Ecological evidence links adverse biological effects to pesticide and metal contamination in an...

Ecological evidence links adverse biological effects

to pesticide and metal contamination in an urban

Australian watershed

Claudette R. Kellar1*, Kathryn L. Hassell1, Sara M. Long1, Jackie H. Myers1, Lisa

Golding1†, Gavin Rose2, Anupama Kumar3, Ary A. Hoffmann1 and Vincent Pettigrove1

1Centre for Aquatic Pollution Identification and Management (CAPIM), Department of Zoology, The University of

Melbourne, Parkville, Vic. 3010, Australia; 2Agricultural Research Division, Agriculture Group, Department of

Environment and Primary Industries (DEPI), Terrace 4, Ernest Jones Drive, Macleod, Vic. 3085, Australia; and3Centre for Environmental Contaminants Research, CSIRO Land and Water, Private Bag No. 2, Glen Osmond, SA

5064, Australia

Summary

1. Aquatic ecosystems near urban areas are often ecologically impaired, but causative factors

are rarely identified. Effects may be revealed by considering multiple lines of evidence at

different levels of biological organization.

2. Biological impairment is evident in the urban section of the Upper Dandenong Creek

Catchment (Victoria, Australia). We assessed whether episodic sewage spills or other pollutants

were the cause of poor ecological condition in the stream. The evidence evaluated included

chemical and invertebrate assessments, caging studies of mudsnails Potamopyrgus antipodarum,

antioxidant biomarkers and endocrine disruption-related endpoints in fish (Carassius auratus

and Gambusia holbrooki) and toxicological studies with chironomids (Chironomus tepperi).

3. A combination of metals and pesticides is likely to be affecting the aquatic fauna across

all biological levels, with macroinvertebrate communities, P. antipodarum and C. tepperi pop-

ulations and C. auratus individuals all ecologically impaired. Adverse alterations to aquatic

fauna were consistently seen in Bungalook Creek and persisted downstream of this confluence

into Dandenong Creek.

4. In addition, chemical assessments and toxicity identification evaluation (TIEs) resulted in

several point sources of both metals and pesticides being identified as origins of impairment.

This contrasted with an expectation that adverse effects were likely to be associated with

sewer-related pollution. As a consequence, target areas and specific pollutants were identified

for remediation instead of an expensive sewer upgrade.

5. Synthesis and applications. The results demonstrate that it is important to investigate bio-

logical effects in different taxa, in both the laboratory and field, to understand which stressors

are causing adverse effects on faunal assemblages. When adverse effects are seen across multi-

ple levels of biological organization and caused by the same pollutant from an identifiable

source, there is a clear remedial path for managers.

Key-words: ecological impairment, ecotoxicology, endocrine disruption, episodic sewage

discharges, toxicity, weight-of-evidence

Introduction

Pollution-induced changes in aquatic communities are

particularly prevalent in urban ecosystems. For example,

pollutants such as pesticides (Liess & von der Ohe 2005)

and metals (Clements et al. 2000) that enter waterways

through storm water discharges, road run-off and indus-

trial and mining activities have all been shown to detri-

mentally affect aquatic fauna. Of growing concern are

domestic and industrial wastewater discharges, including

regular treated sewage effluents and episodic overflows of

untreated sewage during rain events. These discharges can

†Present address: CSIRO Land and Water, New Illawarra Road,

Lucas Heights, NSW 2234, Australia

*Correspondence author E-mail: [email protected]

© 2013 The Authors. Journal of Applied Ecology © 2013 British Ecological Society

Journal of Applied Ecology 2014, 51, 426–439 doi: 10.1111/1365-2664.12211

lead to increased nutrients and organic pollutants in

receiving waterways and micropollutants such as pesti-

cides, surfactants and hormonally active compounds

[endocrine disrupting chemicals (EDCs)], all of which can

cause ecological impairment.

Rapid bioassessment of macroinvertebrates and physi-

cochemical data are the most common methods for

stream pollution assessments (Chessman 1995; Bonada

et al. 2006). However, current monitoring regimes cannot

effectively isolate pollution effects, especially when only

field studies are carried out. Hence, there has been consid-

erable effort to quantify the impacts of contaminated sedi-

ments and surface waters on aquatic fauna through

laboratory and field ecotoxicological studies (Burton, Pitt

& Clark 2000). For example, laboratory studies have

measured toxicity using bioassays of cultured taxa

(Choung et al. 2010), and field microcosms have measured

the response of invertebrate communities to contaminated

sediment (O’Brien et al. 2010). In situ bioassays, where

caged aquatic organisms are exposed to in-stream

conditions, measure biological responses over time and

can incorporate impacts from pulse events such as

episodic sewage spills, which are often difficult to assess

in traditional laboratory-based bioassays (Crane et al.

2007).

In addition to these assessments, information on partic-

ular pollutants can emerge from physiological and mor-

phological changes in individual organisms by measuring

different types of biological responses (biomarkers). These

can act as an early warning, signalling that individuals

are responding (usually negatively) to the pollutant, and

if they continue to be exposed, then population decline is

likely to occur, ultimately compromising the ecological

health of the system. Antioxidant enzymes are general

biochemical biomarkers that demonstrate the occurrence

of oxidative stress in aquatic fauna following exposure to

chemicals, providing direct evidence of chemical exposure

(van der Oost, Beyer & Vermeulen 2003). EDCs can

interfere with endocrine signalling in organisms and

adversely impact reproductive viability in aquatic inverte-

brates and vertebrates. Specific biological effects of EDCs

include elevated levels of the blood protein vitellogenin

(Vtg; a biomarker for oestrogenic EDCs) in male and

juvenile fish, masculinization or feminization of the

internal or external genitalia and poor development of

embryos (Jobling & Tyler 2003; Gust et al. 2010). These

effects have been widely linked to exposure to sewage-

related pollutants (e.g. Jobling et al. 2004; Jenkins et al.

2009).

There are numerous eco-epidemiological studies that

have investigated causation of altered biological condition

in aquatic fauna (Clements et al. 2002; Haake et al. 2010;

Cormier et al. 2013). In urban systems there are a num-

ber of stressors, including modified flows, habitat alter-

ation and numerous pollution sources, that contribute to

biological impairment, and it is increasingly difficult to

isolate which stressor is causing the greatest ecological

stress. One way of elucidating causal factors contributing

to aquatic impairment is to apply a weight-of-evidence

approach (WoE), whereby possible ecological impacts are

determined based on the evidence of detrimental relation-

ships between biological, chemical and physical data

(Chapman 1990; Suter, Norton & Cormier 2010).

