A Laboratory Study of Earthworm Mortality to Selected Turf Insecticides
K. A. Delahaut & C. F. Koval, Department of Entomology, University of Wisconsin-Madison

Earthworms play a vital role in the enhancement of soil structure and fertility. In general, the benefits to turf as a result of earthworm activity greatly outweigh any inconveniences (Delahaut and Koval, 1989). Earthworms benefit the soil primarily due to the chemical and physical effects they produce through their casts and burrows.

Earthworm casts are made up of waste excreted after feeding. This waste consists of soil particles and plant residues. Casts modify the soil structure by breaking larger structural units into smaller granules. Cast production is most abundant in the spring and fall; the reason being that the moist soil conditions present in the upper layers of the soil profile at these times is favorable for earthworm activity. In irrigated turf, this activity may occur any time the ground is not frozen since adequate moisture is always present. In the turf industry, the casting behavior of earthworms is not always viewed as a favorable process. Earthworms commonly deposit their casts beneath the soil surface where the casts contribute to pedogenesis. However, in certain situations, these casts may be deposited on top of the soil surface where their presence is not desired as occurs on golf course greens.

Nutrient availability is also enhanced by earthworm activity. As they feed, earthworms deposit partially digested organic matter and minerals in casts along their burrows. Analysis of casts have shown that they contain several times more nitrogen, phosphorus, potassium, magnesium and calcium than surrounding soil. The partially decomposed plant residues are further acted upon by soil micro-organisms to release available nutrients.

The burrowing habit of earthworms is beneficial to the soil structure. Burrows increase aeration and drainage by making the soil more porous. Earthworm burrows can increase a soil's moisture-holding capacity by as much as 400%. Burrowing is also responsible for the translocation of minerals and organic matter from one location to another. This process will increase the amount of plant nutrients located in close proximity to the plant's roots.

Several pesticides have a detrimental effect on earthworms. Historically, many studies have evaluated the toxic effects of pesticides on earthworm populations (Benz and Altwegg,1975; Inglesfield, 1984; Lee,1985; Potter et.al.,1990; Randall, Butler and Hughes,1972; Roberts and Dorough, 1985; Stenerson, 1979; and Thompson, 1971). A majority of the early studies focused on inorganic and organochlorine compounds. Subsequently, there has been a substantial turnover in the types of insecticides available. The inorganic and organochlorine insecticides which were once heavily relied upon, have been supplanted by more specific compounds such as the synthetic pyrethroids and biorationals.

The effect of a pesticide on earthworms may be immediate and acute resulting in the death of the worm. Conversely, this impact may also be sub-lethal or chronic whereby a reduction in reproductive capabilities may occur, or a decreased functioning in life-supporting behaviors such as food-getting may be realized.

This study was conducted to evaluate the acute effects of several insecticides currently used in the management of turf insect pests. A few old standby insecticides were tested as well as new formulations of those traditional chemicals. In addition, new releases were also evaluated. The insecticides tested included diazinon, chlorpyrifos (Dursban), trichlorfon (Dylox), Bacillus thuringiensis var San Diego (M-One), ethoprop (Mocap), isofenphos (Oftanol 2), carbaryl (Sevimol), cyfluthrin (Tempo), and isazophos (Triumph).

The experiment consisted of two individual studies. The first study involved the use of insecticide-impregnated filter paper while the second involved the incorporation of the insecticide in soil samples to which earthworms were added. Both studies were based on procedures evaluated in Heimbach, 1984.

The filter paper test was a method for determining contact toxicity of the insecticides. A piece of filter paper was placed in the bottom of the larger half of a petri plate. Insecticides were dissolved in acetone or deionized water to provide concentrations of 1.0, 0.1, 0.01, lx10 -3, lx10 -4, lx10 -5, and lx10 -6 mg of active ingredient per square centimeter of filter paper. The paper was wetted with the chemical and allowed to air dry. Subsequently, the paper was re-wetted with 2 mi deionized water prior to placment of the worms on the paper. Each concentration was replicated five times and the entire experiment was repeated once.

The earthworms used were a cross between a Canadian variety of nightcrawler and the common red worm. The worms were 2-6 weeks old and 4-5 cm in length.

Prior to exposing the earthworms to the impregnated filter paper, worms were washed and placed between sheets of moistened paper towels for two hours. Initial weights were taken and recorded. A single worm was placed on each plate. Final weights were measured on worms surviving the duration of the study. The plates were covered and placed in an incubation chamber at 20 degrees centigrade and kept in total darkness for 48 hours. Evaluations were made at 24 and 48 hours after treatment.

