A COMPARATIVE STUDY OF THE TOXICITY OF … COMPARATIVE STUDY OF THE TOXICITY OF CHIPPED TIRES AND WOOD CHIPS LEACHATE Submitted To: Minnesota Department of Transportation Office of

A COMPARATIVE STUDY OF THE TOXICITY OF CHIPPED TIRES AND WOOD CHIPS LEACHATE

Submitted To:

Minnesota Department of Transportation Office of Environmental Services

3485 Hadley Avenue North Oakdale, Minnesota 55128

SLI Report #95-10-6161

SLI Study Numbers: 13595.0995.6100.131 13595.0995.6100.124 13595.0995.6101.430 13595.0995.6102.261 13595.0995.6103.630 13595.0995.6104.705

Program Manager: Ronald C. Biever

Springborn Laboratories, Inc. Environmental Sciences Division

790 Main Street Wareham. Massachusetts 02571-1075

November 27. 1995

DRAFT REPORT

Report No . 95-10-6161 Page 2 of 58

TABLE OF CONTENTS

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : . . . . . . . . 6

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 INTRODUCTION 7

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 MATERIALS AND METHODS 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1TestMethods 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2TestMaterials 8

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3TestSpecies 8 2.3.1 Ceriodaphnia dubia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.3.2 Pimephales promelas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.3.3 Selenastrum capricomutum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.4 Lactuca sativa 9

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.5 Eisenia foetida 9 2.4 Leachate Procedure and Dilution Preparations . . . . . . . . . . . . . . . . . . . . : . . . . . . . . 9

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5TestProcedures 10 2.5.1 Ceriodaphnia dubia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2.5.2 Pimephales promelas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2.5.3 Selenasfrum capricomutum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 2.5.4 Lactuca sativa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2.5.5 Eisenia foetida . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2.5.6 n-OctonalkVater Partition Coefficients . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Statistics and Data Evaluation 14

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.0RESULTS 16 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Leachate Preparation 16

3.2 Toxicological Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1 Ceriodaphnia dubia 17

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2 Pimephales promelas 18 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3 Selenastrum capricomufum 18

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.4 Lactuca sativa 19

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.5 Eisenia foetida 19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 n-Octonal/Water Partition Coefficients 19

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.0 DISCUSSION 20

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SIGNATURES AND APPROVAL 58

Springbom Laboratories. Inc .

Report No. 95-10-6161 Page 3 of 58

Table 1.

Table 2. Table 3.

Table 4. Table 5. Table 6.

Table 7.

Table 8.

Table 9.

Table 10.

Table 11.

Table 12.

Table 13.

Table 14.

Table 15.

Table 16.

Table 17.

Table 18.

Table 19.

Table 20.

Table 21.

LIST OF TABLES Page

A summary of the toxicity test results performed with dilutions of leachate from chipped tires and wood chips. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 ~ o s ~ o s i t i o n of algal growth medium (AAP medium) used in this study. . . . . . . 25 Monitoring equipment and methods used during the conduct of the toxicity tests. - ,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LO

