Determination of the Effects of Contaminant Mixtures on Aquatic Macrophytes

Transcription

1 Determination of the Effects of Contaminant Mixtures on Aquatic Macrophytes R A M O N A D. S M I T H 1, P. C H R I S W I L S O N 1, A N D S A M I R A D A R O U B 2 1 S O I L A N D W A T E R S C I E N C E D E P A R T M E N T, U N I V E R S I T Y O F F L O R I D A / I F A S - I N D I A N R I V E R R E S E A R C H A N D E D U C A T I O N C E N T E R, F O R T P I E R C E, F L, U S A 2 S O I L A N D W A T E R S C I E N C E D E P A R T M E N T, U N I V E R S I T Y O F F L O R I D A / I F A S - E V E R G L A D E S R E S E A R C H A N D E D U C A T I O N C E N T E R, B E L L E G L A D E, F L, U S A

2 Introduction

3 More than 600 million pounds of pesticide were used in both 2006 & Class Millions Pounds a.i. % of Total 2006 Herbicides/PGR Insecticides/Miticides Fungicides 46 7 Nematicides/Fumigants Other 25 4 Total Herbicides/PGR Insecticides/Miticides 65 9 Fungicides 44 6 Nematicides/Fumigants Other 25 4 Total U.S. agricultural pesticide use by class 2006 and (Fishel 2007)

4 Acres 250,000,000 Total pesticide application on US farms. 200,000, ,000, ,000,000 50,000,000 0 Fungicide Herbicide Insecticide USDA/NASS estimates of pesticide application on U.S. Farms in 2007

5 Agriculture is a valuable industry in Florida

6 Florida Agriculture and Pesticide Application

7 Florida has to balance delicate ecosystems, agriculture and urban areas

8 Pesticides are commonly found in surface water samples nationwide Atrazine Nationally: 85% of 1382 samples from 65 mixeduse streams (USGS). Max. concentration: 41.3 ug/l 3.6% > than 1 ug/l Metolachlor Nationally: 68.13% of 1386 samples from 65 mixed-use streams (USGS). Max. concentration: 16.4 ug/l 1.4% >1 ug/l

9 Florida Surface Waters: Caloosahatchee Study Field sampling sites for related surface water pesticide sampling

10 Results of Caloosahatchee sampling December 2004-April 2006 Number of Detection Highest conc. Lowest conc. Median Pesticide name Use Chemical type MDL n detections Frequency (%) detected (ng/l) detected (ng/l) Conc. (ng/l) % RSD ethoprop insecticide organophosphate phorate insecticide organophosphate nd nd nd nd CIAT herbicide triazine CEAT herbicide triazine atrazine herbicide triazine simazine herbicide triazine acetochlor herbicide chloroacetanilide nd nd nd nd alachlor herbicide chloroacetanilide nd nd nd nd ametryn herbicide triazine metolachlor herbicide chloroacetanilide metribuzin herbicide triazine p,p'-dicofol insecticide organochlorine nd nd nd nd pendamethalin herbicide dinitroaniline cyanazine herbicide triazine nd nd nd nd fenamiphos insecticide organophosphate nd nd nd nd ethion insecticide organophosphate methoxychlor insecticide organochlorine cis-permethrin insecticide pyrethroid nd nd nd nd trans-permethrin insecticide pyrethroid nd nd nd nd Trifluralin herbicide dinitroaniline nd nd nd nd alpha-hch insecticide organochlorine nd nd nd nd diazinon insecticide organophosphate gamma-hch insecticide organochlorine nd nd nd nd heptachlor insecticide organochlorine cyclodiene chlorothalonil fungicide chloronitrile aldrin insecticide organochlorine chlorpyrifos insecticide organophosphate nd nd nd nd malathion insecticide organophosphate chlorpyrifos-oxon insecticide organophosphate fipronil insecticide phenyl pyrazole nd nd nd nd g-chlordane insecticide organochlorine nd nd nd nd trans-nonachlor insecticide organochlorine nd nd nd nd a-chlordane insecticide organochlorine nd nd nd nd a-endosulfan insecticide chlorinated hydrocarbon nd nd nd nd 4,4'-DDE insecticide organochlorine dieldrin insecticide organochlorine cis-nonachlor insecticide organochlorine nd nd nd nd 4,4'-DDD insecticide organochlorine b-endosulfan insecticide chlorinated hydrocarbon nd nd nd nd 4,4'-DDT insecticide organochlorine nd nd nd nd endo-sulfate insecticide chlorinated hydrocarbon Mirex insecticide organochlorine

11 Caloosahatchee River Data Atrazine Metolachlor Detection frequency: 98.7% Maximum concentration detected: 2854 ng/l Minimum concentration detected: 12.9 ng/l Median concentration detected: 72.2 ng/l %RSD: 222.9% Detection frequency: 94.7% Maximum concentration detected: ng/l Minimum concentration detected: 2.5 ng/l Median concentration detected: 17.7 ng/l %RSD: 137.5%

12 It is rare to find only one pesticide in a surface water sample When all 42 analytes were tested: 2-12 compounds were detected (n=75) Average detections: 5.6 Median detections: 6 % relative standard deviation: 38.5% 71 of 75 samples had BOTH atrazine & metolachlor present

13 Summary of Approach

14 Toxic Unit Approach Concentrations of toxicants expressed in units of lethality or in units of effect (LD50, LC50 or EC50) Chemical A has LD50 of 100 μm. So 1 TU=100 μm Chemical B has a LD50 of 50 μm. So 1 TU=50 μm A study examining 0.5TU A TU B = 1 TU A+B So expect 50% mortality (1 TU)with this mixture containing 50 μm A and 25 μm B

