hickey setac seville 2010 - sourcewatch · 2014-01-30 · calculated toxic threshold for bm larvae...

Auckland harbour

National Institute of Water and Atmospheric ResearchHamilton, New Zealand

Chris Hickey and Michael Stewart

Catchment studies in Georges Bay, Tasmania: base-flow water and foam toxicity to

cladocerans and blue-mussels

A case of unintended consequences?

Society for Environmental Toxicology and Chemistry; Seville 24 May, 2010

Background

The Problem

Results

toxicity assessment

characterisation

Forensic eco-toxicology

Integrated risk assessment

Conclusions

Future studies

Overview

Helicopter crash in catchment (2003) – multiple pesticides detectedMass mortality of oysters in Georges Bay, Tasmania was observed in summer 2004 following a record floodsExtensive mortality of other marine species - filter-feeders (clams, mussels, barnacles), prawns, crabs, sea urchins and a variety of fish speciesFreshwater species affected were noted as “rafts of dead frogs and other insects” in the Bay

Insecticides and herbicides have been measured in the catchment soils and waters - though their water concentrations have generally been below effects thresholds

Human health issues – various and often rare at elevated frequencyOyster health decline – “novel” symptomsWildlife issuesHigh ecotoxicity effects measured in foam and storm-flow watersObserved elevated production of foams in river catchment

Plantation forests of Eucalyptus nitens established and developing in catchment over this period

Background

Multiple effects/targets – persistent over time

Background: Site

The Problem: Rivers

Drinking water intake pipe on George River at Priory South George foam collection

Collapsed foam Flocculated particulates in water-treatment system

More river foam: prior to filtration

The Problem: Bay

Transport and wildlife exposure

Study design: EcotoxicityBaseflow stream-water collection – multiple sites, including reference catchment (pre-filtered 50 µm)

River foam collection at 2 sites (pre-filtered 140 µm)

Toxicity assessment: freshwater cladocerans (Ceriodaphnia dubia)

marine bivalve, blue mussel embryo-larvae (Mytilus galloprovincialis)

Chemical characterisation:organic content, suspended solids, particle counts, particle size distribution, pesticide suite

organic extract (EtOH) of foam and E. nitens leaves (catchment & reference site (Victoria))

bioassay directed fractionation: HPLC/mass spec � Molecular fractionation (LH20) � ms & NMR

Toxicity identification:filtration; add-back

screening and definitive bioassays of foam and leaf extracts (toxic units, TUs)

chemical standards

Foam characterisation:quantitative foam assay procedure

applied to chemical fractions

Results: Foam concentration-response

Filtered

Unfiltered

Blue-mussel: Water Intake (WI) foam

Concentration (%)

Filtered

Unfiltered


Filtered

Unfiltered


Concentration (%)

Filtered

Unfiltered

Blue-mussel: South George (SG) foam

Concentration (%)

Filtered

Unfiltered

Blue-mussel: South George (SG) foam

Concentration (%)

Concentration-response relationships for filtered and unfiltered foam samples from Georges River catchment (South George & Water Intakes) to blue mussel larvae

Higher particle-associated toxicity in foams

75 µm75 µm

Normal blue-mussel larvae at 48 h Abnormal blue-mussel larvae at 48 h Abnormal larvae in foam debris

Results: foam specific toxicity

Blue-mussel larvae markedly more sensitive

Source of foam and toxicity?

Calculated toxic threshold for BM larvae is 3x above base flow SS concentration

Forensic: Study design

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18

CRUDE ETHANOL EXTRACT

FRACTIONATION BY PREPARATIVE HPLC

TOXIC FRACTIONS COMBINED

FRACTIONATION BYSIZE EXCLUSION CHROMATOGRAPHY

F7c-1

F7c-2

F7c-3

F7c-4

F7c-5

F7c-6

F7c-7

F7c-8

F7c-9

F7c-10

F7c-11

F7c-12

F7c-13

F7c-14

F7c-15

F7c-16

F7c-17

F7c-18

F7c-19

F7c-20

FIRST FRACTION SERIES

SECOND FRACTION SERIES

F7c-4-1

F7c-4-2

F7c-4-3

F7c-4-4

F7c-4-5

F7c-4-6

F7c-4-7

F7c-4-8

F7c-4-9

F7c-4-10

F7c-4-11

F7c-4-12

F7c-4-13

F7c-4-14

FRACTIONATION BY ANALYTICAL HPLC

THIRD FRACTION SERIES

Preparative HPLC

Size exclusion LH20

Analytical HPLC

Concurrent analyses:

1. Foam from St George River, Tasmania2. Tasmania leaves – new growth E. nitens3. Victoria leaves (reference)

Freeze dry � EtOH extracts

Bioassay analyses:

