The purpose of this experiment is to investigate the
safety of using salicylic acid as an algaecide in a real ecosystem by testing
the effects of the acid on four species of fresh water invertebrates.
It is hypothesized that 0.2% salicylic acid added to the simulated
pond water habitats will result in a drastic decline in pH that will have
a detrimental effect on the test specimens.
Secondly, it is hypothesized that the salicylic acid concentrations
of .03%, .01% and .005% will be well tolerated by the invertebrates and that
the LD50 for each of these species will be greater that .03%.
Three
sets of five jars are labeled - Control (0% Salicylic Acid), 0.2%, .03%, .01%
and .005% - and 200ml of pond water is added to each jar. Equal numbers of snails are added to one set
and the same for Daphnia and
planaria. The crayfish are divided
among five plastic bowls with 300ml pond water, aquarium gravel, and plastics
vials for hiding caves. Twenty-four
hours later, the salicylic acid is added and daily observations are made on
death loss for the 96-hour test period.
Turbidity and pH measurements are taken and the LD50 for each
invertebrate is determined.
The results of this experiment indicate that the 0.2% salicylic acid did cause a drop in pH in all four habitats and had a deadly effect on the snails, Daphnia, and planaria. The hardy crayfish survived all four levels of salicylic acid, which means the LD50 for this species is greater than 0.2%. Except for the crayfish, the data supported the first hypothesis that 0.2% salicylic acid added to the pond water habitats will result in a drastic drop in pH and will therefore have a detrimental effect on the fresh water invertebrates. The data showed that the salicylic acid LD50 for the pond snails and Daphnia is .01% and the LD50 for the planaria is less than .005%. Therefore, the second hypothesis is not correct.
For decades poultry producers in northwest Arkansas and eastern Oklahoma have applied phosphorus rich chicken litter to pastures as fertilizer as a means of disposing of the litter. Currently, poultry farms within the endangered watershed of Lake Eucha in northeast Oklahoma have the capacity to produce more than 84 million birds and 1500 tons of chicken litter each year (3). The Oklahoma Conservation Commission, Oklahoma State University, and the Oklahoma Water Resources Board have evaluated Lake Eucha and Lake Spavinaw, another drinking water reservoir fed by Lake Eucha. All three studies indicate that the chronic taste and odor problems in the drinking water supplied to the large city of Tulsa, Oklahoma has been caused by an overload of phosphorus resulting in eutrophication of these two lakes. (3)
The city of Tulsa has invested hundreds of millions of dollars since 1924 to secure the rights to these reservoirs and to build a state-of-the-art water treatment plant and filtering system. However, last fall, the taste and odor problems were so severe that Tulsa was forced to draw water from its emergency sources. Unfortunately, the quality of these lakes has also declined due to phosphorus pollution in the Illinois River, which feeds these emergency water sources (3).
Oklahoma legislators passed the Oklahoma Poultry Bill in 1998 to regulate the spread of poultry litter fertilizer and to require poultry producers to develop and follow an approved animal waste management plan (6). The Natural Resources and Conservation Service has worked with poultry producers and other landowners in the endangered Oklahoma watersheds to establish riparian buffer zones around ponds, lakes and creeks (7) and to preserve natural wetlands that serve to filter pollutants and provide flood control (4).
In response to pressure from northeast Oklahoma residents, the State Board of Agriculture has proposed new regulations that prohibit poultry houses from being built within a half mile of an occupied home or within 300 feet of a body of water or public drinking well. Under these new guidelines, poultry houses cannot be built in a flood plain (1). These regulations help prevent further pollution of watersheds, but do not address the problem Oklahoma faces now of finding a cure for eutrophication. Furthermore, much of the pollution in the Illinois River and the nutrient overload in Lake Eucha and Lake Spavinaw has come from sources in Arkansas (2). Oklahoma’s laws have no effect on Arkansas’ poultry producers and Arkansas water waste treatment facilities that dump into the Illinois River.
In last year’s experiment entitled “A Cure for those Eutrophication Headaches? The Effects of Salicylic Acid on the Growth of Closterium Algae in Poultry Litter Runoff Water,” salicylic acid is tested as an algaecide. Salicylic acid is an allelochemical that occurs naturally in the oils of birch trees and gaultheria shrubs and prevents the uptake of potassium in other plant species (8). Salicylic acid has keratolytic properties and is used in corn removal preparations and acne medications. Salicylic acid cannot be taken internally as it is irritating to the mucous membranes and inner lining of the gastrointestinal tract (9). However, the acetylated form of salicylic acid is the active ingredient in aspirin. Allelopathic studies revealed that salicylic acid could block the uptake of phosphorus by algae (8), which is required by algae cells to grow and multiply. The results of this experiment supported the hypothesis that adding salicylic acid to a nutrient rich media like poultry litter runoff will inhibit the growth and survival of Closterium algae.
