labelled sample jar and were preserved in 70% alcohol. Macroinvertebrates

from permanently inundated cobble banks were sampled on nine sampling dates (Table

2.2) from four sites in the Mitta Mitta River and one site on Snowy Creek (Figure 2.1).



Macroinvertebrates from newly inundated cobble banks on cobble banks were sampled on

four sampling dates (2, 3, 5 and 6 in Table 2.2) from only the four sites in the Mitta Mitta

River (Figure 2.1). Four replicates were collected from each site on each sampling date.

7.2.2 Field methods-Littoral habitats

Macroinvertebrates in littoral areas at each site were sampled qualitatively using a sweep

net. Sampling involved vigorously sweeping the net through the water column around

overhanging vegetation and snags. Following sampling, net contents were emptied into a

labelled sample jar and were preserved in 70% alcohol.

7.2.3 Laboratory methods

All invertebrates were removed from each sample using a dissecting microscope and were

stored in 70% ethanol. Fauna from the surber samples were counted and identified to

family level. Fauna from qualitative sweep samples were identified to family level.

7.2.4 Data Manipulation and Analyses

The raw data were used to calculate the following attributes for each habitat (permanently

inundated cobble, newly inundated cobble) at each site on each sampling date:

• Mean and standard error of number of Families;

• Mean and standard error of number of individuals;

• SIGNAL scores based on Chessman (1995)

The mean number of families and mean number of individuals were compared between

dates within each site using single-factor ANOVA. The following planned comparisons

were undertaken:

1. Date 1 (end of first variable flow release) to date 4 (end of second variable flow release) and date 1 to date 7 (end of third variable flow release) for each of the five sites to examine the individual and cumulative effects of the second and third variable flow releases.

2. Permanently inundated cobble to newly inundated cobble habitat for sites 1 to 4 in the

Mitta River for each of the sample date 2 (flow peak of second variable flow release),



date 3 (day 8 of second variable flow release), date 5 (flow peak second of flow release) and date 6 (day 8 of third variable flow release).

3. Date 7 (end of flow release 3 and start of constant flows) to date 9 (end of constant

flow period) to examine the effect of 37 days of constant flows.

Macroinvertebrate community composition was analysed using non-metric

multidimensional scaling (NMDS), analysis of similarities (ANOSIM) and species

contributions to similarities (SIMPER) analyses available in the software package

PRIMER (Clarke and Warwick 1994). The Bray-Curtis distance measure was used to

generate the similarity matrix using square root transformed data. NMDS is a method of

summarising multivariate species data where the similarity between sites is represented

graphically in an ordination. The more similar the sites are in species composition, the

closer they will group together within the ordination. The ANOSIM routine in Primer

(Clarke and Warwick 1994) was used to carry the same three a priori comparisons as used

in the ANOVA. ANOSIM analysis computes an R statistic that reflects the differences

between treatments. Clarke and Gorley (2001) advise the following levels of R

interpretation:

R = 1 complete separation of treatments

R > 0.75 treatments are well separated

R > 0.5 treatments may be overlapping but are still clearly different

R < 0.25 treatments are barely separable

The SIMPER routine was used to identify the families that contributed to the significant

differences between groups identified by the ANOSIM analysis.

7.3 Results

7.3.1 Overview of macroinvertebrate data

There were a total of 53 families of macroinvertebrates collected in the cobble habitat

during this study. Twelve families were in the order Coleoptera, ten families in the order

Trichoptera (caddis flies), eight families in the order Diptera (two winged flies), four

families in the order Ephemeroptera (mayflies) and three families in the order Plecoptera

(caddis flies).



In general, site one had fewer macroinvertebrate families than all of the other sites (Figure

7.1). The mean number of families in the permanently cobble habitat at site one increased

by 175% between dates one and nine, whereas other sites showed relatively smaller

increases (eg site 2) or very little change at all (eg. site 3). The mean number of families in

the newly inundated cobble was generally lower than that in the permanently inundated

cobble at the beginning of the second variable flow release, but was similar to that in the

permanently inundated cobble by date 6 (Figure 7.1)

The numbers of macroinvertebrate families in littoral habitats varied considerably between

sample dates at all sites, including the reference site (Figure 7.2). This suggests that factors

other than flow may influence this parameter and that the qualitative sweep method used to

sample the littoral habitats may not be as reliable for the assessment of CRP as the

quantitative surber sample method used in the cobble habitat.

There was considerable variation in the number of macroinvertebrate individuals in the

cobble habitat on different sampling dates. During the 37 days of constant flows following

the CRP there was an increase in the number of individuals at sites two and three (Figure

7.3).

The SIGNAL scores for the macroinvertebrate assemblage in the cobble habitat were

lowest at site one (Figure 7.4). The SIGNAL score at site one increased from 4.00 on

sample date one to 6.18 on date nine. There was not a similar increase in SIGNAL scores

at the other sites. The three sites in the Mitta Mitta River had scores that fluctuated around

6.0 (range 5.3 to 6.6) and Snowy Creek generally had a slightly higher SIGNAL score

ranged from 6.3 and 7.15.

In general, the macroinvertebrate community assemblage in the newly inundated cobble

habitat was different to that in the permanently inundated cobble on sampling dates 2 and 3

(Figure 7.5). The macroinvertebrate assemblage in newly inundated cobble tended to

become more similar to that in the permanently inundated cobble by date 6 (day 8 of the

second variable flow release) (Figure 7.5). The two-dimensional ordinations (Figure 7.5)

all had low stress values (range 0.06 to 0.14) which suggests that these ordinations reliably

summarise the relationships between sample dates.



Figure 7.1: Number of macroinvertebrate families in permanently inundated and newly inundated

cobble habitat at four sites in the Mitta Mitta River (sites 1-4) and one site in Snowy Creek (site 5).

(mean ± SE, n = 4).

Site 1

0

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Site 5

02468

101214

2/12/01 16/12/01 30/12/01 13/01/02 27/01/02



Figure 7.2: Number of macroinvertebrate families in littoral habitat at four sites in the Mitta Mitta

River (sites 1-4) and one site in Snowy Creek (site 5).

Site 4

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Figure 7.3: Number of macroinvertebrate individuals in permanently inundated and newly inundated cobble habitat at four sites in the Mitta Mitta River (sites 1-4) and one site in Snowy Creek (site 5). (mean ± SE, n = 4)

Site 2

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Figure 7.4: SIGNAL scores in permanently inundated and newly inundated cobble habitat at four sites in the Mitta Mitta River (sites 1-4) and one site in Snowy Creek (site 5).

Site 1

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Site 2

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Figure 7.5: NMDS ordination of macroinvertebrate assemblage from permanently inundated (P)

and newly inundated (N) cobble in the Mitta Mitta River (sites 1-4) and Snowy Creek (site 5) over

nine sampling dates. For details of sampling dates see Table 1.2.

1P

2P

2N

3P

3N4P

5P

5N

6P6N

7P

8P

9P

Stress: 0.06

1P

2P

2N3P 3N

4P5P

5N

6P6N

7P

8P

9P

Stress: 0.07

1P

2P

2N

3P

3N

4P

5P 5N

6P

6N

7P8P

9P

Stress: 0.1

1P

2P

2N3P3N4P5P5N6P6N7P8P9P

Stress: 0.01

1P3P

3N4P5P

5N

6P

6N

7P

8P

9P

Stress: 0.14

1P 2P

3P

4P

5P6P

7P

8P

9P

Stress: 0.11

Site 1

Site 2

Site 3

Site 4

Site 5



Comparison of the effect of two variable flows and three variable flows on

macroinvertebrates in permanently inundated cobble habitat.

There was no significant increase in the number of macroinvertebrate families in the

permanently inundated cobble at any of the sites between the beginning and the end of the

second variable flow release (date 1 and 4)(Table 7.1, Figure 7.1). However, by the end of

the third variable flow release (date 7) there was a significant increase in the number of

families at site one. This occurred over a period of time when there was no change in the

number of families at the reference site (site 5 Snowy Creek)(Table 7.1, Figure 7.1). There

was no significant change in the number of individuals at all permanently inundated cobble

sites between the beginning and the end of the second variable flow release (date 1 and

4)(Table 7.2, Figure 7.3). There was a significant decrease in the number of individuals at

site 3 by the end of the third variable flow release (date 7).

The results of the ANOSIM analyses (Table 7.3) show that there were no significant

changes in the composition of the macroinvertebrate community in the reference site

Snowy Creek during the period when the CRP was conducted in the Mitta Mitta River.

However, there were differences in community composition in the permanently inundated

cobble habitat at site four by the end of the second variable flow release (date 4) and at site

one, three and four by the end of the third variable flow release (date 7)(Table 7.3). The

NMDS ordinations (Figure 7.5) demonstrate graphically the changes occurring in the

macroinvertebrate community over time. These ordinations show that at site one, three and

four the macroinvertebrate community in the permanently inundated cobble at the

beginning of the second variable flow (date 1P) is quite different to that at the end of the

CRP (date 7P).

Table 7.4 lists the families that made the greatest contribution to the difference in

community composition between sampling dates. At sites one, three and four in the Mitta

Mitta River between sample date one and seven there was a decrease in the abundance of

Chironomidae and increase in the abundance of Oligochaeta (Table 7.4). At site one there

was an increase in the abundance of the families Caenidae, Coloburiscidae and

Griptopterygidae by date 7, resulting in a 45% increase in SIGNAL score over this period

(SIGNAL score increased from 4.00 to 5.83)(Table 7.5). Whereas, at site three and four

there were decreases in the families Caenidae, Leptophlebidae and Gripopterygidae, but



increases in other families such as Coloburiscidae at site 3 and Glossosomatidae at site 4

(Table 7.4). Consequently, there was little change in the SIGNAL score at sites two, three

and four between dates one and seven (Table 7.5). Over the same period that there was an

increase in SIGNAL score at site one there was a decrease in SIGNAL score in the

reference site Snowy Creek (Table 7.5).

Table 7.1: Probability value and significance levels of one-way ANOVA’s comparing number of macroinvertebrate families in permanently inundated cobble habitats between sample dates 1 and 4 and between dates 1 and 7 at four sites in the Mitta Mitta River (sites 1 to 4) and in Snowy Creek (site 5). Direction of trend is shown in brackets. ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Site Two variable flows

(date 1 to date 4) Three variable flows (date 1 to date 7)

1. Mitta Mitta 0.315 ns 0.000 *** (1<7) 2. Mitta Mitta 0.031 * (1>4) 0.620 ns 3. Mitta Mitta 0.293 ns 0.293 ns 4. Mitta Mitta 0.560 ns 0.138 ns 5. Snowy Creek 0.176 ns 0.437 ns

Table 7.2: Probability value and significance levels of one-way ANOVA’s comparing number of macroinvertebrate individuals in permanently inundated cobble habitats between sample dates 1 and 4 and between dates 1 and 7 at four sites in the Mitta Mitta River and in Snowy Creek. Direction of trend in shown in brackets. ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001.



