Use of re-exposures to examine the effect of watercress derived isothiocyanates on Gammarus pulexMelanie Dixon

Why use re-exposures?

• watercress farm & wash/pack house factory

• chalk stream headwater

• variable factory discharge ~8h per day

• weight/type products being washed

Watercress farm outfall (Spring 08)

Isothiocyanates

• glucosinolates occur naturally; watercress and other crucifers

• enzyme released when plant wounded

• isothiocyanates produced as secondary metabolites

• important in defence of plant against herbivorous invertebrates Parabolic screen above

outfall

Hypothesis

• Gammarus pulex shows an increased sublethal response following repeated exposure to pure and watercress derived phenethyl isothiocyanate (PEITC).

Gammarus pulex (L.)

Method Development

• selection of test organism; depletion of G. pulex

• acute juvenile test

• sublethal endpoint, precopular separation

• time constraints, degradation rate

G. pulex precopular pairs

Method

• prepare wash water or PEITC solution

• expose pairs for 2h

• return to clean water

• count pairs re-formed

• re-expose for 2h in fresh wash water

Results• response during 2h

exposure

• comparison of pairs separated: exposure & re-exposure

• pairs reforming after 24h in clean water

• ET50 values

Response during 2h exposure

Pairs separated

%

PEITC (1µl/l)Wash waterControlRe-exposures

0

20

40

60

80

100

0 15 30 45 60 75 90 105 120Time (mins)

Comparison of pairs separatedafter 2h (exposure/re-exposure)

Control Upper 95% CI

Control Lower 95% CI

0

20

40

60

80

100

Washwater 2

Washwater 4

WashWater 5

PEITC 3 PEITC 5

%

Pairs re-forming after 24h in clean water

0

10

20

30

40

50

60

70

80

Washwater PEITC Control

Mea

n %

pairs re-formed after 24h

pairs at exposure end

ET50 (95% CI) values

Mean81±9 mins

020406080

100120

Mean71±14 mins

Individual Tests

Min

ute

s

Washwater

020406080

100120140

PEITC

Mean ET50 (± SE)

Wash water

WW re-exp. PEITC

(1ug/l) PEITC re-exp.

0

20

40

60

80

100

Min

ute

s

• Environmentally relevant concentration used.

• Baseline sensitivity /variability in mate guarding phase.

• Potential pairs ‘lost’ during exposure; 3rd exposure possible?

Watercress (Nasturtium officianale (R.Br.))

Conclusions

• re-exposures could be successfully carried out

• re-exposures resulted in slight (not statistically significant) increase of impact on most occasions

• transient impact?

Watercress in flower

We gratefully acknowledge the support of the Vitacress Conservation Trust and use of facilities at the National Oceanographic Centre and Environment Agency Biological Effects Laboratory.

Vitacress Salads Ltd, Hampshire, UK