Stock solutions used to formulate the nutrient solution for lettuce. . . . . . . . . . . 27 Results of the characterization of the leachate samples and dilution water. . . . 28 Chemical analyses performed on the chipped tires and wood chips leachates and the control water. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 The water quality ranges measured during the short-term static-renewal exposure of Ceriodaphnia dubia to chipped tires and wood chip leachates. . . . 31 The number of organisms surviving at 48-hours during the short-term static- renewal exposure of Cenodaphnia dubia to chipped tires and wood chip leachates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 The number of organisms surviving at test termination during the short-term static-renewal exposure of Ceriodaphnia dubia to chipped tires and wood chip leachates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 The total number of offspring produced at test termination of the short-term static-renewal exposure of Ceriodaphnia dubia to chipped tires leachate. . . . . . 34 The number of organisms surviving at 48-hours during the short-terrn static- renewal exposure of Cenodaphnia dubia to chipped fires leachate. . . . . . . . . . 35 The number of organisms surviving and offspring produced during the short-term static-renewal exposure of Ceriodaphnia dubia to chipped tires leachate. . . . . . 36 The water quality ranges measured during the short-term static-renewal exposure of Pimephales promelas to chipped tires and wood chip leachates. . . 37 Percent survival of fathead minnows (Pimephales promelas) at 48-hours exposed to chipped tires and wood chip leachate during the short-term static- renewaltest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Percent survival of fathead minnows (Pimephales promelas) at test termination (day 7) exposed to chipped tires and wood chip leachate during the short-term static-renewal test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Mean weight of fathead minnows (Pimephales promelas) exposed to chipped tires and wood chip leachate during the short-term static-renewal test. . . . . . . . 40 Conductivity, pH, temperature and light intensity measured during the 96-hour exposure of Selenasfrum capricomufum to chipped tires leachate. . . . . . . . . . . 41 Conductivity, pH, temperature and light intensity measured during the 96-hour exposure of Selenasfrum capricomufum to wood chips leachate . . . . . . . . . . . 42 Cell densities (x l o L cellslmL) of Selenasfrum capricomufum after 96 hours of exposure to chipped tires and wood chips leachate. . . . . . . . . . . . . . . . . . . . . . 43 Percent emergence of lettuce (Lactuca sativa) seeds exposed to chipped tires and wood chip leachate during the seedling emergence test. . . . . . . . . . . . . . . 44 Shoot length of Lettuce (Lactuca sativa) seeds exposed to chipped tires and wood chips leachate during the seedling emergence test. . . . . . . . . . . . . . . . . 45

Springbom Laboratories, Inc.


Page

Table 22. Soil pH and temperature measured during the 14-day exposure of Eisenia foetida . . . . . . . . . . . . . . . . . . . . to chipped tires and wood chips leachate. .-. . . . . . .46

Table 23. Percent survival observed on day 7 of the toxicity test exposing earthworms (Eisenia foefida) to chipped tires and wood chips leachate. . . : . . . . . . . . . . . . 47

Table 24. Percent survival observed on day 14 of the toxicity test exposing earthworms . . . . . . . . . . . . . . . (Eisenia foetida) to chipped tires and wood chips leachate. 48

Table 25. HPLC analytical results for the reference substances used to prepare the calibration curve. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Table 26. Calculated n-OctonallWater Coefficients for chipped tires and wood chips leachate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50



LIST OF FIGURES Page

Figure 1. The dissolved oxygen and pH of the chipped tire and wood chips leachate during the7-dayleachingperiod . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Figure 2. The mean dissolved oxygen concentrations from the fathead minnow beakers of the chipped tire and wood chips leachates afler the first 24 hours of exposure . 54

Figure 3. A representative chromatogram from the n-octanollwater coefficient analysis of the chipped tire leachate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Figure 4. A representative chromatogram from the n-octanollwater coefficient analysis of the wood chips leachate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Figure 5. Historic zinc toxicological responses compared to the toxicity observed in the chipped tire and wood chips leachate, expressed as ~ g l L zinc. . . . . . . . . . . . . 57

Springborn Laboratories, Inc.

Report No. 95-1 0-6161 Page 6 of 58

SUMMARY

Representative aquatic and terrestrial plants and animals were exposed to leaihates from chipped tires and wood chips to assist in evaluating the relative safety or risks associated with using chipped tires in road beds. A series of quantitative and qualitative analyses were also performed on each of the leachates in an effort to identify potential sources or mechanisms of toxicity. A water flea (Ceriodaphnia dubia), the fathead minnow (Pimephales promelas) and a green algae (Selenasfrurn capricomuturn) were the aquatic organisms used for the exposures. Lettuce (Lacfuca sativa), and an earthwon (Eisenia foetida) were the terrestrial organisms used for the exposures. The two leachates and the dilution water were analyzed for baselneutral extractables (EPA Method 625) and selected metals. The n-octanollwater partition coefficients log(P,) were estimated for each resolved peak from an HPLC chromatogram of each leachate. -