The results of the filter paper test are summarized in Table 1. Trichlorfon, ethoprop, carbaryl, cyfluthrin, isazophos, and diazinon were very toxic at all rates. In the cases of ethoprop, trichlorfon, diazinon, and cyfluthrin all worms were dead within 48 hours; many dying within the first several hours of exposure to the chemical. Worms exposed to trichlorfon had several blood-filled herniations and death appeared to be a result of hemorrhage. Worms exposed to diazinon exhibited the same symptoms of blood-filled herniations. In addition, ulcerated sores such as those characteristic of contact with carbaryl, were also apparent. Fluid-filled blisters are common on worms exposed to carbaryl. The blisters are relatively large, often covering several segments. These blisters will eventually rupture and blood and body fluid are extruded.

Worms exhibited a writhing behavior when placed on the cyfluthrin-treated filter paper. After 24 hours, several constrictions were present along the worm's body almost as if a worm twisted its anterior segments so many times that the distal segments were broken off. Many times, body segments may have been completely segregated or merely held together by a thin band of tissue. This segregation began distally and progressed toward the anterior segments. In addition, several worms had their genital setae exposed - this was not observed in any other worms prior to, or during the study. Ethoprop resulted in a violent seizures of the worms immediately upon contact with the impregnated filter paper. After 5-10 minutes the seizures stopped and many of the worms were dead. The worms remained intact and there were no herniations or constrictions.

Isazophos and carbaryl had a similar effect on the earthworms as trichlorfon. Ulcers and herniations were present on many worms. A few worms remained alive at the 48 hour evaluation, particularily those exposed to lower concentrations of the chemical, however, all worms were dead after 72 hours.

Chlorpyrifos had a lower immediate toxicity than the previously described materials. Many worms were still responsive to light and touch after 48 hours. However, many of these same worms showed the blood-filled herniations characteristic of trichlorfon, diazinon, and isazophos exposed worms.

Finally, Bacillus thuringiensis var. San Diego and isofenphos appeared to be relatively non-toxic at all rates other than l x and O.lx in the case of isofenphos. All other worms treated with these materials were very reactive to light and touch after 48 hours. These worms were placed into containers filled with a 80:20 sand: loam mixture and evaluated 7 and 14 days later. Many of these worms remained active for the duration of the study.

The second study involved the same insecticides. In this experiment insecticides were added to individual containers holding 500 mg of soil. The soil was comprised of 80% sand and 20% loam by volume. The purpose of this study was to evaluate the effects of contact with the compounds as well as potential ingestion. The insecticides were mixed in 75 mi of deionized water to achieve concentrations of 0.1, 1.0, 10, 100, and 1000 mg active ingredient and added to the soil containers. Normal field rates for each insecticide tend to fall approximately in the center of the test range. Each rate was replicated three times. Six worms were added to each container. Worm survival/activity was evaluated on days 1, 2, 3, 7, 10 and 14. All worms were removed after 14 days.

The results of this experiment are given in Table 2. Results of the soil test are similar to those of the filter paper test. Again, trichlorfon, ethoprop and carbaryl were very toxic at all rates while cyfluthrin, chlorpyrifos and isazophos were toxic at all but the lower rates. As demonstrated in the previous trial, Bacillus thuringiensis var. San Diego and isofenphos were relatively non-toxic to earthworms. This study illustrates the acute effects of several insecticidal compounds on earthworms. It did not however, evaluate such chronic effects as long-term weight loss due to an alteration in feeding behaviour or physiologic processes.

This study also did not examine the effects on reproductive and developmental processes. Further investigation is necessary in these areas to fully understand the adverse effects on earthworms of insecticides frequently applied to turfgrass. Also, the effects of a particular insecticide once it is applied to different soil types, moisture conditions, and temperatures must be examined.