In this study, we applied multiple lines of evidence at

the individual, population and community levels from

both laboratory and field data as evidence of biotic stress,

as well as chemical assessments within an urban waterway

(Upper Dandenong Creek) south-east of Melbourne,

Australia. Urbanization has led to several modifications

of this natural waterway which has caused a decline in

the ecological health of the creek, including loss of

macroinvertebrate communities, native fish and platypus

(Melbourne Water, unpublished data). Contributory fac-

tors include channelling, modified flows, urban storm

water, discharges of wastewater, barriers to migration of

aquatic life and a lack of streamside vegetation. A study

by Marshall et al. (2010) showed that macroinvertebrate

communities were poorest in the urban areas of the

Upper Dandenong Creek Catchment before showing some

recovery in the urban areas in the Lower Dandenong

Creek Catchment. Physicochemical parameters, nutrients,

flow alteration, impervious catchment and habitat altera-

tions were similar throughout the urbanized catchment,

and the study identified that the likely stressors to the

macroinvertebrate community in the Upper Dandenong

Catchment were stream pollutants. Our study therefore

only focusses on isolating the effects of stream pollutants

in the Upper Dandenong Catchment. Poor water quality

from urban and industrial sources and occasional episodic

sewer spills during storm events may all contribute to the

observed stress.

We included a variety of biological assessments with

the overall aim of investigating the potential effects of epi-

sodic sewage spills via an emergency relief structure

(ERS) on resident aquatic fauna and separating these

effects from other sources of pollutants present within the

catchment. In addition, we also considered the most

appropriate management actions required to enhance

aquatic ecosystem health in the catchment using a WoE

approach.

Materials and methods

DESCRIPTION OF THE UPPER DANDENONG CREEK

CATCHMENT AND STUDY AREA

The Upper Dandenong Catchment is steep and narrow and

drains a 94-km2 catchment area in the south-eastern suburbs of

Melbourne, Australia. The top of the catchment is a largely

intact native forest. The forested catchment gives way to pasture

before the creek flows into a retarding basin, is piped under-

ground for 4 km and resurfaces in the urban (both residential

and industrial/commercial) suburbs of Melbourne. Major tribu-

taries entering into Dandenong Creek are Old Joes, Bungalook

and Heatherdale Creeks. During high rainfall events raw sewage

© 2013 The Authors. Journal of Applied Ecology © 2013 British Ecological Society, Journal of Applied Ecology, 51, 426–439

Linking biological impacts to pollution 427

may enter Dandenong Creek via an emergency relief structure

(ERS) in the urban area (Fig. 1).

This study was conducted in Dandenong Creek between the

suburbs of The Basin (�37�843360° 145�334925°) and Vermont

(�37�845496° 145�210813°). A total of ten sites were examined,

including two reference sites (Dandenong Creek, Dobsons Lane,

DN1; the Liverpool Retarding Basin, LRB), four sites upstream

(Old Joes Creek, King Street, OJC; Bungalook Creek, Bungalook

Road East, BNG; Dandenong Creek, King Street, DN2 and

Ricdanic Drive, DN3) and four sites downstream of the ERS

(Dandenong Creek, Wantirna Road, DN4; Shilbor Drive, DN5

and Boronia Road, DN6; Heatherdale Creek, Shilbor Drive,

HEA) (Fig. 1).

ENDPOINTS MEASURED

This study utilized a holistic approach to gather evidence and elu-

cidate the likely pollutant stressors causing the decline of aquatic

fauna in the urban catchment. A recent assessment of macroin-

vertebrate communities was conducted prior to this study, and

this information was used in the overall WoE assessment

(Marshall et al. 2010). To determine whether pollutants were

impacting resident fauna, invertebrate and fish conditions were

assessed. Toxicological tests were conducted to assess whether

sediment pollutants were responsible for a depauperate fauna,

and toxicity identification evaluation (TIE) assessment was

conducted to elucidate the class of chemicals causing the stress.

Biological responses in two fish species, a short- and long-lived fish,

were investigated to determine effects of episodic sewage spills.

SEDIMENT COLLECTION AND CHEMICAL ANALYSIS

Depositional stream sediments were collected from ten sites

between September and November 2010 following Marshall et al.

(2010). Sediment samples were analysed for total metals, total

petroleum hydrocarbons (TPH), total organic carbon (TOC%),

total Kjeldahl nitrogen (TKN) and total phosphorus (TP) by the

ALS Laboratory Group (Springvale, Melbourne), and 84 pesti-

cides were analysed by the Department of Environment and

Primary Industries (Macleod, Victoria) (see Appendix S1 in Sup-

porting information). Results were compared to the freshwater

sediment quality guidelines by MacDonald, Ingersol and Berger

(2000) (metals and organochlorine pesticides), ANZECC/ARM-

CANZ (2000) (silver) and Pettigrove & Hoffmann (2005) (TPH).

Only metals that are part of the sediment quality guidelines and

pesticides above the limits of detection are presented (see Table

S1 in Supporting information). Toxicity units were calculated and

summed for synthetic pyrethroids (see Appendix S1, Supporting

information).

SEDIMENT TOXICOLOGY

Six litres of fine (<64 lm) sediments were collected from six sites

(DN1, LRB, BNG, DN3, DN5 and DN6) and 2 L from an

Fig. 1. Location of sampling sites and sewer release site (ERS) in the Upper Dandenong Creek Catchment.


428 C. R. Kellar et al.

external reference site (Glynns Wetland, �37�739167°145�194167°) and sent to CSIRO Land & Water (Adelaide) for

toxicological analyses. Standard sediment toxicity tests using the

chironomid Chironomus tepperi Skuse (Diptera: Chironomidae)

were based on the procedures of Stevens (1993) and USEPA

(2000) with modifications (see Appendix S1, Supporting informa-

tion). Survival and length of chironomid larvae were recorded for

the growth assays, and the number of emerging adults was

counted daily for the emergence assay.

Toxicity identification evaluation (TIE) tests were based on

USEPA (2007) procedures with modifications and conducted on

sediments from the same sampling locations and external refer-

ence site as the other sediment toxicity assays to assess the toxic-

ity of organic pollutants (using pyrolized, activated coconut husk,

PCC) and metal pollutants (using SIR 300) (see Appendix S1,

Supporting information).