1. Mitta Mitta 0.857 ns 0.285 ns 2. Mitta Mitta 0.411 ns 0.144 ns 3. Mitta Mitta 0.126 ns 0.009 ** (1>7) 4. Mitta Mitta 0.082 ns 0.390 ns 5. Snowy Creek 0.318 ns 0.535 ns

Table 7.3: R values and significance levels of ANOSIM analyses comparing macroinvertebrate faunal assemblages on permanently inundated cobble between sampling dates at five sites in the Mitta Mitta River. ns = not significant and *= P<0.05.



1. Mitta Mitta -0.063 ns 0.594 * 2. Mitta Mitta -0.052 ns 0.063 ns 3. Mitta Mitta 0.354 ns 0.719 * 4. Mitta Mitta 0.869 * 0.979 * 5. Snowy Creek 0.313 ns 0.583 ns



Table 7.4: Abundance of macroinvertebrate families that contributed significantly to the SIMPER analyses comparing permanently inundated cobble habitat between dates 1 and 4, and 1 and 7. The % contribution to the SIMPER analyses is shown for each comparison. Comparisons shown to be significant by the ANOSIM analyses are highlighted.

Average abundance of

macroinvertebrate Families on each sample date

% contribution to SIMPER

Family Date 1 Date 4 Date 7 Date 1 vs 4 Date 1 vs 7 Site 1 Chironomidae 25.0 8.2 7.0 37.3 17.8 Oligochaeta 3.8 9.2 34.0 20.1 24.2 Caenidae 1.8 2.0 2.5 15.9 8.7 Coloburiscidae 0 0 2.5 0 9.5 Griptopterygidae 0 1.5 5.2 11.8 17.9 Site 2 Chironomidae 8.0 8.0 2.0 40.6 33.6 Oligochaeta 1.2 1.2 2.2 8.1 11.6 Caenidae 6.0 2.0 2.0 21.9 22.8 Leptophlebiidae 3.0 2.5 2.2 13.0 8.9 Site 3 Chironomidae 48.2 14.5 1.0 58.6 64.0 Oligochaeta 0.5 2.0 1.2 3.0 2.1 Caenidae 7.2 3.8 0.2 6.8 9.1 Coloburiscidae 0.2 1.5 2.8 2.6 3.4 Griptopterygidae 2.5 3.0 1.2 3.7 2.2 Leptophlebiidae 5.5 6.5 4.0 7.8 6.8 Site 4 Chironomidae 21.0 33.8 6.0 37.5 26.9 Oligochaeta 1.8 6.8 13.0 15.0 19.8 Caenidae 1.8 0.5 0.2 3.3 2.8 Coloburiscidae 2.2 0 0.8 6.4 4.0 Glossosomatidae 0 2.0 11.0 5.7 19.0 Leptophlebiidae 6.0 0.5 2.2 15.9 7.8 Griptopterygidae 1.5 3.0 0.8 4.6 2.1 Site 5 Chironomidae 17.0 17.5 16.0 16.5 15.7 Coloburiscidae 0 1.0 1.2 4.2 7.2 Leptophlebiidae 6.5 8.8 10.8 5.7 6.6 Glossosomatidae 1.2 1.5 0 5.6 4.9 Baetidae 2.0 2.2 11.0 4.8 11.6 Gripopterygidae 1.8 2.2 0.2 4.4 6.7

Table 7.5: SIGNAL scores for macroinvertebrate samples from four sites in the Mitta Mitta River and in Snowy Creek for sample dates 1, 4 and 7.

Site Date 1 Date 4 Date 7 1. Mitta Mitta 4.00 4.71 5.83 2. Mitta Mitta 6.60 6.00 6.56 3. Mitta Mitta 6.20 6.31 6.00 4. Mitta Mitta 6.00 6.08 6.14 5. Snowy Creek 7.00 6.63 6.35



Comparison of macroinvertebrates in permanently inundated and newly inundated cobble

habitat

On the peak of the second variable flow release (date 2) there were significantly fewer

macroinvertebrate families in the newly inundated cobble habitat than in the permanently

inundated cobble habitat at three of the four sites in the Mitta Mitta River (Table 7.6,

Figure 7.1). However, by the eighth day of the third variable flow release (date 6) there

were a similar number of families in the newly inundated and permanently inundated

habitat at three of the four sites (Table 7.6, Figure 7.1).

On the peak of the second variable flow release (date 2) there were significantly fewer

individuals in the newly wetted cobble habitat than in the permanently inundated cobble

habitat at all of the four sites in the Mitta Mitta River. However, by the eighth day of the

third variable flow release (date 6) at all sites there was no difference in the number of

individuals in the newly inundated and permanently inundated cobble habitat (Table 7.7,

Figure 7.3).

The community composition of macroinvertebrates in the newly inundated cobble habitat

was, in general, different to that in the permanently inundated cobble habitat at all of the

four sites in the Mitta Mitta River on dates 2, 3 and 5 (Table 7.8, Figure 7.5). By the eighth

day of the third variable flow release (date 6) at all sites there was no significant difference

in the community composition between habitats (Table 7.8, Figure 7.5). With only one

exception (site 4, date 3), SIGNAL scores were the same or higher in the permanently

inundated cobble than in the newly inundated cobble on these dates (Table 7.9, Figure 7.4).

Several macroinvertebrate familes increased their abundance in the newly inundated

cobble habitat during the CRP. At site one there were several families (eg. Caenidae and

Coloburiscidae) absent from the newly inundated cobble on dates two, four and five that

had colonised this habitat by date 6. Table 7.10 lists the families that made the greatest

contribution to the difference in community composition between newly inundated and

permanently inundated cobble habitat.



Table 7.6: Probability value and significance levels of one-way ANOVA for differences in number of macroinvertebrate families between permanently inundated (P) and newly inundated (N) cobble habitats at four sites in the Mitta Mitta River on four sample dates. Direction of trend in shown in brackets. ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Site Date 2 Date 3 Date 5 Date 6 1 0.168 ns 0.031 * (N<P) 0.445 ns 0.260 ns 2 0.020 * (N<P) 0.219 ns 1.000 ns 1.000 ns 3 0.001 *** (N<P) 0.027 * (N<P) 0.039 * (N<P) 0.025 * (N<P) 4 0.010 ** (N<P) 0.339 ns 0.003 ** (N<P) 0.689 ns

Table 7.7: Probability value and significance levels of one-way ANOVA for differences in total number of macroinvertebrate individuals between permanently inundated (P) and newly inundated (N) cobble habitats at four sites in the Mitta Mitta River on four sample dates. Direction of trend in shown in brackets. ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Site Date 2 Date 3 Date 5 Date 6 1 0.010 ** (N<P) 0.006 ** (N<P) 0.172 ns 0.236 ns 2 0.001 ** (N<P) 0.185 ns 0.288 ns 0.731 ns 3 0.043 * (N<P) 0.503 ns 0.031 * (N<P) 0.244 ns 4 0.000 *** (N<P) 0.819 ns 0.001 ** (N<P) 0.218 ns

Table 7.8: R values and significance level of ANOSIM analyses comparing macroinvertebrate faunal assemblages between permanently inundated and newly inundated cobble at four sites in the Mitta Mitta River. All comparisons were based on 25 permutations, with the exception of site 3, date 2 which had 15 permutations. ns = not significant and *= P<0.05.

Site Date 2 Date 3 Date 5 Date 6 1 0.396 * 0.313 * 0.500 * 0.240 ns 2 0.857 * 0.047 ns 0.365 * 0.063 ns 3 0.969 * 0.885 * 0.573 * 0.219 ns 4 No test 0.813 * 0.906 * 0.260 ns

Table 7.9: Comparison of SIGNAL scores for macroinvertebrates collected from permanently inundated and newly inundated cobble habitat in the Mitta Mitta River on four sample dates. P = permanently inundated cobble, N = newly inundated cobble.

Date 2 Date 3 Date 5 Date 6 Site P N P N P N P N 1 4.83 4.25 4.71 3.00 5.00 3.33 5.83 5.07 2 7.00 7.00 6.40 5.78 5.86 4.78 5.73 5.73 3 6.25 4.33 6.53 5.82 6.18 4.38 6.19 5.92 4 6.50 No data 5.38 5.50 5.88 4.33 6.15 5.43



Table 7.10: Average abundance of macroinvertebrate families that contributed significantly to the SIMPER analyses comparing permanently inundated and newly inundated cobble habitat on four sample dates. P = permanently inundated cobble, N = newly inundated cobble, % = percent contribution to difference. Av Dissim = Average Dissimilarity. Comparisons shown to be significant by the ANOSIM analyses are highlighted.

Site 1 Date 2 (Av Dissim 79.3)

Date 3 (Av Dissim 84.5)



Family P N % P N % P N % P N % Chironomidae 5.5 1.2 28.6 21.5 0.8 36.0 21.0 3.8 22.5 20.8 14.2 15.5 Oligochaeta 0.8 0 10.9 4.0 0.5 12.9 34.2 3.2 26.4 10.5 63.8 33.5 Caenidae 2.2 0 25.6 5.8 0 21.8 2.8 0 11.5 9.0 1.5 10.9 Coloburiscidae 0 0 0 0.5 0 7.0 1.0 0 10.0 0.5 0.2 2.4 Griptopterygidae 0.2 0 3.4 1.5 0 8.8 0 0.2 2.0 0.8 5.8 10.5 Site 2 Date 2

(Av Dissim 100) Date 3 (Av Dissim 60.0)



Family P N % P N % P N % P N % Chironomidae 1.8 0 14.4 7.0 16.8 17.9 6.5 2.2 16.7 64.8 55.5 22.8 Oligochaeta 3.8 11.5 16.9 1.8 3 14.1 31.8 67 24.6 Caenidae 1 0 10.7 1.2 6.0 12.9 1.2 0.5 9.3 2.2 10 11.0 Coloburiscidae 0.2 0 5.8 2 0 10.0 0.2 0 3 1.2 6.8 Griptopterygidae 0 0.5 6.5 0.5 0.2 4.1 0 2.2 17.0 0.8 1.0 5.5 Leptophlebiidae 6.8 0 57.6 2.2 2 7.2 4.5 1 18.0 5.8 1.8 7.9 Site 3 Date 2

(Av Dissim 69.6) Date 3 (Av Dissim 71.0)