Table 1. Earthworm Mortality Resulting from Exposure to Selected Insecticides at Varying Rates Applies to Filter Paper.
Insecticide	mg Active Ingredient	Average Ratinga Trial 1	Average Ratinga Trial 2
dazinon	1.0	5.0 c b	5.0 c
-	0.1	5.0 c	5.0 c
-	0.01	5.0 c	5.0 c
-	0.001	4.8 c	4.8 c
-	1x10-4	5.0 c	5.0 c
-	1x10-5	3.4 b	4.6 c
-	1x10-6	2.4 b	4.6 c
-
chlorpyrifos	1.0	5.0 c	5.0 c
-	0.1	5.0 c	5.0 c
-	0.01	2.6 c	3.0 b
-	0.001	3.4 b	3.3 bc
-	1x10-4	2.4 b	3.4 bc
-	1x10-5	4.6 c	4.6 c
-	1x10-6	4.6 c	3.8 bc
-
Bacillus thuringiensis	1.0	4.4 bc	3.4 bc
San Diego	0.1	0.2 a	0.6 a
-	0.01	0 a	0.4 a
-	0.001	0 a	0.2 a
-	1x10-4	0 a	0 a
-	1x10-5	0 a	0 a
-	1x10-6	0 a	0 a
-
ethoprop	1.0	5.0 c	5.0 c
-	0.1	5.0 c	5.0 c
-	0.01	5.0 c	5.0 c
-	0.001	5.0 c	5.0 c
-	1x10-4	5.0 c	5.0 c
-	1x10-5	5.0 c	5.0 c
-	1x10-6	5.0 c	5.0 c
-
isofenphos	1.0	5.0 c	5.0 c
-	0.1	5.0 c	5.0 c
-	0.01	0 a	1.0 a
-	0.001	0.2 a	0.2 a
-	1x10-4	0 a	1.4 ab
-	1x10-5	0 a	0 a
-	1x10-6	0 a	0.2 a
-
carbaryl	1.0	5.0 c	5.0 c
-	0.1	5.0 c	5.0 c
-	0.01	4.8 c	4.4 c
-	0.001	4.6 c	3.6 bc
-	1x10-4	5.0 c	3.8 bc
-	1x10-5	4.6 c	5.0 c
-	1x10-6	4.6 c	4.4 c
-
cyfluthrin	1.0	5.0 c	5.0 c
-	0.1	5.0 c	5.0 c
-	0.01	5.0 c	5.0 c
-	0.001	5.0 c	5.0 c
-	1x10-4	5.0 c	4.8 c
-	1x10-5	5.0 c	5.0 c
-	1x10-6	5.0 c	5.0 c
-
isazophos	1.0	5.0 c	5.0 c
-	0.1	5.0 c	5.0 c
-	0.01	5.0 c	4.4 c
-	0.001	5.0 c	5.0 c
-	1x10-4	5.0 c	5.0 c
-	1x10-5	4.4 bc	4.8 c
-	1x10-6	4.6 c	4.2 c
-
check 1	-	1.0 a	1.0 a
check 2	-	1.0 a	0.0 a
LSD (P=0.05)	-	1.05	1.07
a Rating Scale: 0-5 where 0 = no weight loss, reactive to light and touch, integument intact and 5 = dead or unresponsive to light and touch, herniations or constrictions present.
b Numbers within each column followed by the same letter do not differ significantly according to Fisher’s least significant difference (P=0.05)

Table 2. Earthworm Mortality Resulting from Exposure to Selected Insecticides at Varying Rates Applied to a Soil Mixture.
Insecticide	mg Active Ingredient	Rating a Average
diazinon	0.1	0 a b
-	1.0	0.7 a
-	10	2.0 c
-	100	4.0 d
-	1000	4.0 d
-
chlorpyrifos	0.1	0 a
-	1.0	2.3 c
-	10	3.0 d
-	100	4.0 d
-	1000	4.0 d
-
thrichlorfon	0.1	4.0 d
-	1.0	4.0 d
-	10	4.0 d
-	100	4.0 d
-	1000	4.0 d
-
Bacillus thuringiensis	0.1	0 a
San Diego	1.0	0 a
-	10	1.0 ab
-	100	0.3 a
-	1000	2.7 cd
-
ethoprop	0.1	3.0 cd
-	1.0	3.7 d
-	10	4.0 d
-	100	4.0 d
-	1000	4.0 d
-
isofenphos	0.1	0.7 a
-	1.0	0 a
-	10	2.7 cd
-	100	3.0 d
-	1000	2.0 bc
-
carbaryl	0.1	4.0 d
-	1.0	3.3 d
-	10	4.0 d
-	100	3.3 d
-	1000	4.0 d
-
cyfluthrin	0.1	0 a
-	1.0	2.3 c
-	10	3.0 cd
-	100	3.7 d
-	1000	4.0 d
-
isazophos	0.1	0.7 a
-	1.0	2.7 c
-	10	3.0 cd
-	100	3.0 d
-	1000	4.0 d
-
check	-	0 a
LSD (P=0.05)	-	0.88
a Rating Scale: 0-4 whereby 0 = alive at 2 week check, 1 = alive at 1 week but dead at 2 weeks, 2 = alive at 48 hours but dead at 1 week, 3 = alive at 24 hours but dead at 48 hours, and 4 = dead within 48 hours.
b Numbers within each column followed by the same letter do not differ significantly according to Fisher’s least significant difference (P=0.05)

Literature Cited

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