AQUATIC FIELD CAGING EXPERIMENTS

A total of 80 field-collected mudsnails Potamopyrgus antipodarum

Gray from a non-contaminated site were assigned to three repli-

cate cages and deployed at each site (DN1, LRB, OJC, BNG,

DN3, DN4, DN5 and DN6) in December 2010 (see Appendix

S1, Supporting information). Sites DN2 and HEA were excluded

as cages were not able to be fully submerged. The exposures

lasted 42 days (except at site BNG where exposure lasted

28 days). Mudsnail survival across all sites was compared to the

reference site DN1.

MORPHOLOGICAL, PHYSIOLOGICAL, B IOCHEMICAL

AND HISTOLOGICAL MEASUREMENTS OF CONDIT ION

IN FISH

Two fish species, goldfish Carassius auratus Linnaeus and eastern

mosquitofish Gambusia holbrooki Girard, were used in this study

and were collected upstream (LRB) and downstream (C. auratus:

DN4 and DN6; G. holbrooki: DN5) of the ERS in September

and November 2010 and January and February 2011. The

upstream site drains into a subterranean pipe, creating a barrier

to upstream movement by downstream fish.

GOLDFISH CARASSIUS AURATUS

Goldfish were captured from sites DN4 and DN6 by electrofish-

ing, and a seine net was used to capture fish from the LRB site.

All fish were euthanized in clove oil followed by cervical transec-

tion, then weighed, measured and dissected (blood sample,

gonads, liver, bile and gills) (See Appendix S1, Supporting infor-

mation).

MORPHOLOGICAL AND PHYSIOLOGICAL

BIOMARKERS

External condition was assessed by recording the incidence of

ulcerations and lesions and calculating physiological tissue indi-

ces: gonadosomatic index (GSI) [Gonad weight (g)/Total body

weight (g)] 9 100; the liver somatic index (LSI) [Liver weight

(g)/Total body weight (g)] 9 100; and the condition factor (CF)

[Total body weight (g)/Fork length (cm3)] 9 100 for each

goldfish.

BIOCHEMICAL BIOMARKERS

Liver and gill samples were prepared according to the method of

Oliva et al. (2010) (see Appendix S1, Supporting information).

Glutathione S-transferase (GST), glutathione reductase (GR) and

catalase (CAT) activities were measured. All biochemical biomar-

kers are expressed as lmol min�1 mg�1 protein. Vitellogenin

(Vtg) concentrations in the plasma of all goldfish were measured

and reported as relative absorbance units (%) (see Appendix S1,

Supporting information).

GONAD HISTOPATHOLOGY

Male gonadal sections were examined for the presence of plaques

and fibrous tissue, testicular oocytes, altered spermatogenesis and

increased testicular degeneration, while female gonadal sections

were examined for plaques, increased oocyte atresia, altered

oogenesis and reduced post-ovulatory follicles, as described in the

OECD document (Johnson, Wolf & Braunbeck 2009) (see

Appendix S1, Supporting information).

EASTERN MOSQUITOFISH GAMBUSIA HOLBROOKI

A minimum of 50 eastern mosquitofish were caught at each site

(see Appendix S1, Supporting information). A suite of comple-

mentary ratios of anal fin morphology were measured, including

gonopodium length (GP4), length (GP4)/standard body length

ratio, the index of elongation (4:6 ratio), gonopodium extension

(GPx) and percentage of fish with hooks (Game et al. 2006).

STATISTICAL ANALYSIS

For all field data, means and 95% confidence intervals (CIs),

assuming normal distribution, were calculated for each endpoint.

Confidence intervals for percentage data were obtained from

analyses on angular-transformed proportions which were then

backtransformed for graphical presentation. Survival, growth and

emergence of chironomids were compared between sites and

between treatments using two-way ANOVAS. Chironomid survival

and emergence data were arcsine-transformed. Statistical analyses

were performed using SPSS 20.0 (Pearson Education, SPSS Inc.

IL, USA).

WEIGHT-OF-EVIDENCE APPROACH

We applied a logic system WoE determination for establishing

causality based on the sediment quality triad approach as outlined

by Chapman (Chapman, McDonald & Lawrence 2002) and Bur-

ton (Burton et al. 2002) (see Table S2 in Supporting information).

Results

BIOLOGICAL HEALTH OF THE DANDENONG

CATCHMENT

The overall ecological condition of the reference site is

very good, with macroinvertebrate endpoints all above the

Environmental Protection Authority of Victoria State

Environmental Protection Policy Objectives (EPA SEPP)



(EPA Victoria 2004) (with exception of AUSRIVAS), a

high percentage of native fish and the presence of crayfish

and platypus (Table 1). Biological impairment arises in the

urban area of the Upper Dandenong Creek Catchment,

both upstream and downstream of the ERS and in Bunga-

look Creek, with all macroinvertebrate endpoints failing the

EPA SEPP Objectives (2004), the dominance of exotic fish

and the disappearance of crayfish and platypus (Table 1).

SEDIMENT CHEMISTRY

Sediments from reference sites DN1 and LRB contained

concentrations of metals and TPH below threshold values,

and only one pesticide was detected in low concentrations

at site DN1 (Table 2). Across all other sites, stream sedi-

ment was contaminated with hydrocarbons, metals and

pesticides. OJC contained toxic concentrations of syn-

thetic pyrethroid insecticides (bifenthrin and cypermeth-

rin) and metals (Cd, Cr, Ni, Cu, Pb, Ag and Zn)

compared to the reference sites above this tributary, sug-

gesting that OJC is the source of these pollutants entering

Dandenong Creek (Table 2 and Fig. 1). High concentra-

tions of these metals were also found in Dandenong

Creek downstream of the confluence of OJC. At site

DN2, Cu, Pb, Ni and TPH exceeded the threshold effect

concentration (TEC), and Zn exceeded the probable effect

concentration (PEC) and contained toxic concentrations

of synthetic pyrethroids (bifenthrin, cypermethrin and per-

methrin). These chemicals were sources of pollution enter-

ing the catchment, as they were not found in high

concentrations at the reference sites. At all sites down-

stream of DN2 and in the tributaries BNG and HEA

TPH was above the TEC (Table 2). Sediments at BNG

had toxic concentrations of permethrin and bifenthrin

(4�8 TUs) and elevated concentrations of the fungicides

tebuconazole and propiconazole compared to sites in

Dandenong Creek upstream of this tributary (Table 2).