Family P N % P N % P N % P N % Chironomidae 12.2 14.5 11.6 10.0 20.8 12.9 5.0 6.0 5.4 19.2 12.2 15.3 Oligochaeta 1 1.2 4.0 1.0 12.0 9.9 3.2 4.5 9.4 7.5 6.2 8.9 Caenidae 2.2 0.2 6.4 0.5 0.5 2.4 0.8 0 5.1 0.8 1.8 4.5 Coloburiscidae 7.8 0 13.7 11.0 0 16.0 3.5 0 12.2 8.8 0 15.4 Griptopterygidae 3.2 0.2 8.5 3.8 0.5 7.8 1.5 0.2 7.3 4.5 2.2 7.0 Leptophlebiidae 5.8 0.2 12.4 5.0 2.2 6.8 3.0 0 11.0 4.0 6.7 3.4 Hydropsychidae 3.8 0 11.3 2.5 0.2 6.3 4.5 0 14.4 1.0 0 3.9 Conoescucidae 2.0 0 6.1 3.8 0 9.0 1.0 0 7.0 1.2 0 5.1 Glossosomatidae 1.0 0 4.2 1.5 0 4.7 0.8 0 4.4 2.5 0 7.4 Site 4 Date 2

(Av Dissim 69.6) Date 3 (Av Dissim 71.0)



Family P N % P N % P N % P N % Chironomidae 0.8 0 32.3 22.0 16.8 10.5 49.5 6.8 21.8 20.5 14.8 10.9 Oligochaeta 1.2 5.8 10.2 33.2 5.2 18.9 8.0 39.2 10.9 Caenidae 0 1.8 5.5 1.5 0 6.3 0.5 0.5 3.8 Coloburiscidae 2.2 0 4.8 0 0.2 1.4 0.8 0.5 4.0 Griptopterygidae 0.8 0 22.9 1 4.5 8.0 2.8 1.5 5.6 4.8 6.2 8.7 Leptophlebiidae 0.5 0 19.8 4.2 0.5 9.6 3.2 0 9.9 2.0 3.2 7.2 Hydropsychidae 1.5 0 6.4 0.8 0 4.0 0.2 0 1.2 Conoescucidae 0.2 0 25.0 0.5 0 3.0 0.5 0 1.5 0.8 0 4.9 Glossosomatidae 1.2 0 3.2 1.2 0 4.6 1.0 0 4.9 Baetidae 1.2 0 6.8 1.2 0 5.0 2.5 4.8 7.5



Effect of 37 days of constant flows on macroinvertebrates in permanently inundated cobble

At sites two and three there were significant increases in number of families in the

permanently inundated cobble over the 37 days of constant low flows following the CRP

(Table 7.11). At site two the increase resulted in a larger number of families recorded than

at any other time in the study (Figure 7.1). At site three the increase during the 37 days

constant flows returned the number of families to a previously observed level (Figure 7.1).

At sites two and three there were significant increases in number of individuals in the

permanently inundated cobble over the 37 days of constant low flows following the CRP

(Table 7.11, Figure 7.3). The increase in number of individuals was particularly marked at

site three.

There were significant changes in the community composition between sample dates seven

and nine at all five sites, including the reference site (Table 7.13). Accompanying these

changes the SIGNAL score at site one continued to increase. However, the SIGNAL score

at sites two and five decreased from date 7 to date 9 (Table 7.14).

The changes in community composition at sites one to four do not parallel that in the

reference site (Table 7.15). All four sites in the Mitta Mitta River showed increases in the

abundance of Chironomids and decreases in the abundance of Coloburiscidae between

dates seven and nine, whereas there was a decrease in the abundance of Chironomids and

increase in the abundance of Coloburiscidae in Snowy Creek between these dates (Table

7.15). All sites, including Snowy Creek, showed increases in Hydropsychidae during this

period (Table 7.15). The SIMPER data shows that the significant increases in the numbers

of individuals at sites two and three between dates 7 and 9 are due to increases in

Chironomidae and Oligochaeta.



Table 7.11: Probability value and significance levels of one-way ANOVA’s comparing number of macroinvertebrate families between the end of the CRP (date 7) to the end of the 37 days constant flows (date 9) in permanently inundated cobble habitats at four sites in the Mitta Mitta River and in Snowy Creek. ns = not significant, * = P < 0.05. Direction of trend is shown.

Site Probability value Significance level Post hoc test 1 0.346 ns 2 0.020 * 7 < 9 3 0.016 * 7 < 9 4 0.876 ns 5 (ref) 0.147 ns

Table 7.12: Probability value and significance levels of one-way ANOVA’s comparing number of macroinvertebrate individuals between the end of the CRP (date 7) to the end of the 37 days constant flows (date 9) in permanently inundated cobble habitats at four sites in the Mitta Mitta River and in Snowy Creek. ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Site Probability value Significance level Post hoc test 1 0.916 ns 2 0.021 * 7 < 9 3 0.0002 *** 7 < 9 4 0.511 ns 5 (ref) 0.718 ns

Table 7.13: Results of ANOSIM analyses comparing macroinvertebrate faunal assemblages on permanently inundated cobble between the end of the CRP (date 7) and the end of 45 days constant flows (date 10) at four sites in the Mitta Mitta River and in Snowy Creek. All comparisons were based on 35 permutations. * = P<0.05.

Site R value Probability value Significance level 1 0.594 0.029 * 2 0.740 0.029 * 3 0.896 0.029 * 4 0.802 0.029 * 5 (ref) 0.698 0.029 *

Table 7.14: SIGNAL scores for macroinvertebrate samples from four sites in the Mitta Mitta River and in Snowy Creek for sample dates 7 and 9.

Site Date 7 Date 9 1. 5.83 6.18 2. 6.56 6.21 3. 6.00 6.00 4. 6.14 6.12 5. (ref) 6.35 6.07



Table 7.15: Abundance of macroinvertebrate families that contributed significantly to the SIMPER analyses comparing permanently inundated cobble habitat between dates 7 and 9. The % contribution to the SIMPER analyses is shown for each comparison. Comparisons shown to be significant by the ANOSIM analyses are highlighted.

Site 1

(Av Dissim 52.6) Site 2 (Av Dissim 66.2 )

Site 3 (Av Dissim 76.2)

Site 4 (Av Dissim 54.4)

Site 5 (ref) (Av Dissim 52.7)

Family Date 7

Date 9

% Date 7

Date 9

% Date 7

Date 9

% Date 7

Date 9

% Date 7

Date 9

%

Chironomidae 7.0 20.2 12.0 2.0 31.5 18.2 1.0 160 31.2 6.0 29.5 13.8 16.0 11.5 9.4 Oligochaeta 34.0 12.8 13.5 2.2 19.8 11.3 1.2 34.0 12.9 13.0 10.8 7.5 0.2 0 1.2 Caenidae 2.5 2.5 7.3 2.0 5.2 3.9 0.2 8.2 7.0 0.2 1.2 4.1 0 0 0 Coloburiscidae 2.5 0 7.6 0.8 0.5 3.5 2.8 1.0 3.0 0.8 1.2 6.8 8.1 Griptopterygidae 5.2 1.0 9.9 0.2 6.8 12.2 1.2 1.2 1.7 0.8 0.2 3.1 0.2 1.5 4.7 Leptophlebiidae 0 2.5 9.7 2.2 0.2 7.0 4.0 4.5 1.7 2.2 3.5 5.5 10.8 9.2 4.5 Hydropsychidae 0 0.8 2.5 0 3.0 7.1 0.8 35.0 14.1 1.8 7.8 6.9 0.5 2.5 5.1 Glossosomatidae 0.5 0 4.4 0 2.5 6.2 2.0 0.8 2.4 11.0 0 16.5 0 4.5 8.7 Baetidae 0.2 0.5 3.1 0 1.2 6.6 0.5 1.2 2.2 1.2 0 5.0 11.0 1.5 9.4 Tabanidae 0.2 1.8 4.9 0 1.2 2.9 0 8.5 7.9 0 1.5 4.7 0.2 0.8 3.0



7.4 Discussion This study examined the effects of CRP from Dartmouth Dam to the Mitta Mitta

River on the species richness, abundance and community composition of

macroinvertebrates. The implementation of this release pattern resulted in significant

changes in the macroinvertebrate community, with the greatest effects being observed

at the most upstream site (site 1). Although the full extent of the response to CRP

could not be determined by this study (due to the lack of sampling prior to the CRP),

this study has demonstrated that it is possible to detect a response of

macroinvertebrates to the CRP.

Site one in the upper reaches of the Mitta Mitta River had fewer families and

considerably lower SIGNAL scores than the other sites in the Mitta Mitta River and

the reference site in Snowy Creek. This is consistent with the findings of a previous

study by AWT (2000), which found that SIGNAL scores in the Mitta Mitta River

were between 5.0 and 6.0, whereas the major tributary Snowy Creek, had a SIGNAl

score of 6.7. As site one is at a similar altitude and has similar adjacent land use to

Snowy Creek, this suggests that the poorer ecological condition at this site can be

largely attributed to the management of Dartmouth dam.

Macroinvertebrates were found to respond relatively rapidly to the individual variable

flow releases. After only 14 days there was a significant change in the community

assemblage at site 4. After 28 days there was a significant increase in the number of

macroinvertebrate families at site one in the Mitta Mitta River and significant changes

in the community assemblage at sites 1, 3 and 4. These relatively rapid responses are

consistent with the results of Watts et al (2001), who found that there were highly

predictable associations between macroinvertebrate attributes and hydrological

variables at both short (10 and 30 days) and long (year) temporal scales.

The greatest effect of the CRP was observed at site one, the most upstream site. At

this site there was a significant increase in macroinvertebrate families, a significant

change in community composition and a substantial increase in SIGNAL scores in

permanently inundated cobble by the end of the third variable flow release. Changes

in the composition of the macroinvertebrate assemblage were also observed at sites



three and four by the end of the third variable flow, however there were no significant

changes in the number of families or SIGNAL scores at these sites. It is not surprising

that the observed changes were greatest at site one, as it was most degraded site at the

beginning of the study so had the greatest scope for improvement. Sites 3 and 4 may

have showed less change than site 1, as they are downstream of the confluence with

Snowy Creek. This tributary may play an important role in providing propagules to

these sites. Alternatively, the decreased benefit for macroinvertebrates at sites 3 and 4

may be because the flood peak attenuates as it moves downstream and was shown to

decrease the scouring of cobble at more downstream sites (see section 6).