Blue mussel assays0.1% EtOHScreeningDefinitive – dilution series

Foam assays: agitation

Chemistry

Preparative HPLC with Toxicity Overlaid

Foam

TasmanianLeaves

VictorianLeaves

f1 f2 f3 f4 f5 f6 f7 f8 f9 f10 f11 f12 f13 f14 f15 f16 f17 f18

Blue Mussel: darkest most active

Daphnid: darkest most active

Indicates foam production in fraction: + weak; ++ moderate

+

++ ++

++ ++

+

+

Forensic: Bioassay directed fractionation

Common toxic fractions for size exclusion chromatography

Composited fractions

Toxicity (TUs)Parent (% recovery of all fractions)

36 TUs(78%)

21 TUs(126%)

59 TUs(57%)


Tasmanian leaves

Victorian leaves

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40Tim e (m in)

0

50

100

0

50

100

-50000

0

50000

uAU

0

50000

uA

U

0

50

100

0

100-100

-50

0

1.8316.94 18.19 18.81 23.41 25.76

18.1118.85

21.23 21.49

18.17

18.59

18.2021.2418.45 33.02

23.4023.7317.80

33.0517.82

23.4018.05 21.23

17.77

17.75

16.87

NL:3 .51E4

T otal Scan PDA tnl1 3 -7c-03

NL:7 .16E4


NL:2 .57E5


NL:9 .51E4


NL:7 .53E4


NL:4 .42E5


NL:3 .42E5


c:\xcalibur\...\19-04-10\vol1 2-7c-07 04/20/2010 03:24:52 AM

RT: 0.00 - 40.00

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40Time (m in)

0

200000

400000

uA

U

0

200000

400000

600000

uA

U

0

200000

400000

600000

uA

U

0

200000

400000

uA

U0

200000

400000

uA

U

0

100000

uA

U

-50000

0

uA

U

23.1416.82 21.10 23.7019.7414.92 18.7513.03 24.74

13.0314.92

16.94 23.1417.76 18.75

14.92

13.0316.05

14.23 16.97 21.3418.1221.58

18.14

17.2814.9112.4721.1018.87 21.32 23.68

18.09

17.2618.43

16.92 19.4812.47 22.90 23.1619.8512.98

13.8818.05

17.73 23.5118.52 19.67

22.91

19.4313.96

17.76

22.5720.264.50

NL:6.95E4

Total Scan PDA VOL1 2-7c-02

NL:1.83E5

Total Scan PDA vol1 2-7c-03

NL:4.40E5


NL:5.98E5


NL:7.22E5


NL:7.27E5


NL:5.23E5


Fraction

1

2

3

4

5

6

7

Fraction

1

2

3

4

5

6

7

Molecular weight fractionation (LH20)(Parent: HPLC f7-9, 5x concentrated;UV of HPLC/mass spec)

parent 256 TUs; f3-5 28%

parent 370 TUs; f3-5 15%

TUs

1.4

11

59

TUs

1.0

16

39


Toxic fractions of foam poorly resolved – multiple components

Foam

RT: 0.00 - 20.00

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Tim e (m in)

0

100000

uA

U

0

200000

uAU

0

100000

uAU

0

100000

200000

uA

U

0

100000

uA

U

-50000

0

uA

U

-50000

0

uAU

0

100000

200000

uAU

12.15

11.5311.1810.52

10.63 11.13 12.14

10.75 11.3712.11

11.5310.7110.57

11.80

11.5311.18

10.5211.80

12.21

12.15

11.5311.19

12.16

11.91

12.15

11.90

NL:2.89E5

Total Scan PDA TNF2 1-7c_100215215245

NL:8.34E4

Total Scan PDA tnf2 1-7c-01

NL:7.74E4


NL:1.41E5


NL:2.25E5


NL:1.82E5


NL:3.51E5


NL:1.52E5


TNF2 1/7c f1-f7 tnf2 1-7c-06 #2916 RT: 12.15 AV: 1 N L: 1 .34E6 microAU

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600

wavelength (nm)

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

700000

750000

800000

850000

900000

950000

1000000

1050000

1100000

1150000

1200000

1250000

1300000

uA

U

409 .00

399.00

376.00

327.00239.00

272.00220.00

506.00535.00

475.00

MW 592

f1

f2

f3

f4

f5

f6

f7

parent

RT: 0.00 - 40.00

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40Tim e (m in)

-90000

-80000

-70000

-60000

-50000

-40000

-30000

-20000

-10000

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

110000

120000

130000

140000

150000

160000

170000

180000

190000

uA

U

NL:1.95E5

T otal Scan PDA T NF2 1-7c-04 Fracti onati on 01

NL:1.97E5

T otal Scan PDA tnf2 1-7c-04 fractionation 02

NL:1.98E5


NL:1.99E5


NL:1.99E5


f1 f2 f3 f4 f5 f6 f7 f8 f9 f10 f11f12 f13 f14

Molecular weight fractionation (LH20)(Parent: HPLC f7-9; UV of mass spec)

TUs50

(24%)

4.0

5.3

2.6

Further fractionation of this fraction looses toxicity


Elimination of known compounds in toxic foam fractions; 400-500 MW

Metabolite Group Simple FPCsa

monoterpene euglobals macrocarpalsb sideroxylonals c

Molecular Weight (MW) 266 252 386 472 500 Extracted m/z (-ve mode) 265 251 385 471 499