Both leachates were toxic to the aquatic organisms and non-toxic to the terrestrial organisms, with the chipped tire leachate being more toxic than the wood chips leachate (Table 1). The toxicity to aquatic organisms was strongly correlated with the zinc concentrations in the two leachates. The zinc concentration of the 100% chipped tire and wood chips leachates were 2.95 mglL and 0.284 mg/L. respectively. The absense of toxicity towards the terrestrial organisms was also supported by the zinc concentrations. Baselneutral extractable organics and the other metals analyzed were found at concentrations at least an order of magnitude below LC50 concentrations. There Ke[e.no constituents in the tire leachate which showed a - . . pgtectial to bioaccu.mulate. The highest calculated P, in the chipped tire leachate was 210. .. ,

T h 6 were three constituents in the wood chips leachate which had a calculated P, greater than 10,000.

~ l ~ a k may have been the most sensitive species tested for both leachates, assuming that the majority of effects seen in the algae were toxicant related and not a result of competition or predation by other organism. In contrast, lettuce growth was stimulated with increasing volumes of leachate. C. dubia were more sensitive than the fathead minnows in the chipped tire leachate and the fathead minnows were more sensitive than the C. dubia in the wood chips leachate. Earthworms were not effected by either leachate. The earthworms were exposed to the highest concentrations of zinc with no observed effect (theoretical soil concentration of 1.1 mglkg).



1.0 INTRODUCTION

Due to decreasing safe land fill sites and natural resources and the increased costs associated with trash disposal, the demand to recycle or reuse rather than dispose of many items is constantly increasing. However, when reused materials are placed back into the environment, managers and regulators have the difficult task of evaluating the safety of using complex conglomerations in systems open to the environment. The Minnesota Department of Transportation has recently had opportunities to use chipped tires for road bed materials, but were unable to confidently evaluate the environmental safety associated with the proposed use. In an effort to further evaluate the safety of using chipped tires in the environment, the Minnesota Department of Transportation proposed to compare the relative toxicity of chipped tires to the relative toxicity of a generally accepted as safe product - wood chips. The testing program included leaching the two materials and exposing aquatic and terrestrial plants and animals to the leachates in standard side-by-side comparison toxicity studies. -

The objective of this study was to determine the effects of leachate generated f rom chipped tires and wood chips on the following: survival and reproduction of the freshwater invertebrate, Ceriodaphnia dubia, survival and growth on the freshwater fish, Pimephales promelas, growth of the freshwater green alga, Selenastrum capncomutum, emergence and shoot growth of lettuce, Lactuca sativa, and survival of earthworms, Eisenia foetida. The n- octanollwater partition coefficients log(P,) of each leachate were estimated by determining the capacity factor (a measure of net retention time) for each resolved peak from an HPLC chromatogram and interpolating from a calibration curve of the capacity factor versus the known .- log(P,) for a series of reference substances. The two leachates and the dilution water were also analyzed for baselneutral extractables (EPA Method 625) and selected metals.

The studies were initiated on 6 October 1995. The experimental phase of each study was conducted between - 6 and 21 October -. 1995 at Springbom Laboratories, Inc., Wareham. Massachusetts. All original raw data and the final report generated during this study are stored by Springborn at the above location.

2.0 MATERIALS AND METHODS

2.1 Test Methods The studies were performed according to the following Springbom test methods:

"Modified Short-Term Static Renewal Toxicity Test with Ceriodaphnia dubia" Springbom Test Method #: 062895NlrAT-Cp-323, "Modified Short-Term Static Renewal Larval Growth Toxicity Test Using Faihead Minnows (Pimephales promelas)" Springbom Test Method #:062895/WAT-Pp-334, "96-Hour Toxicity Test with the Freshwater Green Alga, Selenastn~m capricomufum, to meet ASTM Standard Guideline # El218 and U.S. EPA Guidelines for Screening Hazardous Waste Sites" Springbom Test Method #:Dl 1695WAQT-Sc-380, "14-Day Lettuce (Lactuca sativa) Seedling Emergence Screening Toxicity Test of Leachate in Silica Sand" Springbom Test Method #:062795/LEACHATE-Ls-2614, "14-Day Earthworm Subacute Toxicity Test of Leachates in Soil" Springborn Test Method :: 062795lLEACHATE-Ef-201" and

Springbom Laboratories, lnc.