There was a different macroinvertebrate assemblage and fewer macroinvertebrate

families and individuals in newly inundated cobble than in permanently inundated

cobble on day two of the second variable flow release (date 2). However, by day 8 of

the third variable flow (date 6) there was a similar macroinvertebrate assemblage and

a similar number of families and individuals in the newly inundated cobble to the

permanently inundated cobble. This suggests that newly inundated cobble provides

suitable habitat for macroinvertebrates and that they were able to colonise this habitat

over time. The cumulative effect of the variable flow releases on the

macroinvertebrate assemblage parallels that observed in the biofilm in littoral areas

(section 6), which continued to develop over time despite being dried between the

individual variable flow releases.

Despite the fact that there was a similar macroinvertebrate assemblage and a similar

number of families and individuals in the newly inundated cobble by the end of the

third variable flow release (date 6), the SIGNAL scores in newly inundated cobble

were lower or identical to that in permanently inundated cobble on all four sample

dates (2, 3, 5 and 6). This suggests that while there are many macroinvertebrate

families colonising the newly inundated cobble, some of the sensitive taxa have not

colonised this habitat by the end of a third variable flow release.

The 37 days of constant flows following the CRP produced different responses of

macroinvertebrates at different sites. At site one there was no significant change in the

number of families or individuals, however there was an increase in SIGNAL score.

In contrast, at sites two and three there were significant increases in number of



families, increased abundance of tolerant families such as Chironomidae and

Oligochaeta and decreased abundance of more sensitive families such as

Coloburiscidae. Thus, the more sensitive families were colonising the cobble habitat

at site one during the period of constant flows however the more tolerant taxa were

increasing in abundance at sites further downstream. The reduced level scouring of

the biofilm at sites 3 and 4 relative to site one (see section 6) means there was less

change in the biofilm community during the CRP at these sites relative to site one.

Thus it is possible that the benefits of the CRP were still being realised at site 1 during

the constant flow period.

7.5 Summary of findings • The implementation of the CRP to the Mitta Mitta River resulted in significant

changes in the macroinvertebrate community, with the greatest effects being

observed at the most upstream site (site 1). Macroinvertebrate indices responded

relatively rapidly to the variable flow releases. Significant responses to the flows

were detectable by 14 days after the beginning of the second variable flow release.

Although the full extent of the response to the CRP could not be determined by

this study (due to the lack of sampling prior to the CRP), this study has

demonstrated that it is possible to detect a response of macroinvertebrates to CRP.

• Site one had fewer families and lower SIGNAL scores than other sites, which is

consistent with the findings of a previous study by AWT (2000) in this river.

• The greatest effect of the CRP was observed at site one, where there was a

significant increase in macroinvertebrate families, a significant change in

community composition and a substantial increase in SIGNAL scores in

permanently inundated cobble by the end of the third variable flow release. There

was no change in the number of families or community assemblage in Snowy

Creek during the period when variable flows were released in the Mitta Mitta

River.



• The newly inundated cobble provided suitable habitat for macroinvertebrates and

was colonised by more families during the third variable flow release than during

the second variable flow release, suggesting there is a cumulative effect of the

flows. The macroinvertebrate assemblage in the newly inundated cobble became

more similar to the permanently inundated cobble over time.

• The 37 days of constant flows following the CRP produced different responses of

macroinvertebrates at different sites. At site one the SIGNAL score for the

macroinvertebrate assemblage increased during the 37 days of constant flows. In

contrast, at sites two and three there were significant increases in number of

families, increased abundance of tolerant families and decreased abundance of

more sensitive families.

• The numbers of families in littoral habitats varied considerably between sample

dates at all sites, including the reference site. This suggests that the qualitative

sweep method used to sample littoral habitats is not as reliable for the assessment

of the CRP as the quantitative surber method used in the cobble habitat.



8.0 SUMMARY & RECOMMENDATIONS

8.1 Effects of variable flow releases on the ecological condition of the Mitta Mitta River

Table 8.1 summarises the response of different parameters to the variable flow

releases from Dartmouth Dam to the Mitta Mitta River. Variable flow releases from

Dartmouth Dam to the Mitta Mitta River led to substantial changes in the water

quality and biotic parameters measured in this study. In contrast, over the same period

of time there was generally no change in the parameters measured at the reference site

Snowy Creek.

The water quality in the Mitta Mitta River during the CRP differed from that during

the 37 days of constant low flows. There was lower conductivity, pH and temperature,

and higher POM, TSS and water column Chl-a in the Mitta Mitta River during the

CRP than during the constant low flow period.

There was a slight decrease in the biomass of biofilms following the CRP in the Mitta

Mitta River. This coincided with increased activity of some major water column

bacterial enzymes, changed composition of biofilm algal species and rapid changes in

net productivity at all four sites in the Mitta Mitta River. These data suggest that the

variable flow releases scoured algal biofilms from cobble substrata at all sites along

the Mitta Mitta River. The scouring of the biofilm from the cobble substrata appears

to have triggered a response of bacteria in the water column and changed the

community composition of biofilms by removing filamentous green and blue-green

algae and increasing the relative biovolume of early successional species of diatoms.

The response of macroinvertebrates was more pronounced at site one than at the other

sites in the Mitta Mitta River. There was an increased number of families and

increased SIGNAL scores at site one only, however there were significant changes in

community composition of macroinvertebrates at three of the four sites in the Mitta

Mitta River. These changes in community composition may have been a response to

the changed composition of the biofilm algal species.



Table 8.1 Summary of effects of the CRP on chemical and biological parameters in the Mitta Mitta River and Snowy Creek. = increase, = decrease, =

community composition changed, - = no change in parameter, no data = no data for this parameter at this site.

Mitta Mitta River Parameter Site 1 Site 2 Site 3 Site 4 Site 5 (ref) Water quality DOC - - - - POM Water column Chlorophyll - - Total suspended solids - nutrients - - - - - temperature No data No data No data conductivity No data No data No data Dissolved oxygen - No data No data - No data pH No data No data No data Water column extracellular enzymes α-glucoside (carbohydrate) - β-glucoside (carbohydrate) - Butyrate (fatty acid) - Leucine (aminopeptidase) - - - Xyloside (woody substrate) - - - Biofilm structure and function Total biomass (dry weight) - Organic biomass (ash free dry weight) - Algal biomass (chlorophyll a) - - Biofilm taxonomy No data No data No data Biofilm metabolism No data No data No data Macroinvertebrates Diversity of families - - - -

Number of individuals - - - - SIGNAL scores - - - Community composition - -



8.2 Effects of 37 days constant and low flows on the ecological condition of the Mitta Mitta River Table 8.2 summarises the response of different parameters to the 37 days of low and

constant flows that followed the CRP in the Mitta Mitta River. Many water quality

and biotic parameters displayed substantial changes during the 37 days of low and

constant flows that followed the successive variable flow releases in the Mitta Mitta

River. In contrast, over the same period there was no change in most of the parameters

measured at the reference site in Snowy Creek.

Water quality in the Mitta Mitta River changed substantially during the low flow

period. During this period there was an increase in temperature and conductivity and a

decrease in TSS and POM.

The biomass of biofilms increased during the constant flow period in the Mitta Mitta

River. This coincided with very low activity of water column bacteria, changed

composition of biofilm algal species and decreased net productivity to a point where

biofilms at site 4 were net consumers of oxygen by the end of this period. During the

constant flow period there was no scouring of biofilm from cobble substrata and the

composition of the biofilm at site four changed from one dominated by diatoms and

green algae to one dominated by only two species of filamentous blue-green algae.

The response of macroinvertebrates to the constant flows differed between sites. At

site one there was no change in the number of families but the SIGNAL score

continued to increase, suggesting that the more sensitive families were continuing to

colonise the cobble and any ecological effects of the CRP at this site were still being

realised. In contrast, there was a decrease in the SIGNAL score at site two and

increased abundance of tolerant families such as Chironomidae and Oligochaeta and

decreased abundance of more sensitive families such as Coloburiscidae at sites two

and three by the end of the constant flow period. It is possible that any ecological

effects of the CRP are more short-lived at sites two, three and four when compared to

site one.



Table 8.2 Summary of effects of 37 days of constant flows on chemical and biological parameters in the Mitta Mitta River and Snowy Creek. = increase, = decrease, = community composition changed, - = no change in parameter, no data = no data for this parameter at this site. Mitta Mitta River Parameter Site 1 Site 2 Site 3 Site 4 Site 5 (ref) Water quality DOC - - - - - POM - Water column Chlorophyll - - - Total suspended solids nutrients - - - - - temperature No data No data No data conductivity No data No data - No data Dissolved oxygen No data No data No data pH No data No data - No data Water column extracellular enzymes α-glucoside (carbohydrate) - - - - - β-glucoside (carbohydrate) - - - - - Butyrate (fatty acid) - - - - - Leucine (aminopeptidase) - - - - Xyloside (woody substrate) - - - - - Biofilm structure and function Total biomass (dry weight) - Organic biomass (ash free dry weight) - Algal biomass (chlorophyll a) - - - Biofilm taxonomy No data No data No data Biofilm metabolism No data No data No data Macroinvertebrates Diversity of families - - - Number of individuals - - - SIGNAL scores - - Community composition



8.3 Recommendations for future monitoring programs

• The second and third releases produced substantial changes to the water quality

and biotic parameters measured in the Mitta Mitta River. It is possible that some

ecological changes may have also occurred during the first release, however due

to the timing of the call for tenders no samples could be collected in the period

prior to, or during the first release. This limited the ability of this study to assess

the full ecological impact of the CRP.

Recommendation 1: Future assessments of CRP’s should include at least two sample

dates prior to the first release, followed by two sample dates during each subsequent

release and several samples dates during the constant period of flows following the

end of the release pattern.

• The results suggested that long periods of low and constant flows substantially

alters the ecological condition of the river system, however the current study was

not structured to identify the critical threshold point at which constant flows

become detrimental.

Recommendation 2: Future assessments of CRP’s should include a detailed study of

the period of constant flows prior to and following the release period to identify

thresholds at which constant flows become detrimental and at which point CRP’s

should be introduced.

• This research demonstrated very short-term responses of enzyme activity during

peak flows with minimal changes in activity under constant flow conditions.

Recommendation 3: Future CRP’s in the Mitta Mitta River downstream of Dartmouth

Dam require flood peaks of at least 4800ML/day (at Colemans gauge) to maximise

rates of microbial productivity for a diverse range of water column bacteria. Future

monitoring can target resources at specific flow events to streamline sample collection

and minimise costs.



• Structural and functional responses of biofilms were evident immediately

following the peak flows of each variable flow release, as well integrating

responses to flow regime over longer time periods. This suggests the biofilm

attributes and the temporal scale of their measurement used in this study are

appropriate for monitoring changes in the ecological condition of the Mitta Mitta

River.