Another source of contamination appeared to occur

between DN4 and DN5, with elevated concentrations of

Cu, Pb, Zn, and the organochlorine pesticides DDE and

dieldrin observed at site DN5 compared to DN4. The

most toxic concentrations of synthetic pyrethroids (6�4TUs) were found at site DN6 (September sampling only).

In addition, four organochlorine pesticides were detected

within the urban areas between DN2 and DN6, and some

of these exceeded the TEC (Table 2).

SEDIMENT TOXICOLOGY

Water quality parameters were similar through time and

between sites in the 5-day C. tepperi growth experiment

(see Table S3 in Supporting information). No field sedi-

ments were acutely toxic to C. tepperi, with mean survival

>80% in all treatments and no significant differences

between any field sites compared to the external reference

site sediment (control) (F6,18 = 0�460, P = 0�831) (Fig. 2a).There were significant differences in C. tepperi growth

between sites (F6,63 = 5�298, P < 0�001), and both SIR 300

and PCC increased growth by more than 20% compared to

no manipulation (F2,63 = 43�069, P < 0�001) (Fig. 2b).

There was also a significant site and treatment interaction

(F12,63 = 5�519, P < 0�001). No significant difference

between sites (F2,54 = 0�053, P = 0�998) or interaction

between site and treatment (F2,54 = 0�686, P = 0�733) was

Table 1. Biological health in the Upper Dandenong Creek Catchment, 1994–2007

Metrics Source

Reference

Upstream of ERS

Downstream of ERS

Dandenong Creek Dandenong Creek Bungalook Creek Dandenong Creek

Macroinvertebrates* Melbourne Water

Macroinvertebrate

Database

SIGNAL Meets SEPP Fails SEPP Fails SEPP Fails SEPP

Total no. of families Above SEPP Fails SEPP Fails SEPP Fails SEPP

AUSRIVAS Fails SEPP Fails SEPP Fails SEPP Fails SEPP

Key families Above SEPP Fails SEPP Fails SEPP Fails SEPP

Fish Melbourne Water

Fish reports

% Native fish 67% 50% 33% 20%

Total no. of taxa 3 4 3 5

Central highlands crayfish

Presence/Absence Melbourne Water

Fish report

Present Absent Absent Absent

Platypus Melbourne Water

Waterways report

Presence/Absence Present Absent Absent Absent

*Macroinvertebrate indices are described and measured against the EPA Victoria SEPP Guidelines (2004) for Region B2 (reference) and

Region B4.

SIGNAL: A biotic index of water pollution based on tolerance or intolerance of the biota to pollution.

AUSRIVAS: A predictive model that predicts the macroinvertebrates which should be present in specific stream habitats under reference

conditions.



Table

2.Sedim

entconcentrationsofmetals,nutrients

andpesticides

sampledfrom

reference,upstream

anddownstream

sitesoftheem

ergency

relief

structure

(ERS)in

theUpper

Dandenong

Catchment

Chem

icals

Reference

Upstream

ofERS

Downstream

ofERS

TEC*

PEC†

DN1

LRB

OJC

DN2

BNG

DN3

DN4

DN4‡

HEA

‡DN5‡

DN6

DN6‡

Sedim

entmetalsandnutrients

(mgkg�1dry

weight)

Ag

<2

<2

52†

<2

<2

16†

7†

2<2

34†

4†

13�7

As

<5

<5

98

78

5<5

<5

66

<5

9�79

33

Cd

<1

<1

3*

<1

<1

1<1

<1

<1

<1

<1

<1

0�99

4�98

Cr

25

24

78*

42

33

37

25

16

14

24

26

25

43�4

111

Cu

14

16

268†

91*

54*

91*

50*

26

44*

94*

70*

71*

31�6

149

Pb

18

19

284†

66*

76*

113*

65*

44*

49*

91*

98*

111*

35�8

128

Ni

10

931*

35*

17

20

15

89

15

15

14

22�7

48�6

Zn

74

77

2470†

1080†

1080†

1380†

812†

434*

520†

950†

1020†

749†

121

459

Hg

<0�1

<0�1

<0�1

<0�1

<0�1

<0�1

<0�1

0�2

0�2

0�1

<0�1

0�3

0�15

1

TKN

2400

2540

5010

5330

2830

3660

2900

1860

2880

2340

3750

2180

TP

446

914

949

913

615

681

583

387

470

494

705

490

TOC

(%)

2�24

2�24

4�53

4�5

3�47

3�87

2�75

3�02

3�2

3�51

3�01

2�57

TPH

(sum)

<50

<50

820

1510*

980*

930*

1060*

630

1210*

1140*

930*

480

860

1720

Sedim

entpesticides

(lgkg�1dry

weight)

Bifenthrin

<5

<5

27

29

74

53

36

<5

<5

<5

47

<5

Cyfluthrin

<4

<4

<4

<4

<4

<4

<4

<4

<4

<4

22

<4

Cyhalothrin

<5

<5

<5

<5

<5

68

<5

<5

<5

18

<5

Cypermethrin

<5

<5

25

19

<5

18

15

<5

<5

<5

17

<5

Esfenvalerate

<4

<4

<4

<4

<4

<4

<4

<4

<4

<4

8<4

Permethrin

<20

<20

<20

70

268

23

75

<20

<20

<20

73

<20

Sum

oftoxicity

units(TUs)§

2�6

1�7

4�8

3�4

6�4

Heptachlor

epoxide

<2

<2

<2

<2

<2

<2

<2

<2

<2

<2

<2

22�47

16

p,p′-DDE

<3

<3

4*

3<3

5*

5*

36�5*

6*

6*

4*

3�16

31�3

Dieldrin

<4

<4

18*

6*

14*

13*

9*

10*

13*

11*

18*

28*

1�9

61�8

Totalchlordane

<4

<4

10*

<4

5*

4*

<4

<4

5*

6*

6*

8*

3�24

17�6

Tebuconazole

<4

<4

5<4

28

17

13

6<4

10

11

6

Propiconazole

<4

<4

5<4

25

21

12

6<1

810

4

Trifloxystrobin

1<1

<1

<1

<1

<1

<1

<1

<1

<1

<1

<1

<‘number’meansbelow

limitofdetection.

*Concentrationsexceedingthreshold

effect

concentrations(TEC)are

indicatedformetals,organochlorines

(MacD

onald,Ingersol&

Berger

2000)andsilver

(ANZECC/A

RMCANZ

2000)andthe

proposedTEC

forhydrocarbons(Pettigrove&

Hoffmann2005).