'Test Method for the Detennination of n-OctonaltWater Partition Coefficient Following OECD Guidelines, Section 117" Test Method #: 0629941Kow by HPLC.

2.2 Test Materials J .

The test materials, chipped tires and wood chips, were received from the Minnesota - Department of Transportation. Saint Paul. Minnesota on lS-a~~7-Sep.ternbed'95~ respectively. Upon receipt at Springbom, the test materials were stored at room temperature (approximately 20 "C). Approximately 0.6 m3 of chipped tires and 0.7 m3 of wood chips were received.

2.3 Test Species 2.3.f Ceriodaphnia dubia Cen'odaphnia dubia, r; 24 hours old, were obtained from cultures maintained at

Springbom. The culture system consisted of approximately one hundred 30-mL plastic beakers each containing 15 mL of culture medium and one adult. Six days prior to test initiation, adult daphnids were cultured in laboratory water adjusted to a hardness of 44 mglL as CaCO,. Total hardness was adjusted by addition of reagent grade chemicals (EPA, 1989). Ce'odaphnia dubia cultures were fed suspensions of a unicellular green algae (Selenastnlm capn'comutum) and YCT (yeast, trout chow and cereal leaves) once daily. Prior to test initiation, all immature daphnids were removed from the culture beakers. Offspring produced over the first 8-hour period were culled individually using a glass pipet and were used to initiate the test.

2.3.2 Pimephales promelas Pimephales promelas (fathead minnows), ~ 2 4 hours old at test initiation, were obtained

from the Springbom culture facility. Soft town and well water was continuously pumped into the fish breeding unit and holding tanks. The fish were maintained under a 16:8 light and dark photoperiod. Breeding fish were fed frozen brine shrimp (Arfemia salina) daily. Fertile embryos were obtained by natural spawning of brood stock minnows. Eggs obtained from the culture units 4 to 5 days before the start of the toxicity test were incubated until hatching. The eggs were maintained in oscillating egg cups in a separate aquarium containing continuously flowing water. On the day prior to test initiation, all cups were checked and previously hatched larvae were removed.

2.3.3 Selenastrum capricornutum The alga used in this toxicity test was the freshwater green alga Selenastnlm

capricomutum, strain 1648, Class Chlorophyceae. The alga was originally obtained from the Carolina Biological Supply Company, Buriington, North Carolina, and was maintained in stock culture at Springbom.

The culture medium used was Algal Assay Procedure (MP) medium prepared with sterile, deionized water. The components used to formulate AAP medium are listed in Table 2. The pH of this medium was 7.5 f 0.1. Stock cultures were grown in 125-mL glass flasks containing 50 mL of medium. The flasks were covered with stainless steel caps which permitted gas exchange.

The stock cultures were maintained within the following conditions: shaking rate of 100 f 10 rpm, temperature of 24 f 1 OC with continuous illumination at the surface of the medium of




approximately 300 to 500 footcandles for a minimum of seven days before test initiation (Springbom Algae Culture Daily Log, 1995). Cultures were maintained in a temperature- controlled environmental chamber. Lighting was supplied by ~ u r o - ~ e s @ Vita-LiteD fluorescent lights. Culture flasks were agitated continuously on an orbital shaker.

.

The inoculum used to initiate the toxicity test with each leachate was taken from a stock culture that had been transferred to fresh medium seven days before testing.

2.3.4 Lactuca sativa Lactuca sativa (buttercrunch lettuce) seeds (Lot # RA08) collected in 1994, were

obtained from Park Seed Company, Greenwood, South Carolina. The seeds used for testing were not pretreated with fungicides or insecticides to avoid potential interaction with the leachate. Upon receipt at Springbom, seeds were stored refrigerated at approximately 4 OC in the dark until test initiation.