Recommendation 4: Future assessments of CRP’s should include all the biofilm

attributes sampled to provide a range of structural and functional responses of

ecological communities to variable flow releases. Increased replication may reduce

the large variances found in some attributes and help better identify significant

changes in biofilm assemblages.

• The collection of four replicate water samples at each site on each sampling date

to determine extracellular enzyme activity from the water column provided

minimal statistical power to test hypotheses and examine the significant effects of

the CRP’s on microbial productivity.

Recommendation 5: Replication of this parameter should be increased to improve the

power of the statistical analyses. The suite of enzymes examined represent a range of

naturally occurring carbon sources and should be retained in future monitoring

programs.

• Several macroinvertebrate attributes measured in cobble habitats (number of

families, SIGNAL scores and community composition) responded relatively

rapidly to the variable flow releases. The four quantitative surber replicates taken

from cobble habitats at each site on each sampling date provided sufficient

statistical power to detect significant effects of the release pattern. This suggests

that the attributes and temporal scale of measurement used in this study are

appropriate for future monitoring programs.



Recommendation 6: Future assessments of CRP’s should include a minimum of four

surber sample replicates of cobble habitat per site per sample date to allow ANOSIM

analyses to be performed to detect significant differences in community composition

between sample dates. Both abundance and diversity of macroinvertebrates should be

measured.

• The variability in numbers of families in the littoral habitat at all sites, including

the reference site in Snowy Creek, suggests that factors other than flow may

influence this parameter and it may not be as reliable as the quantitative

assessment of cobble habitat for the assessment of CRP’s.

Recommendation 7: Future assessments of CRP’s should carefully consider the cost-

benefits of including the qualitative sweep method in the monitoring program.



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APPENDIX 1

PROJECT BREIF



Call for Tenders Variable Releases from Dartmouth Dam River Murray Water (RMW) propose to trial variable releases from Dartmouth Dam over the period late-November to late-December 2001. The background and details of this proposed release pattern are contained in the attached letter from RMW (dated 14 November 2001). The Commission has been provided with a draft study brief by Dr Terry Hillman, Member of the Expert Reference Panel on Environmental Flows and the Independent Sustainable River Audit Group (ISRAG), and this is also attached. Further, a letter to RMW from the North East CMA (John Riddiford) commenting on the proposal is attached as background. Please note that this letter should be treated as commercial-in-confidence correspondence between NECMA and RMW and is to be used only in the preparation, and if successful, execution of this work. Tenders Tenders are invited from suitably qualified suppliers to perform monitoring in the Mitta Mitta Valley during November and December 2001. Tenders will define a proposed sampling program consistent with the draft study brief prepared by Dr Hillman, and may include further work with appropriate justification. Geomorpholocigal issues may also be consuideredn in this program. Please allow in your tender for provision of a full report on the monitoring including recommendations for future monitoring of such events in the Mitta Mitta River. While the planned releases are detailed in the attached documentation, such releases can be changed significantly in response to weather conditions. Accordingly please include the basis for variations in contract arrangements. Please advise by close of business Monday 19 November 2001 of your intention, or otherwise, to proceed with a tender. Tenders are welcome as soon as practicable. Contacts Tony McLeod, Environmental Flows, 02 6279 0127 0409 908 015 Brian Lawrence, Manager Riverine, 02 6279 0160 Bruce Campbell, River Murray Water, 02 6279 0169 ________________________________ Tony McLeod Murray-Darling Basin Commission GPO Box 409, Canberra ACT 2601 Tel: 02 6279 0127 Fax: 02 6248 8053 Mobile: 0409 908 015 Email: [email protected] Web Page: www.mdbc.gov.au ________________________________



10 November 20051 Mac Paton Chairperson Mitta Mitta Water Services Committee RMB 1135 Tallangattta Vic 3700 Fax (02) 6071 7260 Dear Mac

Proposal for Pattern of Variable Release from Dartmouth Dam We are writing to seek your comments regarding a proposal to introduce a cycle of variation in the release rate from Dartmouth Dam for environmental benefit. For your background, harmony releases at Colemans have been maintained at 4 000 ML/day since 18 October 2001. This is despite the fact that harmony transfer rules show that based on current storage levels in both Dartmouth and Hume Reservoirs, the calculated harmony transfer volume for November would require releases at close to channel capacity in November, with relatively little volume transferred in December. However, River Murray Water, in consultation with Goulburn-Murray Water and the North East Catchment Management Authority, has determined that in the current circumstances, it would be more appropriate to average the harmony transfers in November and December. This is expected to reduce the potential for erosion caused by higher flow rates. With this in mind, River Murray Water presently estimates that the current requirements for transfer to Hume will be maintained until about the end of December, however, if wetter conditions occur in the Murray catchment, a lower average rate of transfer may be required. In addition to this, rather than continuing to release at a constant rate over the next several weeks, it is proposed that a minor flow “pulse” be trialed to simulate a natural rainfall event. This variable flow pattern is consistent with the recommendations of the Commission’s River Murray Scientific Panel on Environmental Flows1. The proposed cycling of flow includes a variation in water level of about 0.25 m at Colemans and 0.25 at Tallandoon (see Attachment A). The size of the proposed variation is similar to what may occur during power station entitlement releases, or which occur during natural flow events in tributaries downstream of Dartmouth. The trial flow pattern is expected to benefit riparian vegetation and biofilms in the riparian zone, fish migration, and reduce the impact of erosion caused by constant water levels. The MDBC Natural Resources section is arranging for some monitoring to be undertaken to gauge the environmental response to the trial. This proposal is not expected to cause inconvenience to diverters along the lower Mitta Mitta River. However, diverters are advised that the flow rises could increase leaf litter and other debris. Flow rises will be timed to take place during the week to reduce the potential for these problems, as well as providing some small benefit to electricity generation.



If the Water Services Committee has any concerns regarding the proposed trial, please contact Bruce Campbell, River Management Engineer, on 02 6279 0100 by 18 November 2001. River Murray Water is intending to issue a media release late this week confirming arrangements if no significant obstacles are identified. If there is no initial concern regarding this trial, we will also seek feedback from landholders via the Water Service Committee after any trial is carried out. Yours sincerely David Dole General Manager 1 Thoms, M. C., Suter, P., Roberts, J., Koehn, J., Jones, G., Hillman, T. and Close, A., 2000. Report of the River Murray Scientific Panel on Environmental Flows: River Murray - Dartmouth to Wellington and the Lower Darling River. Scientific Panel on Environmental Flows, Murray-Darling Basin Commission, Canberra, June 2000.



Attachment A

Dartmouth Release – Trial Flow and Water Level Pattern

November/December 2001 The trial flow “pulse” is proposed on the following basis:- Duration of each pulse cycle 14 days Flow Rise 2 days Flow Recession 12 days First pulse proposed to commence Week commencing 19/11/01 Colemans Gauge Average flow required 4 000 ML/day Maximum Flow 4 800 ML/day Minimum Flow 3 200 ML/day Average Water Level (Colemans gauge) 2.08 m Water Level Variation 0.25 m total Tallandoon Gauge * Average flow 5 000 ML/day Maximum Flow 5 800 ML/day Minimum Flow 4 200 ML/day Average Water Level (Tallandoon gauge) 2.43 m Water Level Variation 0.25 m total

* Assumes constant inflows of 1 000 ML/day from Snowy Creek and other minor tributaries. These flows may be slightly higher if there is significant rainfall.

If the variation on flow coincides with a significant flush caused by inflows from tributaries downstream of Dartmouth, the program may be modified to prevent inconvenience to diverters.



November 2001 Mr Bruce Campbell River Management Engineer River Murray Water GPO Box 409 Canberra ACT 2601 Dear Bruce Re : Dartmouth operations season 2001/2002 Thank you for your fax dated 11 October 2001 requesting comments on the water transfer from Dartmouth Dam. In general, the CMA supports the proposed program during spring/early summer of 2001 because it creates the opportunity to periodically inundate areas of the Mitta Mitta riparian zone for the benefit of wetland ecology. However, I qualify my support with the following comments and recommendations. a) The Harmony program is essentially a form of 'pre-release' strategy that aims to

minimise the chance of physical spill from Dartmouth storage. As such, the program does not benefit the floodplain on ecological grounds because it minimises floodplain inundation (as also stated by the River Murray Expert Panel for Environmental Flows, 2000).

b) However, given that the chance of spill is very low in both storages (currently around

10%), then the alternative is for 'normal' transfer rules to be maintained that causes constant in-channel flow rates to be passed from Dartmouth Dam. The disadvantage of constant flow rates have been highlighted by the Expert Panel as causing: - Loss of stimulation for fish movements; - Reduction in the range of riverbank habitat and bed habitat (i.e., reduced wet/dry area); - Instability of the river channel banks and subsequent reduction of in-channel complexity and habitat diversity due to erosion and sedimentation. The basis of the current RMW proposal is to fluctuate the rate of the transfers to cause variability in Mitta Mitta River running levels. Given this being the alternative to stable transfer rates, then the opportunity for variable transfer rates is supported.



c) However, concern exists for low water temperature arising from Dartmouth release being inappropriate for native fish breeding response or success, and reducing other biological response. Variable water flow management may instead be generally more beneficial to improved riparian habitat and diversity, with native fish benefiting only from anticipated improved food resource being washed and transferred into the lower river systems.

d) In releasing variable flow rates, the following hydrograph and associated issues are

recommended: - Aim for cycle of 2 peaks per month (though four per month, as may be preferred by

the power station, could instead be accommodated, though remains NECMA's secondary preference);

- Have rapid rise followed by slower recession; - Current proposal is fluctuation within river channel capacity. Explore options to

increase flow rates within this range that will connect the river to adjoining anabranches, backwaters and associated floodplain wetland depressions;

- Have greatest flows in October/November, decreasing in December, so as to more closely emulate natural hydrograph. However, having said this, it may be pertinent to shift the hydrograph by a month to assist in alleviating impact of cold water pollution (reservoir thermal effect).

- That significant flow pulses in Snowy Creek be reflected as a flow pulse down the Mitta Mitta River to Hume Dam.

e) Implement a monitoring program to validate the effectiveness of variable flow

management in the Mitta Mitta River. At a minimum, monitoring should include: - Hydrographic monitoring (i.e., to record river running height variations); - Basic mapping (recording) of inundation extent (especially sites where water escapes

from the main river channel); - Basic phys-chemical properties (i.e., pH, temperature, turbidity, conductivity, D.O.,

etc); - Basic survey of flora response (i.e., general growth and flowering). Plus, where possible, include more detailed, scientifically defensible, monitoring such as: - Biofilm development and fate through literal zone; - Fish movement and breeding success; - Flora response between sites (e.g., billabongs) that have been influenced by

increased frequency of flood fluctuations compared to sites without; - Invertebrate response; - Amphibian response; - Waterbird response; - Carbon cycling.

f) The regulated flow rate of ≤5,000 ML/day should be targeted whenever possible to

prevent the long-term saturation of the upper bank caused by the bank full-regulated flow of 10,000 ML/day. - When River Murray Water is required to exceed 5,000 ML/day to meet water

transfer demands, flow releases up to 10,000 ML/day can be used. Releases up to 10,000 ML/day must be designed to mitigate impacts associated with the long-term saturation of the upper bank.