HPLC Retention Time Not

detected 9.9 min

10.4 min

12.6 min

13.5 min 11.5 min

12.1 min

12.8 min

Sampled Tasmanian Leaves (crude extract) - + + + ++ ++ ++ - F7 - ++ ++ - - + - - F12 - - - - ++ - - - F13 - - - - - - ++ - F14 - - - - - - - - Victorian Leaves (crude extract) - ++ ++ + ++ ++ ++ + F7 - ++ ++ - - ++ ++ - F10 - - + + - + ++ ++ F11 - - ++ ++ ++ + ++ ++ F12 - - - - ++ - ++ - F13 - - + - - - ++ - F14 - - ++ - - + ++ - Tasmanian Foam (crude extract) - - - - - - - - F6 - - - - - - - - F7 - - - - - - - - F8 - - - - - - - - F9 - - - - - - - - F10-F14 - - - - - - - -

a Includes jensenone (MW 266) and grandinol/homograndinol (MW 252). b Includes 12 macrocarpals with MW 472. c Sideroxylonals A-C (MW 500). d Only toxic fractions included, with the exception of F10-F14 for foam, which are included for comparison- not detected; + low level detection; ++ high level detection

Fractions coded red are most toxic

Indicated common toxic fraction

Mass spectrometer molecular weight comparisons for HPLC fractions

Forensic: Foam

Marked difference in foam characteristics

Foam decay half-life:

Foam = 2.8 h

Tasmanian leaves = 12 h

Victoria leaves = 2.4 h

Primary (EtOH) extracts

0

1

2

3

4

5

6

7

8

0 1 2 3 4 5

Foam (mm)Tasmanian leaves (mm)Victorian leaves (mm)

y = 5.5074 * e^(-0.2888x) R= 0.99452

y = 6.695 * e (̂-0.081618x) R= 0.94537

y = 3.682 * e^(-0.3065x) R= 0.96895

Time (h)

Summary:highly toxic foams – particularly to bivalve larvae

strong particle-associated toxicity

a toxic effects threshold about 3x over base-flow river suspended solids

strong toxicity for the whole extract for foam and both leaf sources

discrete toxicity in only a few foam and Eucalyptus leaf chemical fractions

concordant toxicity in both cladocerans and blue mussels for all toxic chemical fractions

cont...

Summary:higher number of toxic fractions in leaf extracts than foam samples

an unknown mixture of common toxic components (MW 400-500) result in foam toxicity and some leaf toxicity

foam “hump” reversibly looses toxicity with further purification � difficulty for field chemical characterisation

foam-forming ability not concordant with high toxicity fractions

Tasmanian E. nitens are chemically different and have markedly stronger foam-forming ability

Have we enough information for causation of field effects?

Potential causation: Eucalyptus nitens ?Evidence?

extensive plantations in catchment

time concordance with effects and foam occurrence

persistent and ongoing inputs

foam has high organic content with plant debris

foam and stream particulates have ability to reach target species

common toxic fractions in foam and eucalypt leaves

Knowledge about plantations:selectively breed – for pest resistance

chemically different from parent plantation leaves (this study)

Other information from literature…?

Literature

Rosi-Marshall, E.J.; Tank, J.L.; Royer, T.V.; Whiles, M.R.; Evans-White, M.; Chambers, C.; Griffiths, N.A.; Pokelsek, J.; Stephen, M.L. (2007). Toxins in transgenic crop byproducts may affect headwater stream ecosystems. Proceedings of the National Academy of Sciences of the United States of America 104: 16204-16208.

http://www.bloomberg.com/apps/news?pid=email_en&sid=aEHNB_XJRWGU#

Literature

Bleaney,A. 2007 ‘Risk Awareness and Incident Response Capability in Water Catchments in North Eastern Tasmania, Australia – A Community Based Audit’, Upper Catchments Issues Tasmania, Vol 3 No 3, ISSN 1444-9560.

Weight-of-evidence: Human health

Source: Sunday Tasmanian, 20 September 2009

Weight-of-evidence: Wildlife issues

Conclusion: Potential ecotoxic causation: Eucalyptus nitens?

Very highly plausible

Unintended consequence of silviculture practices

i.e., A combination of toxic components and foam-forming ability

Causation of other catchment health effects? � awaiting further studies

Futuresensitive (and simple) chemical analytical methods

field measurements �

Should do:

routine suspended solids within the George River catchment and Georges Bay � concentrations and mass loads

toxicity monitoring of storm-flow waters � toxicity thresholds exceedance during events

oyster “health” monitoring studies along exposure gradients relative to riverine inputs

This issue needs a lot more investigation!

Alison Bleaney

Marcus Scammell

Rick Krassoi

Jim Harris

Pete Long

Graham

Acknowledgements

Contact: [email protected]

Questions?

ABC documentary reference: www.abc.net.au\austory “Something in the Water?”

Results: Foam characteristics

High particle content of foams

hickey setac seville 2010 - sourcewatch · 2014-01-30 · calculated toxic threshold for bm larvae...

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