2.3.5 Eisenia foetida The earthworms (Eisenia foefida) (SLI Lot #95A80) were received from Carolina

Biological Supply Company, Burlington, North Carolina on 21 September 1995. Upon receipt at Springbom, the earthworms were added to seven cultures each of which consisted of 4.8 kg worm bedding, 1.5 kg cattle manure and 10 L of reagent grade water which provided a moisture content of approximately 75%. The pH of the culture soils ranged from 6.3 to 6.5. The temperature of the cultures was 20 i 2 OC. The earthworms were maintained in the culture soil for 13 days. Twenty four hours prior to test initiation, earthworms were transferred from culture soil to acclimation soil (70% sand, 20% peat moss and 10% Kaolin clay moistened to approximately 25% with distilled, deionized water) and were fed 1 kg of composted cattle manure.

2.4 Leachate Procedure and Dilution Preparations Following recornmendations made in "Ecological Evaluation of Proposed Discharge of

Dredged Material into Ocean Waters" (EPNUSACE. 1977), a leachate was prepared for each test material (chipped tires and wood chips). Each leachate was prepared by combining a 25 L volume of a test material and 100 L of laboratory water (test material to water volume ratio of I : ) . The test materials were leached for 7 days prior to test initiation in separate fiberglass tanks. Each leachate was stirred daily for ten minutes with a polypropolyene rod. Dissolved oxygen, pH, temperature and conductivity were also measured in each leachate daily. After seven days, approximately 80 L of leachate were siphoned from each of the tanks through a 400 vm nitex screen and used for testing. The leachates used for toxicity testing were not filtered.

Each toxicity study was conducted with a dilution water control and five leachate treatments (6.3, 13, 25, 50, and 100%). The leachate treatments for the daphnid, fathead minnow, lettuce and earthworm studies were prepared in common, by mixing the appropriate amount of leachate with laboratory water. Leachate used for the C. dubia and P. promelas renewals were refrigerated (approximately 4 'C). The dilulions were prepared daily. ---- - . .- - - -. - . . - . .. ..

Leachate treatments for the algae studies were prepared by mixing the appropriate amount of leachate with AAP medium. The AAP nutrients and trace metals were added to each



of the 100% leachates (nutrified) prior to making the dilutions to assure that the leachate dilutions were not nutrient deficient. Each leachate (100%) without AAP nutrients and trace metals was also tested.

Total hardness (as CaCO,), lotal alkalinity (as CaCO,), dissolved oxygen, pH, specific conductance, salinity, total residual chlorine, and ammonia (as N) were measured on the dilution water and each of the leachates (100%). A list of monitoring equipment or methods used during the toxicity studies is presented in Table 3.

A sample of each leachate (100%) was taken for n-octanollwater partition coefficient detennination. Samples of each leachate and the laboratory dilution water were also sent to Aqua Air ( A ~ ) Analytical, Weymouth, Massachusetts, were they were analyzed for baselneutral extractables, arsenic, barium, cadmium, chromium, hexavalent chromium, lead, mercury, selenium, silver, and zinc. -

2.5 Test Procedures i

2.5.1 Ceriodaphnia dubia.-- ~ '

Plastic cups (30-m~),.lhach containing 15 mL of test solution and one daphnid, were placed izan insulated foam board (2.5 cm thick), floated in a waterbath and were covered with a sheet of plastic wrap. The board included five leachate treatments plus a dilution water control (laboratory water), with ten replicates per treatment. The test solutions were renewed daily, by transfenihg the adult organisms to freshly prepared solutions. During the renewal process, each cup was examined and the number of offspring produced over a 24-hour period was recorded. C. dubia were fed 100 pL of algae (S. capricornutum) and 100 pL of YCT suspension. . ( . . . .

. . . . . ,. .. f'c 7 . . < .. .

