Biomarkers

by heather seavolt and stephanie mCPHERSON

Types of biomarkers

• DNA, RNA or protein adducts and enzymes

• cytogenic markers• tissue markers and whole body

alterations• behavioral markers• measure toxicants in body fluids &

tissues• death of marker animals

Ld50 & LC50

• Ld50 - median lethal dose (amount of a chemical in one dose that will kill 50% of the population)

• Lc50 - concentration of material in air that will kill 50% of the test subjects

Initial Purpose

• To observe any effects of the fungicide on the red worms

• To determine ld50 for our for our population of red wormspopulation of red worms

SOIL ECOTOXICOLOGY EXPERIMENT

• Materials: • 24 pint mason jars with lids • Worms • Soil mixture - composed of silica sand,

kaolin clay, peat moss, CaCO3, water • Litmus paper • Pesticide - 0.88%

1-(4-CHLOROPHENOXY)-3,3-DIMETHYL-1-(1h-1,2,4-TRIAZOL-1-YL)-2-BUTANONE)

Experiment layout

• 10 worms added to each jar

• Worms allowed to adjust to new soil habitat

• pesticide added after week 1

• Data collected weekly

• worms sorted and analyzed for lesions, swelling, writhing, Number on and below surface

fungicide concentration series

• Control series (3 jars)• 6 series run with fungicide (3

jars/series)• first fungicide series 4 ml

fungicide :128 ml of water• diluted 5 times for each successive

5 series

• 1st dilution: 1/2 the fungicide concentration of series 1 (2:128 ml)

• 2nd dilution: 1/4 the concentration of series 1 (recommended concentration)

• 3rd dilution: 1/8 the concentration of series 1

• 4th dilution: 1/16 the concentration of series 1

• 5th dilution: 1/32 the concentration of series 1

• Samples of lindsey and jason’s soil• 1. sample from the copper pipe

area• 2. sample from site 1• 3. sample from around suspicious

oil container

mortality

• Mortality is defined in this experiment as lack of movement in response to stimulus or absence, since the worms disintegrate rapidly

Further objectives

• Additional objectives of this experiment:

• 1. examine effects of pollution on non-target organisms

• 2. Accuracy of results, if any, in real world situations

• 3. Consider effects of toxin on different organisms

• 4. How this experiment would be improved if carried out for a longer time and replicated

• 5. Ethical issues involved in animal experimentation

dataNumber of worms aliveWeek 2 Week 3 Week 4 Week 5

Jar 1 Jar 2 Jar 3 Jar 1 Jar 2 Jar 3 Jar 1 Jar 2 Jar 3 Jar 1 Jar 2 Jar 3Control Series 9 10 10 9 10 10 6 7 9 6 6 10Original Series 6 4 8 6 5 8 6 5 5 5 5 51st Dilution 9 10 10 8 10 11 8 10 13 8 9 122nd Dilution 10 11 9 7 7 7 8 7 9 7 6 103rd Dilution 9 8 10 8 7 10 8 8 9 7 9 74th Dilution 8 0 9 8 0 9 9 0 10 8 0 95th Dilution 11 9 0 11 8 0 9 4 0 8 6 0Soil - Copper Pipe area9 N/A N/A 7 N/A N/A 6 N/A N/A 5 N/A N/ASoil - Oil Container 3 N/A N/A 3 N/A N/A 3 N/A N/A 2 N/A N/ASoil- Site 1 10 N/A N/A 6 N/A N/A 6 N/A N/A 6 N/A N/A

Concentration vs. Percent Mortality

• pH of each soil was taken after soil was mixed and again after the fungicide was added

• Initial pH of the soils were about 6.0 - 6.5

• pH after addition of fungicide ranged from 4.5 (in the more highly-concentrated soils) to 5.5 (in the less highly-concentrated soils)

results• Slight trend in mortality rates and

increased fungicide concentration

• in soil samples from jason and lindsey, mortality rate low except in soil taken from oil container site

conclusions

• The highest fungicide concentration used (4 times the suggested concentration) appears to have a fairly strong impact on the worms

• The soil sample from the oil container was not a good habitat for the worms since there was a fairly immediate drop in the worm count two weeks after the worms were introduced into this soil

• The other soils with varying concentrations of fungicides provided no worse a habitat for the worms than did the control series

• overall reproductive ability did not appear to be altered