†Concentrationsexceedingprobable

effect

concentrations(PEC)are

indicatedformetals,organochlorines

(MacD

onald,Ingersol&

Berger

2000)andsilver

(ANZECC/A

RMCANZ2000).

‡Samplescollectedin

Novem

ber

2010.Allother

samplescollectedduringSeptember

2010.

§Toxicityunits(TUs)

forsynthetic

pyrethroidsare

basedontheHyalellaazteca10-d

sedim

entLC50provided

byAmweg

etal.(2006).



found after the removal of the external reference site, sug-

gesting that all sediments were adversely affecting growth

as evident from the graph where growth was consistently

increased by 20–30%. There was an increase in growth fol-

lowing SIR 300 and PCC treatments compared to the treat-

ment without manipulation (F2,54 = 57�794, P < 0�001),suggesting that both metals and organic compounds

affected the growth of C. tepperi within the catchment

(Fig. 2b). Emergence was significantly different between

sites (F6,62 = 2�918, P = 0�014) and between the no manipu-

lation, PCC and SIR 300 treatments (F2,62 = 9�814,P < 0�001). No significant differences between sites

(F2,53 = 1�484, P = 0�211) were found after the removal of

the external reference site, suggesting that sediment at all

sites adversely affected C. tepperi populations, with a

decrease in emergence of 30% or more for some treatments

(Fig. 2c). A difference remained between the no manipula-

tion and PCC treatment (F2,53 = 10�585, P < 0�001),

(a)

(b)

(c)Fig. 2. Results of Chironomus tepperi tox-

icity and toxicity identification evaluation

(TIE) tests. Graphs show means and confi-

dence intervals; n = 4 replicates per site;

for (a) midge survival (expressed as %),

(b) midge length (mm) with no manipula-

tion of sediment, manipulation of sediment

with an acid cation-exchange resin (SIR

300) and with pyrolized, activated coconut

husk (PCC), (c) midge emergence (%) with

no manipulation of sediment, manipula-

tion of sediment with SIR 300 and with

PCC in sediment from a reference site and

sites within the Upper Dandenong Creek

Catchment.



suggesting that organic compounds rather than metals

mainly affected the emergence of C. tepperi (Fig. 2c).

AQUATIC FIELD CAGING EXPERIMENTS

Dissolved oxygen, pH and temperature were similar

between sites, while electrical conductivity was higher at

BNG relative to all other sites (see Table S4 in Support-

ing information).

Mudsnail survival was similar between the laboratory

reference and the field reference (DN1). Survival of caged

mudsnails decreased over time at all sites except the

reference site (DN1) and was reduced at BNG (14 and

28 days), DN4 (28 and 42 days) and DN6 (28 days) com-

pared to the upstream reference site (DN1). There was

high variability in mudsnail survival between cages across

all sites in Dandenong Creek downstream of the conflu-

ence of Bungalook Creek (Fig. 3).

MORPHOLOGICAL AND PHYSIOLOGICAL RESPONSES

IN GOLDFISH CARASSIUS AURATUS

Both male and female goldfish were, on average, longer

and heavier at the reference site compared to downstream

of the ERS. Goldfish collected downstream of the ERS

(DN4 and DN6) displayed a greater incidence of lesions

and ulcerations than goldfish collected from the reference

site (LRB) (Table 3).

While there was no difference in female physiological

indices (GSI, LSI, CF) between sites, GSI was higher in

downstream males compared to reference males (Table 3).

BIOCHEMICAL RESPONSES IN GOLDFISH CARASSIUS

AURATUS

Results for liver and gill GST activity, liver GR and CAT

and plasma VTG are reported together as biochemical

biomarkers of exposure. Goldfish collected downstream of

the ERS and the reference site had similar concentrations

of liver GR and plasma VTG (Table 3). Liver and gill

GST activity and liver CAT activity were on average

higher in downstream fish compared to reference. Gill

GST activity in females and liver CAT activity in males

were higher in downstream fish compared to reference.

GONAD HISTOPATHOLOGY IN GOLDFISH CARASSIUS

AURATUS

Atresia (mostly vitellogenic follicles containing yolk) was

observed in all female goldfish, and there was substantial

variation in the incidence between individuals. Post-ovula-

tory follicles were observed in most fish from both refer-

ence and downstream sites indicating that spawning was

occurring in both locations. Some differences were

observed in the proportions of different ovarian cell types

between reference fish compared to downstream fish. The

incidence of ovarian plaques was higher in downstream

compared to reference females (Table 3).

In the male goldfish interstitial spaces were more preva-

lent in downstream fish than upstream fish (Table 3). Tes-

ticular degeneration refers to abnormal or degenerating

male germ cells (Johnson, Wolf & Braunbeck 2009). Most

male goldfish from both reference and downstream loca-

tions displayed some abnormal germ cells, yet there was

no evidence of an increase in testicular degeneration in

downstream males compared to reference male goldfish

(Table 3). While testicular oocytes were observed in two

downstream and one reference male goldfish, it was con-

sidered unremarkable as they were a single, primary or

cortical alveolus oocyte surrounded by normal spermato-

genic tissue (Table 3). Most of the male goldfish collected

in this study had testes containing spermatogenic cells at

all stages of development (spermatogonia, spermatocytes,

spermatids and spermatozoa), and while the proportions

Fig. 3. Survival of Potamopyrgus antipoda-

rum caged at sites over 42 days in the

Upper Dandenong Creek Catchment.

Graphs show means with confidence inter-

vals; n = 3 replicates per site. Survival of

snails was compared at 14, 28 and

42 days. ^no cages remained at this time

point.