- When dropping flow rates from 10,000 to 5,000 ML/day, it is important that drawdown rates do not exceed those that would have occurred naturally after similar



periods of bank full flow. That is, the longer flows are held at bank full, the slower the drawdown rate needs to be to mimic natural. For example, if a transfer is held at 10,000 ML/d for 1 month then the drawdown rate between 10,000 and 5,000 ML/d should be no greater than that recorded from a historical flood event that exceeded 10,000 ML/d for 1 month. In this way the transfer “event” is matched as closely as possible to a “natural” flood event. However, it needs to be noted that if the frequency of bankfull flow events of a specified duration exceeds the natural frequency, elevated erosion rates may occur simply due to increased event frequency (i.e. natural drawdown rates are likely to be associated with some erosion, and increased frequency means an increase in this erosion).

If you require further information please contact Mr Keith Ward on (03) 58 335 947. Yours sincerely John Riddiford Chief Executive Officer CC Keith Ward, Dean Judd



Flow Manipulation in Mitta Mitta River Nov – Dec 2001

NOTES FOR DRAFT STUDY BRIEF Introduction:

• Releases are to be made from Dartmouth Dam during November – December to augment storage in Lake Hume (in line with harmony rules).

• The Murray Darling Basin Commission has decided to make these releases on a variable flow pattern (2 days rise / 12 days fall) and is seeking assistance in assessing the ecological effects of this modification to management practice.

• It is recognised that, because of the timing and lack of opportunity, this study will be exploratory in nature and the Commission therefore intends that the work undertaken will:

Identify components of the ecosystem which might be expected to respond to a change from constant to variable flow patterns on the proposed scale.

Provide measurements indicative of that response Form the basis for preliminary assessment and advice for management of

variable releases Provide data and insights which could support and help direct more rigorous

studies in the future. Components of the Ecosystem to be Assessed Given the time constraints and scale of release the following components of the Mitta Mitta ecosystem have been identified for assessment. WATER QUALITY: Periodic changes in water level may be expected to result in corresponding changes in water quality from a variety of potential causes – wetting/drying, movement to and from riparian zones, resuspension etc. A range of appropriate WQ parameters should be monitored with sufficient intensity to estimate ‘end-of-valley’ effects of the flow manipulation. RIVER PRODUCTIVITY: Short-term variation may influence the rate of photosynthetic production and the relative significance of various primary producers (ie the biodiversity of primary producers). Under the current circumstances, the following observations should be carried out with the desired outcome being measurement of cyclic changes in production/respiration in the river system and indications of areas requiring more intensive study.

1. Biofilm Composition. Static and floating glass-slide samplers exposed to detect effects of flow variation on species diversity and biomass.

2. Benthic production/respiration. Estimations of P/R ratios in wet/dry benthic zones. 3. Water Column Production. Companion measurements to 1 and 2 above on water

column phytoplankton. Aimed at estimating likely changes to the relative contribution (and therefore diversity) of benthic and water column organisms in providing organic carbon to the river system.



4. [ The use of enzyme activity measurements to quantify bacterial activity is also a possibility but I have not been able to assess the relative effort/ payoff or the likely sensitivity to flow fluctuations]

INVERTEBRATES: Observations on invertebrates should address two major questions – Does flow variation stimulate interaction with riparian/floodplain aquatic systems, and, does wetting and drying of in-stream benches change the diversity of macroinvertebrates, increase drift (and therefore mixing, colonization etc) with an overall positive or negative effect on the macroinvertebrate community of the Mitta Mitta.

• Under the present conditions a detailed study of microinvertebrates is not warranted. The deployment of drift nets along the river (see ‘fish’ below also), to cover the range of flow events, and provide the basis for assessing riparian/floodplain inputs is suggested.

• Comparative samples of benthic macroinvertebrates on permanently inundated and cyclically inundated benches again would provide indicative information regarding the likely effects of variable flow.

FISH: The scale, lack of ‘before’ data and/or controls limits the possible value of fish observations. The time of year (post breeding for many of the major species) and significantly changed water temperature further limits the ability to compare observations with other experience. Two sets of observations should be included in this program:

• Use of drift-net catches from Macroinvertebrate survey. If (perhaps because of temperature depression) Murray Cod are breeding drift-net samples may link drift of larval cod to flow pattern providing a basis for further work

• Recent indications that European Carp may be trapped into breeding in highly impermanent waters provides an opportunity for an observational study on backwaters and small riparian waterbodies on the Mitta Mitta during the variable release program. Contribution to Carp control might be incorporated into designing variable flow patterns if a response can be measured during this trial.

Dr Terry Hillman 14 November 2001



APPENDIX 2

JOHNSTONE CENTRE PROPOSAL



JOHNSTONE CENTRE RESEARCH IN NATURAL RESOURCES & SOCIETY

Environmental Consulting Quote No.0047

Ecological Assessment of Variable Flow Releases in the Mitta Mitta River

MDBC

___________ Wagga Wagga 2001



1.0 INTRODUCTION

Current ecosystem management to restore river health in most of the Murray-Darling Basin focuses on providing ‘environmental flows’ from upstream water storages (e.g. EPA 1997). Environmental releases attempt to mimic natural flow variability in regulated rivers and drive ecological processes necessary for river rehabilitation. River Murray Water (RMW) proposes to trial variable releases from Dartmouth Dam into the Mitta Mitta River during November and December 2001. The Murray Darling Basin Commission (MDBC) has decided to make these releases on a variable flow pattern (2 day rises / 12 days fall) and is seeking assistance in assessing the ecological effects of this modification to management practice. This document details a proposal by the Johnstone Centre to undertake the ecological assessment of variable flow releases in the Mitta Mitta River.

2.0 JOHNSTONE CENTRE, CHARLES STURT UNIVERSITY 2.1 CENTRE PROFILE The Johnstone Centre is one of five research centres at Charles Sturt University. The Centre is made up of academics, postgraduate students, consulting staff and support staff. The Johnstone Centre’s mission is to research the fundamental ecological processes involved in the conservation of the diverse range of ecosystems and protected areas in Australia and overseas, crucial to their successful management. Our research contributes to developing a greater understanding of ecological processes on a community and landscape scale and applies the results of such research to challenge current practices, policies and planning. These studies enable us to establish the conditions necessary for ecologically sound management, as well as biodiversity assessment and conservation evaluation. Johnstone Centre - Environmental Consulting and associated academics from Charles Sturt University have worked extensively in the MDB and beyond, with research focused on natural resource management, target survey and management in agricultural systems. Recent projects related to the study of artificial flow variation include ‘Assessment of Environmental Flows for the Murrumbidgee River: Developing biological indicators for assessing river flow management’. 2.2 Assessment of Environmental Flows for the Murrumbidgee River



Dr Robyn Watts et al. (2001) from Charles Sturt University have recently completed the development and assessment of biological indicators for the assessment of environmental flows in the Murrumbidgee River and associated tributaries. Head water tributaries such as the Tumut, Goodradigbee and Goobagandra Rivers were included in this study which analysed the response of indicator species of macroinvertebrates and biofilm metabolism to changes in flow management. A copy of the CD-ROM of this project can be obtained by contacting Dr Robyn Watts on 02 69332329. The Goodradigbee and Goobagandra Rivers are very similar geomorphically, have similar substrata and are cold water streams like Snowy Creek and Little Snowy Creek. The Tumut River is quite similar in many respects to the Mitta Mitta River, in respect to it being downstream of Blowering Dam and having similar substrata and hydrological regimes. The experience, expertise and equipment available to the Environmental Flows Assessment team at Charles Sturt University makes them highly capable of assessing the variable releases proposed for the Mitta Mitta River.



2.0 METHODS

2.1 SITE SELECTION

A recognisance trip will be made to the Mitta Mitta River one week prior to the commencement of sampling to confirm sampling sites. At present four sampling sites have been confirmed and an two extra sites is being considered. Sites 1, 2, 3 and 5 are at the same location as Sites 502, 505, 503 and 504 as used for the Mitta Mitta River Biological Monitoring Program, respectively (AWT Victoria 2000). Sites 4 and 6 are to be located upon Little Snowy Creek u/s of the Mitta Mitta confluence and a site downstream of the Tallandoon gauge, respectively.

2.2 WATER QUALITY (WQ)

Water quality parameters to be measured include; Dissolved Organic Carbon (DOC), Particulate Organic Carbon (POC), Suspended Solids (SS), Conductivity (Cond), Temperature (temp), Turbidity (turb), DO (mg/L), velocity, light intensity, water column chlorophyll. Water samples will be taken from each of the six sites following the proposed sampling regime (Table 5). At each sampling site and on each sampling day, five filtered water samples (300mL) will be collected. These samples will be analysed in the laboratory for DOC, POC, SS and water column chlorophyll. The remaining water quality parameters will be recorded on site using a multimeter and flow meter. Three additional samples (30mL) will also be collected for the analysis of Total Phosphorus (TP), Total Nitrogen (TN) and ortho Phosphate.

2.3 RIVER PRODUCTIVITY

2.3.1 Biofilm Composition

The project team have recommended the use of cobble stones for the analysis of biofilm composition, as apposed to static and floating glass slides. Artificial growing strata such as glass slides act as a reference that are used to gauge the response of biofilms to changes in flow regime. Given that the 1st variable release has started, colonising biofilms would represent a community composition that developed during variable conditions. We propose to use cobbles in the existing substrata as a guide to biofilm composition prior to variable flow release. The project will then assess biofilm response to changes in flow regime over a forty day period following the return to constant conditions. Cobbles will be removed from the water column at two different depths, deep and newly inundated. Two cobbles will be removed at each depth, one will be placed in a cold box at 4



degrees C and taken to the laboratory for live identification and one will be scrubbed and the biofilm will be preserved in alcohol. This process will be conducted upon day 0, 2, 8 and 14 during both variable flow periods and then every ten days for forty days following the return to constant river heights. Sampling of biofilm composition will occur at each of the six study zones. Sampling will be conduced on days 0 and 2 to gauge the response of biofilms to scouring. Dr Adrienne Burns will complete the biofilm identifications to the lowest taxonomic resolution possible.