15 Toxic Units (TU) Given a mixture of 0.5 TU A TU B, The joint toxic effect of A and B is then defined as: Additive if EC50 mix = 1 TU More than additive if EC50 mix < 1 TU Less than additive if EC50 mix > 1 TU

16 Toxic Units in a mixture study It is expected that 0.5 TU A TU B = 1 TU A+B The mixture additivity approach uses the additive index (S) of Marking & Dawson (1975) : S= A m /A i + B m /B i Where A m & B m are the incipient EC50 of toxicants A & B when present in mixture, and A i & B i the toxicity of A & B when tested separately. Additivity Antagonism Synergism Mixture additivity approach illustration

17 Objective To determine the effects of atrazine and metolachlor on aquatic macrophyte growth, reproduction, and health

18 Methods

19 Duckweed was used as the test subject Lemna minor in culture

20 Study Overview EC50 Atrazine EC50 Metolachlor If slopes parallel Test Mixtures of Atrazine + Metolachlor Multiple Exposure Concentrations

21 Test conditions Glass vessels with lids 150 ml: 20% stock Hoaglands media 80% MHW Pesticide dilutions added directly to vessel n = 4, 12 fronds per vessel Moved every other day 12 h light-12 h dark cycle Approximately 25 C 6 days exposure

22 Concentrations Confirmed by GC-TSD Percent recoveries in individual tests: Atrazine: 88% - 113% Metolachlor: 80% Percent recoveries in mixture tests: Atrazine: % Metolachlor: %

23 Measured End Points Frond count (every 2 days) Root length (end of exposure only) Fresh weight (end of exposure only) Photosynthetic efficiency (Fv/Fm; end of exposure only) Chlorophyll & Carotenoid concentration (end of exposure only) Culture dishes growing Lemna minor.

24 Results

25 Individual EC50 values based on frond count Atrazine ppm Figure 9. Atrazine Day 6 Transformed logit y = ln(x) R² = TU = ppm Metolachlor ppm Figure 10. Metolachlor Day 6 Transformed logit y = ln(x) R² = TU = ppm

26 Slope Analysis Source of Variation df SS Y SP XY SS X b Y X SS Ŷ df SS Y X MS Y X Atrazine Metolachlor Sum of Groups Among b i s Pooled within Fs = ns Conclusion: Slopes are not statistically different; we can compare the individual compounds in a mixture study

27 Corrected Toxic Units for Mixtures Combination Measured Concentration Measured Concentration Total TU mix Atrazine TU (0 TU) 0 (0 TU) TU (0.5 TU) (0.25 TU) TU (1 TU) (0.53 TU) TU (1.5 TU) (0.79 TU) TU (2 TU) (1.04 TU) Metolachlor 0 (0 TU) (0.27 TU) (0.54 TU) 0.11 (0.83 TU) (1.18 TU) 0 TU 0.52 TU 1.07 TU 1.62 TU 2.22 TU

28 Toxic Units and the mixture study It is expected that 0.5 TU atrazine TU metolachlor = 1 TU mixture (50% growth rate of control) Growth rate of Lemna minor as a percentage of controls in a mixture of atrazine and metolachlor.

29 Toxic Units and the mixture study It is expected that 0.5 TU atrazine TU metolachlor = 1 TU mixture (50% growth of control) S was calculated using the mixture additivity approach and the additive index of Marking & Dawson (1975) S= A m /A i + B m /B i Where A m & B m are the incipient LC50 of toxicants A & B when present in mixture, and A i & B i the toxicity of A & B when tested separately. For atrazine and metolachlor mixture: S= 1.05 indicating a synergistic relationship between the toxicants.

30 Mean Count Final Frond Number Frond Count (Day 6, n=4) A B 40 C CD D Mixture Toxic Units (α=0.05, P = <0.001, power=1.000)

31 Mean Root Length (cm) Root Length (Day 6, n= 4) 2.5 A B B B B Mixture Toxic Units (α=0.05, P = <0.001, power=1.000)

32 Mean Final Fresh Weight (g) Fresh Weight (Day 6, n=4) A B BC C C Mixture Toxic Units (α=0.05, P = <0.001, power=1.000)

33 Mean Fv/Fm Final Fv/Fm values (Day 6, n=4) A A A B AB Mixture Toxic Units (α=0.05, P = 0.007, power=0.781)

34 ug/ml plant extract solution Chlorophyll a and Chlorphyll b values (Day 6, n=4) Chl a Average Chl b Average Mixture Toxic Units p= and p=0.211

35 ug/ml plant extract solution Total Chlorophyll and Total Carotenoid Content (Day 6, n=4) Total Chl Average A AB AB AB B Total carotenoids Average Mixture Toxic Units

36 Morphology effects Control culture In 0.75 ppm Metolachlor

37 Conclusions

38 Conclusions: Mixture effects of Atrazine & Metolachlor on Lemna minor Synergistic effects Growth rate Significant effects Frond count Root length Fresh weight Carotenoid content No trend effects Chlorophyll a & b content Fv/Fm

39 Acknowledgements Dr. P. Chris Wilson Dr. Samira Daroub Dr. Cathleen Hapeman Dr. Zhenli He Dr. Steve Roberts Youjian Lin, Ryan Hamm Jennifer Harman-Fetcho, Chris Lasser Any Questions?