The effects of salicylic acid on other organisms in a pond, lake, or stream must be evaluated before it can be considered for use as an algaecide. The purpose of this experiment is to investigate the safety of using salicylic acid in a real ecosystem by testing the effects of salicylic acid on four species of fresh water invertebrates. The concentration of salicylic acid that kills 50% or more of the test specimens within a 96-hour testing period known as the LD50 (lethal dose 50%) will be determined for each invertebrate tested. The same concentrations of salicylic acid tested last year are used in this experiment (.2%, .03%, .01%, and .005%).
Four different animals from three different phyla are used in this experiment. From the phylum Arthropoda, the crayfish (Cambarus) and a primitive crustacean (Daphnia magna) are used. Representing the flatworms from the phylum Platyhelminthes are the planarians from the genus Dugesia. A mixture of pond snails representing the phylum Mollusca is also used as test specimens. These animals are commonly found in ponds, lakes, streams, and slow-moving rivers in North America.
The results of algaecide testing in last year’s project indicate that salicylic acid is a very effective algaecide at a concentration of 0.2%. However, this concentration caused a drastic drop in pH levels in the poultry litter runoff water (from 7.3 to 2.3). Therefore, for this experiment, it is hypothesized that 0.2% salicylic acid added to the pond water habitats will result in a drastic decline in pH that will have a detrimental effect on the test specimens.
Salicylic acid concentration of
0.03% exhibited similar algaecidal actions as 0.2% salicylic acid, but it did
not cause a drop in pH in the runoff water.
.01% and .005% salicylic acid took a longer period of time to kill the Closterium
algae tested in last year’s experiment.
A gradual kill of an algae bloom by an algaecide could be considered a
good characteristic, as it would prevent a sudden massive kill of all algae in
the treatment area. This in turn would
prevent a deadly decline in dissolved oxygen.
A second hypothesis for this year’s experiment is that salicylic acid
concentrations of .03%, .01%, and .005% will be well tolerated by the fresh
water invertebrates tested and that the LD50 for each of these
species will be greater than .03% salicylic acid.
Materials and
Procedures
Salicylic acid
Daphnia
Planaria
Pond snails
Crayfish
5 large plastic bowls
Aquarium gravel
Large plastic bucket
Pond water
15ct. –10-dram dark plastic vials
16ct –8-dram plastic vials or test tubes
Stir sticks or tongue depressors
Metric measuring cup
16ct. –480ml clear glass jars
100ml-graduated cylinder
Pipettes
Digital gram scale
Wide range pH test strips
Liver
Yeast
Lettuce
Metric ruler
12cc syringe barrel
a. sedimentation of solids
b. the water to reach room temperature (22° C.)
4. Plastic bowls are set up to receive the crayfish-
a. with a mound of clean aquarium gravel
b. 3-4 plastic vials laid horizontally for hiding “caves”
c. 300ml of pond water are added to each bowl
d. 4 crayfish are placed in each bowl
e. bowls are labeled – Control, .2% salicylic acid, .03%, .01% or .005%
f. pieces of fresh liver or earthworms are used to feed the crayfish
5. 200ml of pond water is added to fifteen - 480ml glass jars.
6. 3 sets of 5 jars are labeled – Control, .2% salicylic acid, .03%, .01% or .005%