2.3.2 Benthic metabolism

Benthic metabolism will be measured at sampling sites 3 and 6. Eight chambers described below will be available at each site during each sampling period and will be placed on racks and held in the water column of the Mitta Mitta River. Four randomly selected cobbles (of suitable size) will be removed from the river and placed into a chamber for a period of 24 hours. During this period an Orion 835A DO datalogger will monitor changes in DO concentration (mgL -1 ) within the chamber at 8 minute intervals. Sampling will be conducted on the day prior to variable flow event 2 and then every 24 hours over the first three days of variable flow release. Metabolism of biofilms will then be assessed on day eight and day 14 for release periods 2 and 3, then every ten days for forty days following day 14 of the 3rd variable flow period. A new cobble will be selected within the same area for each 24 hour assessment. At the completion of the incubation, each block will be removed and sealed in a plastic bag for determination of organic and inorganic biomass. Chambers have been specifically designed for biofilm metabolism assessment. Each chamber consists of a 20cm length of 20cm inner diameter clear Perspex tubing (with a volume of 4L) and two end caps made of clear perspex. Cobbles will be held in place using a nylon screw into a perspex pedestal attached to the base of the chamber that centred the block horizontally. Water is recirculated within each chamber using a variable speed, submersible 12V pump located on the outside of the chamber and connected to each end cap using nylon tubing. Recirculated water entered the chamber through an outlet that disperses water evenly across the block surface and was directed across the chamber to the recirculating pump inlet valve on the other end cap. This simulates the direction and velocity of flow experienced by in situ biofilms by varying the speed of the recirculating pumps. A bilge pump located at the base of one end cap vented the contents of the chamber every 90 minutes. At the top of the other end cap, a one way valve allows the chambers to be refilled with river water via the pressure created from the bilge pump operation. A port in the end cap allowed an Orion 08310A DO probe to be positioned 3cm above the horizontal surface of the colonised block.



At each sampling date, underwater irradiance (_E/cm/sec) will be measured at 15 minute intervals for a minimum of 24 hours at 15cm below the surface using a LICOR LI-92SA Underwater Quantum Sensor attached to a 6004-21 Starlogger System. Water temperature will be recorded by an Orion 835A DO datalogger at 8 minute intervals for the 24 hour incubation. Water column pH and conductivity are to be taken at 15cm depth below the water surface using a Yeo-Kal muliprobe meter at 0, 12 and 24 hour time intervals. Water samples (500mL) will also be taken from 15cm below the water surface at 0, 12 and 24 hour time and filtered through a pre-weighed GFF filter paper, dried at 80°C for 48 hours and reweighed to determine Total Suspended Solids (TSS) in mg/L.

2.3.3 Water Column Metbolism

Water column metabolism will be assessed using Perspex chambers with no cobbles inside. During the benthic metabolism sampling periods the remaining four chambers at each site will be used to measure water column metbolism. River water is pumped through each chamber and the Orion datalogger will record DO concentration at 8 minute intervals for a period of 24 hours. Water column production will be measured at the same times as the benthic metabolism.

2.3.4 Enzyme Activity

Five enzymes including; Butyrate, Alpha and Beta glucoside, Beta xyloside and Leucine will be analysed during the assessment project. These enzymes are involved in the degradation of polysccharides, fatty acids and proteins derived from a range of carbon sources in rivers. Enzyme activity will be assessed at each site, on days 0, 1, 2, 8 and 14 during both variable flow periods 2 and 3, and then every ten days for forty days following to resumption of constant flows. Because of the rapid response of enzyme activity to changes in flow, water samples will be taken every 12 hours during the 1st 2 days of variable flow release. A 30mL vial of river water will be taken at each sampling site on each of the sampling times noted above. The analysis of enzyme activity is an expensive process, however at present Dr Darren Ryder is analysing enzyme activity for another project. It is suggested that samples collected during this assessment could be piggy backed onto these samples for analysis and thus reduce the overall price. 2.3 MACROINVERTEBRATES Macroinvertebrates will be sampled according to the National Protocol defined in the River Bioassessment Manual (MRHI 1994). And in the EPA (1998) publication on Rapid



Bioassessment of Victorian Streams. The widest possible diversity of macroinvertebrates will be assessed by sampling both the riffle and littoral habitats. All sampling will use ISO DIS/7828 250um mesh nets (ISO 1983). Nets will be washed thoroughly between sampling events to prevent cross contamination of samples. Sampling will be conducted using the following methods (Section 2.3.1 and 2.3.2). Sampling will be conducted at each of the six study sites on days 0, 2, 8 and 14 of each of the two variable flow periods and then every ten days for a forty day period following the return to constant flow conditions.

2.3.1 Littoral Habitats Littoral areas will be sampled using sweep nets in shallow areas with little or no current. Vigorous sweeping techniques that disturb the substrate sufficiently to suspend benthic animals will be employed. The nets will be swept through any vegetation, snags and logs in backwaters. This method will be carried out over a random ten metre section at each site. Net contents will then be emptied into white sorting trays and animals will be collected using forceps for a period of 30 minutes. Wide-mouthed pipettes will be used to handle fragile animals. Collected invertebrates will be preserved in 70% ethanol and identified in the laboratory.

2.3.2 Riffle Habitats

Riffle habitats will be sampled using a 250µm mesh kick net held perpendicular to the substrate with its opening facing upstream. Sampling will start at the downstream end of the riffle, the streambed directly upstream of the net will be disturbed by kicking and agitating the stones. This method will continue over ten metres, working upstream against the flow. Net contents will be sorted in white trays, preserved and identified in the same manner as the littoral samples.

2.3.3 Macroinvertebrate Identification

Macroinvertebrates will be identified to key families using keys listed in Hawking (2000). Sample and identification data will be entered into a MS-Access database. Macroinvertebrate data will be assessed using the SIGNAL-95 (Stream Invertebrate Grade Number – Average Level) (Chessman 1995), as used by AWT Victoria (2000). 2.4 Fish

Given the small rise in water levels (~25cm) it is unexpected that the variable flow release will trigger significant fish breeding responses. Our project team proposes to ignore a targeted assessment of fish movement within the Mitta Mitta River during this period. However, as part of the riffle habitat component of our proposal we intend to analyse drift samples for fish larvae. These incidental samples will provide a coarse indication of



breeding and may provide some useful information for future assessments within the Mitta Mitta system.



3.0 BUDGET AND TIME ALLOCATION

Table 1. Summary of budget, time allocation and key personnel. Task Key Personal Time Allocated Total Cost

Field Work Robyn Watts

Darren Ryder

Lachlan Sutherland

Martin Asmus

1 day

1 day

18 days

15 days

Laboratory Work

Biofilm ID

Adrienne Burns 5 days

Macroinvertebrate ID Robyn Watts

Lachlan Sutherland

1 day

10 days

Water Quality Analysis Lachlan Sutherland 21 hours

Enzyme Analysis Darren Ryder 7 hours

Data Entry Lachlan Sutherland 2 days

Data Analysis Darren Ryder

Robyn Watts

Lachlan Sutherland

2 days

1 day

3 days

Prepare and submit assessment report

Alistar Robertson

Robyn Watts

Darren Ryder

Lachlan Sutherland

Bruce Mullins

½ day

1 day

1 day

5 days

3 days

Travel 8400 km at

$0.65/km

Stationary and printing,

Sub Total

CSU Competitive Neutrality Levy**(10%)

Total (excluding GST)

GST

Total (including GST)

* The project team is committed to an entire day on site during biofilm metabolism assessments, because of the security of equipment and monitoring the chamber pump system. Combinations of the individual sampling programs on the same day as biofilm metabolism assessment will not incur an additional fee because of our commitment on site.



** It is a legal requirement that the University charges an infrastructure levy in the interests of competitive neutrality.



4.0 TIMESCALE AND WORK PLAN

The project will commence on the 2nd of December with the start of the sampling period. It is roughly estimated that a draft assessment report will be completed by late February (Table4). Sampling will commence on the 2nd of December, one day prior to the 2nd flood period and continue for forty days after the last day of the 3rd flood event to the 8th of February, 2002 (Table 5). Table 2. Gantt Chart

Conduct sampling

Biofilm ID

Macroinvertebrate ID

Nutrient Analysis

Enzyme Analysis

Water Quality Analysis

Data Analysis

Report

Month Dec Jan Feb

Table 3. Sampling regime Date Day Cumulative days WQ Enzyme Biofilm comp. Benthic metab. Column prod. Macroinvertebrates

2/12/01 0 0 3/12/01 *1 *1 4/12/01 2 2 5/12/01 3 3 6/12/01 4 4 7/12/01 5 5 8/12/01 6 6 9/12/01 7 7

10/12/01 8 8 11/12/01 9 9 12/12/01 10 10 13/12/01 11 11 14/12/01 12 12 15/12/01 13 13 16/12/01 14 14 17/12/01 1 15 18/12/01 2 16



19/12/01 3 17 20/12/01 4 18 21/12/01 5 19 22/12/01 6 20 23/12/01 7 21 24/12/01 8 22 25/12/01 9 23 26/12/01 10 24 27/12/01 11 25 28/12/01 12 26 29/12/01 13 27 30/12/01 14 28

9/01/02 10 38 19/01/02 20 48 29/01/02 30 58

8/02/02 40 68

* Commencement of the 2nd variable release period.