Table

3.Summary

ofbiologicalmeasurements

[meanand95%

confidence

intervals

(CI)]forgoldfish

collectedfrom

areference

site

andsitesdownstream

oftheem

ergency

relief

structure

(ERS)

intheUpper

DandenongCatchment

Species,biologicalmeasurements

Reference

Downstream

Reference

Downstream

LRB

DN4&

DN6

LRB

DN5

Fem

ales

Males

Carassiusauratus

nMean

95%

CI

nMean

95%

CI

nMean

95%

CI

nMean

95%

CI

Totallength

(mm)

10

178�8

(150�23

,207�37

)15

141�87

(115�73

,168�00

)20

205�75

(177�63

,233�87

)15

150�13

(122�98

,177�29

)

Weight(g)

10

125�3

(53�76

,196�84

)15

79�73

(24�25

,135�21

)20

197�30

(121�08

,273�52

)15

83�27

(33�54

,132�99

)

Incidence

oflesionsandulcerations(%

)10

015

15�38

20

13�30

15

30�77

Physiology

GSI(%

)10

8�69

(5�15

,12�22

)15

9�99

(7�06

,12�92

)20

3�12

(2�66

,3�57

)15

4�50

(3�72

,5�28

)

LSI(%

)10

1�97

(1�40

,2�54

)15

2�09

(1�47

,2�72

)19

1�70

(1�40

,2�00

)14

1�94

(1�68

,2�20

)

CF

(%)

10

2�35

(2�24

,2�47

)15

2�38

(2�26

,2�5)

20

2�30

(2�23

,2�37

)15

2�21

(2�11

,2�30

)

Biochem

istry

GillsGSTactivity(nmolmin

�1mg�1protein)

926�16

(23�22

,29�11

)12

38�72

(33�32

,44�12

)19

31�87

(27�96

,35�78

)14

37�96

(30�7,

45�22

)

Liver

GSTactivity(nmolmin

�1mg�1protein)

817�26

(10�06

,24�47

)15

26�16

(20�8,

26�46

)19

21�99

(18�68

,25�3)

15

26�24

(23�29

,29�2)

Liver

GR

activity(nmolmin

�1

mg�1protein)

10

36�24

(26�34

,46�13

)15

30�78

(27�14

,34�42

)20

36�14

(32�48

,39�8)

15

36�33

(31�72

,40�94

)

Liver

CAT

activity(lmolmin

�1mg�1protein)

10

1073�26

(833�68

,1312�84

)15

1554�29

(1246�66

,1861�92

)20

1465�01

(1307�21

,1622�8)

15

2168�83

(1790�4,

2547�26

)

Vtg

relativeabsorbance

(%)

10

60�27

(33�37

,87�17

)14

51�39

(32�42

,70�35

)20

4�19

(0�49

,7�89

)13

2�27

(0�00

,5�98

)

Histology(Incidence

score)*

Plaques

10

0�63

(0�00

,1�31

)13

1�47

(0�97

,1�97

)12

0�72

(0�47

,0�97

)15

0�56

(0�19

,0�92

)

Atretic

follicles(fem

ales)

10

1�35

(0�98

,1�72

)13

1�53

(1�09

,1�96

)

Post-ovulatory

follicles(fem

ales)

10

1�27

(0�00

,2�33

)13

1�05

(0�38

,1� 72

)

Testiculardegeneration(m

ales)

12

0�75

(0�56

,0�94

)15

0�77

(0�54

,1�01

)

Interstitialspacesbetweenlobules(m

ales)

12

0�98

(0�52

,1�44

)15

1�73

(1�48

,1�99

)

Fibroustissue(M

ales)

12

1�40

(1�14

,1�66

)15

1�77

(1�56

,1�98

)

Testis–Ova(M

ales)

1/8

2/13

GSI,gonadosomaticindex;LSI,liver

somaticindex;CF,conditionfactor;GST,GlutathioneS-transferase

activity;GR,glutathionereductase

activity;CAT,catalase

activity;Vtg,Vitellogenin.

*Incidence

score

ismeansemiquantitativegonadhistologyassessm

ent,where0=noincidence,1=low

incidence,2=highincidence.



Table

4.Summary

ofweight-of-evidence

approach

–inference

basedonthesedim

entquality

triad(m

odified

from

Chapman,McD

onald

&Lawrence

2002;Burtonet

al.2002)

Reference

Upstream

oftheERS

Downstream

oftheERS

DandenongCreek

(DN1,LRB)

Old

Joes

Creek

(OJC

)

DandenongCreek

(DN2,DN3)

Bungalook

Creek

(BNG)

Heatherdale

Creek

(HEA)

DandenongCreek

(DN4,DN5,DN6)

Chem

icalspresent

Metals

Low

High:Cd,Cr,

Cu,Pb,Ni,Ag,Zn

High:Cu,Pb,Ni,Ag,Zn

High:Cu,Pb,Zn

High:Cu,Pb,Ag,Zn

Pesticides

Low

High:Synthetic

pyrethroids,

Organochlorines

High:Synthetic

pyrethroids,

Organochlorines

High:Synthetic

pyrethroids,

Fungicides,

Organochlorines

High:

Organochlorines

High:Synthetic

pyrethroids,

Organochlorines

Hydrocarbons

Low

High

High

High

High

High

Summary

ofeffect

�++

++

++

+++

Toxicity

Survival

Notim

pacted

Notim

pacted

Notim

pacted

Notim

pacted

Chironomus

tepperi

Growth

and

emergence

Impacted

Impacted

Impacted

Impacted

TIE

growth

Organicsandmetals

Organicsandmetals

Organicsandmetals

Organicsandmetals

TIE

emergence

Organicsandmetals

Organicsandmetals

Organics

Organics

Summary

ofeffect

++

++

Speciesalterations

Macroinvertebrates*

Indices

High:most

meetSEPP

Low:allfailSEPP

Low:allfailSEPP

Low:allfailSEPP

Potamopyrgus

antipodarum

Survival

Notreduced

Notreduced

Notreduced

Significantlyreduced

Reduced

Morphological

Low%

lesions,Heavier

High%

lesions,

Lighter

C.auratus

Physiological

Biochem

ical

Low

GST,CAT

HighGST,CAT

Summary

ofEffect

��

++

++

++

Endocrinedisruption

C.auratus

Vtg

Notelevatedin

males

Notelevatedin

males

Histopathological

Capable

ofreproducing,

Someim

mature

cells

inlargefish

Capable

of

reproducing

Gambusia

holbrooki

Notissuedamage

Tissuedamage

Gonopodial

Norm

al

Norm

al

Summary

ofeffect

++

Overallassessm

ent

Potentialadverse

effects

predicteddueto:tw

oor

more

toxicological

endpoints

reduced;some

histopathologicalchanges

ingoldfish.

Chem

icalsmeasurednot

Potentialadverse

effectspredicted

dueto:elevated

chem

istry

(particularly

metals).No

biologicalim

pact

detected.