7. Equal numbers of pond snails are added to one set of jars.
a. lettuce leaves are added to feed the snails
8. Equal numbers of Planaria are added to another set of jars.
a. pieces of liver are added to each of these jars
9. Daphnia are added to the third set of jars.
a. a warm water and yeast mixture is made
b. several drops of this mixture are added to each jar
10. All test specimens are observed for 24 hours in the new habitats.
11. Remaining lettuce and liver is removed to prevent fouling the water.
12. The amount of salicylic acid to be added to each container is calculated.
13. Example calculation:
a. .2% = 2mg/ml
b. 2mg/ml = Xmg/300ml
c. X = 600mg
14. For the 3 sets of specimen jars:
a. Control = no salicylic acid added
b. .2% = 600mg
c. .03% = 90mg
d. .01% = 30mg
e. .005% = 15mg
15. For the crayfish bowls, the total volume of water in each bowl will be 400ml.
16. To calculate the mass of salicylic acid for the crayfish bowls:
a. .2% = 2mg/ml
b. 2mg/ml = Xmg/400ml
c. X = 800mg
17. For the crayfish bowls:
a. Control = no salicylic acid added
b. .2% = 800mg
c. .03% = 120mg
d. .01% = 40mg
e. .005% = 20mg
18. The salicylic acid is weighed and measured on a digital scale and poured into labeled plastic vials.
19. Each vial of salicylic acid is first added to 100ml of pond water in a jar with a lid and shaken vigorously.
20. The salicylic acid solution is slowly poured into the corresponding container.
21. Daily observations are made on each test jar and bowl
22. 24 hours after addition of salicylic acid, the pH of each habitat is tested.
23. At 96 hrs. post-salicylic acid addition the number of live and dead specimens is counted and recorded.
24. Turbidity measurements are taken on each jar using a modified Secchi disk procedure developed in last year’s experiment.
25. The LD50 for salicylic acid for each invertebrate is determined from the data collected.
|
Salicylic Acid LD50 Trial |
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|
|
|
|
|
|
|
Snails |
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|
|
pH |
Turbidity (cm.) |
No. Live/Total
No. |
Death Rate |
Comments |
|
Control |
7 |
4.2 |
4 out of 4 |
0% |
|
|
0.2% Salicylic
Acid |
2 |
4.1 |
0 out of 4 |
100% |
dead within 24
hours, cloudy, reddish water, shells breaking
apart |
|
0.03% Salicylic
Acid |
4 |
6 |
0 out of 4 |
100% |
dead within 48
hours |
|
0.01% Salicylic
Acid |
6 |
4.8 |
2 out of 4 |
50% |
LD50
for snails |
|
0.005% Salicylic
Acid |
7 |
4.5 |
4 out of 4 |
0% |
|
|
|
|
|
|
|
|
|
Daphnia |
|||||
|
|
pH |
Turbidity (cm.) |
No. Live/Total
No. |
Death Rate |
Comments |
|
Control |
7 |
7 |
2 out of 10 |
80% |
didn't loose any
till 4th day, pH rechecked and
dropped to 6 |
|
0.2% Salicylic
Acid |
2 |
6.6 |
0 out of 10 |
100% |
dead within 24
hours |
|
0.03% Salicylic
Acid |
4 |
5.3 |
0 out of 10 |
100% |
dead within 24
hours |
|
0.01% Salicylic
Acid |
7 |
5.2 |
4 out of 10 |
60% |
LD50
for Daphnia |
|
0.005% Salicylic
Acid |
7 |
4.2 |
10 out of 10 |
0% |
|
|
|
|
|
|
|
|
|
Planaria |
|||||
|
|
pH |
Turbidity (cm.) |
No. Live/Total
No. |
Death Rate |
Comments |
|
Control |
7 |
3.9 |
11 out of 12 |
8% |
1 died on day 4 |
|
0.2% Salicylic
Acid |
2 |
5.2 |
0 out of 12 |
100% |
dead within 12
hours |
|
0.03% Salicylic
Acid |
4 |
6.6 |
0 out of 12 |
100% |
dead within 12
hours |
|
0.01% Salicylic
Acid |
7 |
3.5 |
0 out of 12 |
100% |
dead within 12
hours |
|
0.005% Salicylic
Acid |
7 |
5 |
0 out of 12 |
100% |
LD50 for
Planaria is less than 0.005% |
|
|
|
|
|
|
|
|
Crayfish |
|||||
|
|
pH |
Turbidity (cm.) |
No. Live/Total
No. |
Death Rate |
Comments |
|
Control |
7 |
|
4 out of 4 |
0% |
unable to measure
turbidity in plastic bowls |
|
0.2% Salicylic
Acid |
5 |
|
4 out of 4 |
0% |
LD50
for crayfish is greater than 0.2% |
|
0.03% Salicylic
Acid |
7 |
|
4 out of 4 |
0% |
|
|
0.01% Salicylic
Acid |
7 |
|
4 out of 4 |
0% |
|
|
0.005% Salicylic
Acid |
7 |
|
4 out of 4 |
0% |
|
Discussion of Data
The snails, Daphnia, and planaria jars had similar pH measurements for each salicylic acid concentration with the 0.2% salicylic acid causing a drastic drop in pH from 7 to 2 and the .03% jars dropped from 7 to 4. The death rate for all three species was 100% for these two concentrations. The Daphnia and snails reached a 50% or greater death loss at .01% salicylic acid resulting in a LD50 of .01%. The planaria were quickly killed by all four concentrations tested. So, the LD50 for the planaria must be less than .005% salicylic acid.