5.0 PROJECT TEAM

The project team will be comprised of: • Professor Alistar Robertson, BSc (Hons), PhD • Dr Robyn Watts, BSc (Hons), PhD • Dr Darren Ryder, BSc (Hons), PhD • Dr Adrienne Burns, BSc (Hons), PhD • Mr Bruce Mullins, BSc, MSc • Mr Lachlan Sutherland, BAppSc (Hons) • Mr Martin Asmus, BAppSc (Hons) Professor Alistar Robertson Professor Alistar Robertson is an ecologist with 22 years experience studying ecological and geochemical processes in aquatic habitats, including trophodynamics, nutrient cycling, fish biology and vegetation dynamics in seagrass, mangrove and freshwater wetlands in Australia, Papua New Guinea, south east Asia and Mexico. His present research targets include the integration of agricultural and ecological systems at a range of spatial scales. As well as, the impact of introduced carp and domestic grazing herds on the ecology of riparian zones and off-river waterbodies and the role of flood pulses in the ecology of floodplain-river systems. Dr Robyn Watts Dr Watts’ current research at CSU is focussed on developing biological indicators of river health for the assessment of environmental flows. Dr Robyn Watts has recently completed a research project entitled ‘Assessment of Environmental Flows for the Murrumbidgee River: developing biological indicators for assessing river flow management’. This work is at the forefront of environmental flows research in Australia as we examined the association between instream and riparian biological indicators and hydrological variables. This work follows on from her previous studies of the population genetics of golden perch, silver perch and eel-tailed catfish within the Murray-Darling Basin. Robyn has also completed a consultancy project for Ok Tedi Mining Limited in Papua New Guinea, examining the population genetics and morphological variation in two freshwater fishes, Nematalosa flyensis and N. papuensis, from the Fly River. Dr Darren Ryder Dr. Ryder is an aquatic ecologist with extensive experience in the field of freshwater ecology, specifically dealing with the microbiology/biogeochemistry of wetland and



riverine systems. His current research at CSU as an Australian Research Council Post Doctoral Research Fellow is focussed on developing biological indicators of river health for the assessment of environmental flows across many NSW river systems. This research specifically examines biofilm structure and function and the response of consumers to changes in biofilm attributes, and builds on a previous Post Doctoral position at CSU that developed biofilms and macroinvertebrates as biological indicators for the assessment of environmental flows in the Murrumbidgee River. Dr. Ryder has a thorough knowledge of riverine hydrology and instream processes and has been a member of many scientific panels including method design team for the NSW Integrated Monitoring of Environmental Flows and 'river health report card' committees. Dr Adrienne Burns Dr Burns is an aquatic ecologist with extensive experience in the field of freshwater ecology, specifically dealing with the biogeochemistry of wetland and riverine systems. Her current research at CSU as a Post Doctoral Research Fellow examines the role of river floodplain interactions during high flows. Dr. Burns has an extensive background in biofilm structure in riverine systems and has developed a thorough knowledge of freshwater algal taxonomy through her doctoral research. Dr Burns and Dr. Ryder have recently published the first Australian based research paper documenting bacterial activity during flood events using a recently developed fluorometric technique. Bruce Mullins Bruce is the Manager of Johnstone Centre – Environmental Consulting and will act as Project Manager. Bruce has extensive experience in planning and conducting flora and fauna surveys as well as planning, costing and organising large-scale operations. He has planned, managed and written numerous reports for the government and private sectors. He has comprehensive knowledge of Federal, State and local government legalisation and planning. Lachlan Sutherland Lachlan is a consultant with Johnstone Centre - Environmental Consulting. He is an ecologist with experience in ecological assessment, riverine ecosystems and GIS. He has recently completed an Honours degree studying the effects of cattle on microinvertebrate diversity in selected billabongs on the Murrumbidgee River Floodplain. He also possesses excellent data management, report writing and liaison skills and has experience in the review of biodiversity literature. MARTIN ASMUS Martin is a Technical Officer and has been involved in a wide range of research projects in aquatic systems, including research on environmental flows in the Murrumbidgee River and review of weirs in the Murrumbidgee and Murray rivers.



6.0 SUPPORTING INFORMATION

6.1 Johnstone Centre

The Johnstone Centre is one of five research centres at Charles Sturt University. The Centre is made up of academics, postgraduate students, consulting staff and support staff. The Johnstone Centre’s mission is to research the fundamental ecological processes involved in the conservation of the diverse range of ecosystems and protected areas in Australia and overseas, which is crucial to their successful management. Our research contributes to developing a greater understanding of ecological processes on a community and landscape scale and applies the results of such research to challenge current practices, policies and planning. These studies enable us to establish the conditions necessary for ecologically sound management, as well as biodiversity assessment and conservation evaluation. Johnstone Centre - Environmental Consulting and associated academics have worked extensively in the riparian zones of the Murrumbidgee Catchment, with research focused on biodiversity, flora and fauna surveys, environmental flows and the impacts of agricultural practices on riparian zones. 6.2 Insurances The Johnstone Centre is covered by insurance policies held by Charles Sturt University. A

Workers Compensation Policy is held with MMI Insurance Group (Policy No. MWR

0024606) and Public Liability and Professional Indemnity Policies are held with Uni

Mutual (Policy No. AU CSU 990002).

6.3 Management System

Quality assurance is a key part of work in a consulting environment. Charles Sturt University has processes in that place ensure proper management and project function. The Office of Research and Consultancy (ORC) contains records of contracts, budgets, reports and other key documents and is independently audited. ORC has an established policy for Outside Professional Activity by staff, which adheres to a number of steps critical in establishing outside consultancies.



Within the Johnstone Centre, the Environmental Consultancy Management Committee is made up of the Centres Director, Professor Alistar Robertson, Associated Director, Dr Allen Curtis and other senior researches. Documents are reviewed prior to release for correct spelling and grammar, appropriate use of scientific methods and whether conclusions, recommendations or proposed strategies are sound. A Directors Project Review Form can be inserted into a document to certify that the document has been reviewed to the satisfaction of the review committee and what it has been reviewed for.



Ammendments Requested by Murray Darling Basin Commission Brian Lawrence Manager, Rivers Program Murray Darling Basin Commission Dear Brian, Following are response to the seven questions provided in your fax. Question 1. Could you state more explicitly the hypotheses – the questions that are being addressed in each of the monitoring themes. Water quality Hypothesis. 2. The concentration of DOC, POC and suspended solids will increase during the variable

flow releases compared to constant flows as a result of increased riverbank and floodplain inundation and in channel resuspension. We predict there will be increased loading of carbon with distance downstream.

Water quality parameters listed in the tender will be used as covariates for the interpretation of biofilm composition and productivity and macroinvertebrate data. Physical habitat stability Hypothesis. 1. Peak flow releases will lead to bed-load movement in cobble habitats and result in the

erosion of fine sediments in habitats with increased water velocity. Biofilm composition Based on the results of Watts et al (2001) we predict that if there are forty days of constant flow releases after the final harmony release, the biofilms will reach a stable state for biomass, composition and productivity. These data will be compared to the data collected during the harmony releases. Hypotheses. 3. Algal and total biomass from cobble substrata will decrease following peak flow

releases compared to the biomass prior to the release due to scouring from increased velocity.

4. Peak flow releases will change the community composition of algal biofilms and promote early successional algal taxa on cobble substrata due to scouring from increased water velocity.

Benthic Metabolism Hypotheses. 3. Peak flow releases will increase carbon respiration of biofilms on cobble substrata from

deep habitats due to scouring from increased water velocity and light deprivation from increased water depth.



4. Newly wetted cobble with established biofilm communities will have increased carbon production relative to those newly wetted cobbles that do not have an established biofilm community.

Enzyme activity Hypotheses. 3. Peak flow releases will increase the overall enzyme activity of biofilms (specifically

increase the activity fatty acids and proteins) on cobble substrata due to scouring from increased velocity.

4. Variable flow releases will increase the overall enzyme activity in the water column (specifically increase the activity fatty acids and proteins) due to increased riverbank and floodplain inundation and in channel resuspension. We predict there will be increased overall enzyme activity with distance downstream.

Macroinvertebrates The samples collected during the forty days of constant flow releases after the final harmony release will be compared to the data collected during the harmony releases. Hypotheses 3. Variable flow releases will increase algal diversity on cobble substrata and will result

in a higher diversity of macroinvertebrates in cobble habitats. 4. Variable flow releases will increase algal diversity on cobble substrata and increase the

relative abundance of primary consumers on cobble habitats. Question 2. Can you include continuous turbidity measurements, as well as advise on constraints or difficulties with continuous monitoring equipment and suggest responses? Yes. We have three Yeo-kal multiprobes that can be used to continuously monitor turbidity, pH, Conductivity, DO (mg/L) and Temperature. These multiprobes can be set up at sites 1, 3 and 6 to continuously monitor parameter trends in the Mitta Mitta over the entire assessment period. The multiprobes will be calibrated and the data downloaded regularly during the collection of water quality samples to ensure quality of data. There are no foreseen constraints of difficulties with the use of this equipment. Question 3. Would Dr Watts be available for greater input into the preparation of the report? Dr Robyn Watts, Dr Darren Ryder and Professor Alistar Robertson are available for greater input into the preparation of the report. Dr Watts will analyse and interpret the macroinvertebrate and water quality data. Dr Ryder will analyse and interpret the biofilm and water column metabolism data and the enzyme activity results. Prof Robertson will calculate the POC and DOC loads. The time allocated to Lachlan Sutherland will be reduced to allow for increased input of the above members. Question 4. Would you be able to include some direct measurements of the impact on physical habitat? Yes. Direct measurements of the impact on physical habitat can be measured in both the upper and lower reaches of the Mitta Mitta River. These measurements will include the movement of cobbles in response to the variable flow releases.



Method: Cobbles of small, medium and large diameters will be selected at each of the three Mitta Mitta River sites, 1, 3 and 6. One third of the cobbles from each of the size classes will be recovered following flow period 2, one third following flow period 3 and one third forty days after the end of the harmony releases. Presence or absence of marked cobbles will determine the impact of single flood events and combined flood events on cobble movement. The second measurements will be based upon removal / accrual of sediment in the lower reaches, within the bed and on the banks at certain sites along the Mitta Mitta River. Method: This method will be used at sites 3 and 6 (depending upon sediment type). Metal rods will driven into the sediment across the channel of the Mitta Mitta River. These rods will cover the river bed and banks. Metal discs (with centre punched holes) will then be placed onto each rod. The disks will be free moving. The rod will be marked at the original position of the disk. After each flow event the amounts of sediment accrual and/or removal can be calculated. The technique will quantify the extent of sediment scouring (removal) or deposition. Question 5. Can you advise on how you will maintain awareness of river operations and what flexibility you have in responding to changing weather and release conditions? Dr Watts has spoken to Gary Tuenon (Dartmouth Dam) and he indicated that instantaneous river heights at Colemans and Talladoon gauges are available via telemetry on numbers that will be provided to us. He also indicated that information on the 24hour releases will be faxed to us on request. Question 6. Would you be able to include an external peer review function for your draft report? Yes. Dr Watts has contacted Craig Schiller from Water Ecoscience (Victoria) and Jane Roberts, Independent Consultant, in respect to this request. Both Craig and Jane have agreed to review the report. Craig Schiller has been part of the Mitta Mitta River Monitoring Group responsible for monitoring macroinvertebrates within this system since 1986. Reference was made to their 1999/2000 report in our original quote. (AWT 2000). Craig also peer reviewed Watts et al (2001), ‘Assessment of Environmental Flows on the Murrumbidgee River’. Craig requires has indicated that his fee will be $1000 to review this document. Jane Roberts is an independent consultant, and was previously a Senior Research Scientist with CSIRO. Jane is a member on the Technical Advisory Panel for the Clarence River, has several expert panels for the assessment of river health and is currently part of the Lower Snowy River Rehabilitation Trial Project. Jane also reviewed Watts et al (2001). Jane requires one day to complete the review and has indicated that her fee will be $1100 inclusive of GST. Robyn Watts Charles Sturt University 27/11/2001