Significantadverse

effects

predicteddueto:elevated

chem

istry(m

etals,

hydrocarbonsand

pesticides);tw

oormore

toxicologicalendpoints

reduced;reduced

macroinvertebrate

fauna

Significantadverse

effectspredicted

dueto:elevated

synthetic

pyrethroids(organic

pollutants);tw

o

ormore

toxicological

endpoints

reduced;

Potentialadverse

effectspredicted

dueto:elevated

chem

istry

Significantadverse

effectspredicted

dueto:elevated

synthetic

pyrethroids

(andsomemetals);

twoormore

toxicological

endpoints

reduced;



of the different cell types were not different between refer-

ence and downstream sampling locations, there were five

large fish (> 240 mm total length) collected from the refer-

ence site that contained exclusively immature spermato-

genic cells.

MORPHOLOGICAL RESPONSES IN EASTERN

MOSQUITOFISH GAMBUSIA HOLBROOKI

A total of 126 fish were collected from the reference site

and 49 fish were collected downstream of the ERS. There

were no differences between standard length and weight

of male fish from the reference site [length: mean

19�94 mm (confidence intervals, 19�53, 20�35); weight:

0�16 g, (0�15, 0�17)] compared to the downstream site

[length: 19�44 mm (18�73, 20�15); weight: 0�15 g (0�13,0�16)].Across all of the gonopodial indices, there were no dif-

ferences between the downstream site and the reference

site [gonopodium length (GP4) mm: reference mean 6�68(confidence intervals, 6�56, 6�81), downstream = 6�59(6�38, 6�80); GP4/Standard body length ratio: refer-

ence = 0�34 (0�33, 0�34), downstream = 0�34 (0�34, 0�35);Index of elongation (4:6 ratio): reference = 2�73 (2�71,2�78), downstream = 2�75 (2�67, 2�79) 0�02, gonopodium

extension mm (GPx): reference = 4�16 (4�15, 4�33), down-stream = 4�24 (4�03, 4�29)] for male fish between the refer-

ence site and the downstream site. The percentage of fish

with hooks present on the tip of the gonopodium at the

reference site (84%) was similar to the downstream site

(86%).

WoE

All lines of evidence (chemistry, toxicity, species alteration

and endocrine disruption) are presented in Table 4. The

reference sites, DN1 and LRB contained low levels of

pollutants and overall supported healthy invertebrate pop-

ulations. However, some of the biological endpoints mea-

sured at these reference sites suggested impairment, with

sublethal toxicity observed in C. tepperi and histopatho-

logical changes in some individuals of C. auratus. TIE

manipulations indicated that both metals and organics

were responsible for the sublethal toxicity in C. tepperi,

yet no pollutants were found within the sediment. At this

stage, the source of the observed impairment remains

unknown and requires further investigation.

Across the rest of the sites in Dandenong Creek, a

high number of pollutants were found upstream and

downstream of the ERS. Based on the concentrations of

chemicals present, three main sources were identified:

the tributary Old Joes Creek; the underground section

of Dandenong Creek; and the tributary Bungalook

Creek.

In the urban section of Dandenong Creek, above the

confluence of BNG, a combination of metals and pesticides

was the likely pollutant responsible for an impoverishedTable

4.(continued)

Reference

Upstream

oftheERS

Downstream

oftheERS

DandenongCreek

(DN1,LRB)

Old

Joes

Creek

(OJC

)

DandenongCreek

(DN2,DN3)

Bungalook

Creek

(BNG)

Heatherdale

Creek

(HEA)

DandenongCreek

(DN4,DN5,DN6)

likelyto

bethecause

of

biologicalim

pairment

significantreduction

insnailsurvival;

reduced

macroinvertebrate

fauna;

Metalsless

likely

tobethecause

ofbiological

impairment

reductionin

snail

survival;reduced

macroinvertebrate

fauna;reduced

fitnessofgoldfish;

nosignofendocrine

disruptiondueto

ERSspills

GSI,gonadosomaticindex;LSI,liver

somaticindex;CF,conditionfactor;GST,GlutathioneS-transferase

activity;GR,glutathionereductase

activity;CAT,catalase

activity;Vtg,Vitellogenin.

Bold

indicatesthesourceofthepollutants.

++Significantadverse

effectspredicted;+potentialadverse

effectspredicted;�

noadverse

effectspredicted.

*Macroinvertebrate

indices

are

described

andmeasuredagainst

theEPA

VictoriaSEPPGuidelines

(2004).



invertebrate fauna and sublethal toxicity effects in

C. tepperi populations. Although no biological impairment

was detected in OJC, there were still high concentrations

of metals present that are likely to cause biological

impairment. The most significant and consistent evidence

that supported organic pollutants, including synthetic

pyrethroids, as the main cause of biological impairment

was in BNG and downstream of this confluence in the

main stem of Dandenong Creek (DN4, DN5, DN6). It

was in this part of the watershed we saw high concentra-

tions of pesticides and significant adverse effects (lethal and

sublethal) across a range of different phyla and across

all levels of biological organization, strongly suggesting

that these sources of pollutants are the main cause of

impairment.

In contrast to the overwhelming evidence that supports

chemical contamination (a combination of metals and

pesticides) as being the stressors causing biological impair-

ment, there was no evidence to suggest that occasional

sewer spills were causing biological impairment to the

fauna living in Dandenong Creek.

Discussion

Petroleum hydrocarbons, pesticides and metals are pres-

ent at several locations within the Upper Dandenong

Catchment and are likely to affect aquatic fauna across

all biological levels, with macroinvertebrate communities,

P. antipodarum and C. tepperi populations and C. aura-

tus individuals all ecologically impaired. We were able to

isolate point sources of several metals and pesticides and

showed that these pollutants persisted downstream of the

point source and into Dandenong Creek. In contrast,

there was no clear evidence that occasional sewer spills

within the catchment had a major impact on the aquatic

ecosystem, with management implications for this

system.

EFFECTS OF EPISODIC SEWAGE SPILLS

Neither the long-lived nor short-lived fish species showed

signs of endocrine-related dysfunction, although some

histological changes were observed in downstream gold-

fish. These histological changes suggest generalized tissue

damage and are likely to be associated with poor water

quality rather than sewage spills. The results contrast

with other studies where both press (continual discharges

of sewage) and pulse (sewage spills) disturbances

adversely affected the reproductive health of a range of

fish and invertebrates (Jobling et al. 2004; Jenkins et al.

2009). While pulse disturbances are often more detrimen-

tal to biological communities than press disturbances

(Canobbio et al. 2009), our study failed to detect any

clear negative effects from this pollution type, suggesting

the current frequency and magnitude of sewage spills in

the Dandenong Creek Catchment are not causing biolog-

ical impairment.