The high death loss in the Daphnia control jar occurred in the last 24 hours of the test period and may have been due to lab error as a pipette used to stir up the Daphnia for the previous day’s count may have been contaminated with salicylic acid. A recheck of the pH in the Control jar after the final count confirmed that the pH had fallen from 7 to 6. The jars with the most survivors were the .01% and .005% containers, and they had a final pH of 7.
The turbidity of each jar was measured with a modified Secchi Disk test. Each jar was placed at the end of a metric ruler. A 12cc syringe barrel is placed directly behind the jar at the edge of the ruler. As the observer looks through the jar, the syringe is moved backwards along the ruler until the numbers on the syringe barrel become fuzzy. A reading in centimeters is made at the point where the syringe barrel stops. The smaller readings are the most turbid jars.
The jars with the greatest death losses generally had the larger turbidity readings, meaning they were much clearer than the jars with surviving creatures. The dead organisms, including the algae, were falling to the bottom of these jars, which cleared the water. One exception is the 0.2% snail jar that was extremely turbid due to rapid decomposition of the snails and their shells. The water in this jar was a reddish color.
The crayfish proved to tolerate the salicylic acid at all concentrations tested, and therefore, the LD50 is greater than 0.2%. The pH drop in the 0.2% crayfish bowl dropped from 7 to 5 compared to a drop from 7 to 2 for the other invertebrates. Something in the crayfish bowls not found in the jar habitats helped to neutralize some of the acid. This could have been the aquarium gravel, the moss from the shipping crates, the plastic vials used for hiding places, or the crayfish themselves.
Conclusions
Except
for the crayfish, the data supported the first hypothesis that 0.2% salicylic
acid added to the pond water habitats will result in a drastic drop in pH and
will therefore have a detrimental effect on the fresh water invertebrates.
The results of this experiment indicated that the 0.2% salicylic acid did cause a drop in pH in all four habitats and had a deadly effect on the snails, Daphnia, and
planaria. The tough exoskeleton or the fact that the crayfish spent less time in the water than the other species tested may be the reasons that the crayfish survived this concentration of salicylic acid. However, if the crayfish had been in the molting stage, they may have been more susceptible to the 0.2% salicylic acid.
The
second hypothesis that .03%, .01%, and .005% salicylic acid will be well
tolerated by the test specimens and that the LD50 for salicylic acid
for each of these fresh water invertebrates will be greater than .03% was
proven incorrect.
The data showed that the salicylic acid LD50 for the pond snails and Daphnia was .01%. The planaria did not tolerate the salicylic acid at any concentration; so the LD50 is less than .005%. The hardy crayfish survived all four levels of salicylic acid, which means the LD50 for this species is greater than 0.2%.
Recommendation for Future Studies
Acknowledgements
I would like to thank the following people for helping me with this project:
Bibliography
1. “Ag Board Toughens Requirements for Poultry Houses.” Vinita Daily Journal 22 Feb. 2002.
2. Canty, Geoffrey. “Water Quality for the Illinois River.” Oklahoma Conservation Commission. August, 1996.
3. Lassek, P.J. “Tulsa to Sue Over Water.” Tulsa World 10 Dec. 2001: A1.
4. “Minnesota Pollution Control Agency-A Citizen’s Guide to Lake Protection.”
<http://www. pca.state.mn.us/water/lakeprotection.html>.
5. Neal, Ken. “Water and Chickens.” Tulsa World 20 May 2001: G6
6. Oklahoma Registered Poultry Feeding Operations Act. Senate Bill 1170. May 1998.
7. “Pond Scum Wipe – Out!! Control of Nuisance Algae in Ponds.” <http://www.wvu.
edu/~agexten/aquaculture/pondscum.html>.
8. Putman, Alan R. “Allelopathic Chemicals.” Chemistry & Engineering. April 1983: 34-36.
9. “Salicylic Acid.” Remington’s Pharmaceutical Sciences. 1970.