BIOLOGICAL IMPAIRMENT WITHIN THE UPPER

DANDENONG CREEK CATCHMENT

High concentrations of pesticides and metals were present

throughout the lower reaches of the catchment, and the

pollutants that appeared to cause the most biological

impairment were synthetic pyrethroids and fungicides.

Most synthetic pyrethroids have only been registered for

use in Australia since 1993 and form constituent ingredi-

ents in a range of insecticides, miticides, parasiticides, seed

treatments and wood preservatives (APVMA 2012), while

the fungicides tebuconazole and propiconazole were first

registered in 1996. At least four different classes of pollu-

tants occur within the Upper Dandenong Catchment at

concentrations likely to cause adverse biological effects,

corroborating reported results in an adjacent catchment

(Schafer et al. 2011).

Our study supports the idea that ecosystem health

should be assessed through several indicators rather than

on chemical concentrations alone (Burton & Johnston

2010). For example, biological impairment was observed

in C. tepperi (growth and emergence) and some goldfish

(gonad histopathology) at the reference sites, which may

be due to both metal and organic compounds. Despite

these observations, sediment chemistry suggested no

expectation of adverse biological effects at the reference

sites.

Evidence of consistent biological impairment in Dande-

nong Creek was first apparent at Bungalook Creek and

persisted in the lower sections of Dandenong Creek, sug-

gesting that the ecological function of this section of the

stream is adversely affected. The TIE manipulation con-

firmed that organic compounds were responsible for

reduced fitness in C. tepperi populations, in agreement

with the sediment chemistry results that indicated high lev-

els of synthetic pyrethroids and fungicides but low levels

of metal pollutants. Pollutants have previously been shown

to adversely affect chironomids by reducing population

fitness (Marinkovic et al. 2011). The sediment concentra-

tions of bifenthrin and permethrin detected here may cause

toxicity to aquatic fauna and may account for the reduced

survival of P. antipodarum in the lower Dandenong Creek

sites. Potamopyrgus antipodarum is quite tolerant to metal

pollution (Laskowski & Hopkin 1996), which may explain

its relatively high survival in Old Joes Creek. In Bunga-

look Creek, all mudsnails died, suggesting sensitivity to

some organic pollutants rather than metals. Synthetic

pyrethroids are potential endocrine disruptors and through

TIE procedures appear particularly toxic to aquatic fauna

(Phillips et al. 2010). Our results are consistent with other

studies that suggest synthetic pyrethroids are toxic to

invertebrates in urban and agricultural waterways (Amweg

et al. 2006; Maul et al. 2008).

In the lower sections of Dandenong Creek, high concen-

trations of pesticides and metals appeared to affect all aqua-

tic fauna, resulting in a loss of community diversity and

reduced fitness of chironomid and goldfish populations. For



example, biological impairment was evident in downstream

goldfish across a number of endpoints measured, including a

high proportion of lesions and ulcerations and elevated gill

GST and liver CAT, suggesting they are in poor physical

condition and experiencing oxidative stress. Other studies

have shown elevated liver antioxidant enzyme activity in

field-collected goldfish following exposure to organic pollu-

tants (Lu et al. 2010). All of the evidence suggests that only

tolerant taxa are present in this section of the stream, ulti-

mately leading to reduced ecological function within the

stream.

WEIGHT-OF-EVIDENCE CATCHMENT APPROACH AND

MANAGEMENT IMPLICATIONS

For effective risk assessment in urban ecosystems like the

Upper Dandenong Creek, it is important to consider both

exposure and effects assessment with causation and eco-

logical relevance adequately addressed. In a recent article,

Burton et al. (2012) acknowledged the need to consider

various measures of environmental health such as chemi-

cal conditions, physical habitat and biological assessment

data in order to make realistic assessments of ecosystem

health. We have demonstrated that effective biological

monitoring programmes need to consider multiple end-

points in a range of organisms which will vary in

responses to different types of pollution. Our WoE

approach, based on the inference approach outlined by

Chapman, Wang and Caeiro (2013) for sediment quality,

includes multiple types of biological tests that identified

the class of pollutants responsible for the observed toxic/

adverse effects in the catchment. Numerous eco-epidemio-

logical studies have used the WoE approach incorporating

chemistry and various biological assessments to under-

stand a particular problem (Neamtu et al. 2009; Wiseman,

LeMoine & Cormier 2010). However, it is difficult to

determine the cause of biological impairment in rivers as

often the same stressors are causing different types of

adverse effects in different organisms. Therefore, it is

important to investigate biological effects in a range of

taxa in both the laboratory and field. When such effects

are seen across all levels of biological organization, caused

by the same contaminant, and when a source is clear,

there is a well-defined remedial path for managers. In this

case, the WoE approach has demonstrated that adverse

effects on aquatic fauna via chemical contamination far

outweighs potential effects on aquatic fauna from occa-

sional sewage spills. Subsequently, managers are currently

evaluating options for remediation by identifying sources

and removing pollutants in Bungalook Creek and Old

Joes Creek.

Acknowledgements

The project was funded by Melbourne Water Corporation, and we thank

Michelle Wotten, Alex Walton, Robert Considine and Erik Ligtermoet for

their contribution. Our thanks to Debra Gonzago and Hai Doan for

C. tepperi toxicity tests and Pei Zhang and AnhDuyen Bui for sediment

pesticide analysis. We appreciate assistance with fishing from John

McGuckin and Tom Ryan, field and laboratory assistance from Daniel

MacMahon, Alexis Marshall, Minna Saaristo, Rebecca Brown, Mayumi

Allinson, Melissa Gamat and Lee Englestad. We also extend thanks to

reviewers who provided valuable comments on this article. Fish were col-

lected under the Fisheries Victoria Collection Permits NP169 and RP998,

and all procedures carried out on fish complied with University of Mel-

bourne Animal Ethics guidelines, under approved project IDs 0911373.1

and 1011590.1.

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Received 29 September 2013; accepted 16 December 2013

Handling Editor: Shelley Arnott

Supporting Information

Additional Supporting Information may be found in the online version

of this article.

Appendix S1. Brief methodologies.

Table S1. Sediment concentrations of some metals and trace

pesticides.

Table S2. Ordinal ranking scheme applied for weight-of-evidence

categorizations.

Table S3. In situ water quality for Chironomus tepperi toxicity

testing.

Table S4. In situ water quality during the caging experiment.



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