The test was conducted at 25 *'I "C, with a 16:8-hour light and dark photoperiod. Conductivity was measured in each new test solution at the beginning of each 24-hour period. Dissolved oxygen, temperature and pH were measured in the aged (approximately 24-hours old) and freshly prepared solutions of each 24-hour renewal period.

2.5.2 Pimephales promelas The test was conducted in triplicate with a dilution water control and five leachate

treatments (6.3, 13, 25, 50, and 100 percent). Test vessels were glass beakers (1000-mL), each containing 800 mL of test solution, which were placed in a ternperature controlled water bath and covered with a sheet of plastic wrap. Each beaker contained 15 post-hatched fish larvae.

Approximately 80 percent of the test solution in each test vessel was renewed daily. Prior to the renewal, unconsumed food and debris were pipetted from the bottom of each beaker. The fish larvae were then counted, the old test solution was siphoned out using a siphon hose with a 6-cm screened funnel attached on the inlet to prevent siphoning fish larvae and the new test solution was gently siphoned into the beaker. Prior to renewal, the

each new test solution was adjusted to 25 i 1 "C. The fish larvae were fed 0.15 suspension of Arfemia nauplii (s 24-hours old) twice daily.



The test was conducted at 25 i 1 OC with a 163-hour light-and dark photoperiod. Conductivity was measured in each new test solution at the beginning of each 24-hour period. Dissolved oxygen, temperature and pH were measured in the aged (approximately 24-hours old) and freshly prepared solutions of each 24-hour renewal period.

At test termination, fish from each test beaker were collected in a net, rinsed with deionized water and were transferred to pre-weighed aluminum pans. The fish larvae were dried to constant weight for 2 hours at 100 OC. The fish were placed in a desiccator until weighed on an American Scientific Products SP 182 electrobalance.

2.5.3 Selenastrum capricornutum The test was conducted in triplicate with a control, five leachate treatments (nutrified)

(6.3, 13, 25, 50 and 100%) and a 100% leachate which contained no AAP medium nutrients. The AAP medium used to prepare the exposure solutions was formulated in the same manner as the culture medium. Several liters of AAP medium were prepared and equilibrated to test temperature. The initial pH of this medium was 7.4 and required no adjustment prior to use.

Sterile 125-mL Erlenmeyer flasks, were conditioned prior to use by rinsing with the appropriate exposure solution. Fifty milliliters of the appropriate test solution were then placed in each of the flasks. The control vessels were prepared under the same conditions as the treatment vessels, but contained no leachate. All test vessels were fitted with stainless steel caps, which permits gas exchange. The test vessels were randomly positioned on a shaking table at test initiation.

Approximately 30 minutes after the test solutions were prepared and added to the test vessels, 0.1 1 mL of an inoculum of Selenastrum capticomutum cells at a density of approximately 1.0 x l o 4 cells/mL, was aseptically introduced into each flask. The inoculum provided a cell density of approximately 50 x 104 cellslmL.

The test was conducted in an environmental chamber adjusted to maintain a temperature of 24 i I "C, with a continuous light intensity of 300 to 440 footcandles, and a shaking table rate of 100 r 10 rpm.

Temperature was measured continuously with a Taylor minimumlmaximum thermometer located in a flask of water adjacent to the test fiasks. The shaking rate of the orbital shaker was recorded daily. Light intensity (footcandles) of the test area was measured with a General Electric Type 214 light meter at 0 hour and each 24-hour interval of the exposure period.

Afler 96-hours of exposure, cell counts were conducted on each test vessel using a hemacylometer (Neubauer Improved) and an Olympus compound microscope. One or more hemacytometer fields, each 0.10 x 0.10 cm in surface area and 0.010 cm deep and containing 0.00010 mL of culture, were examined for each sample until at least four fields or 400 cells were counted. Observations of the health of the cells were also recorded at test termination.

The pH and conductivity of the test and control solutions were measured at test initiation and at the termination of the 96-hour exposure period. Measurements at test initiation were conducted on the remainder of each test and control solution after the individual test flasks had



been filled. At test termination, after cell countswere completed, the three replicate vessels for each test solution or control were individually composited and a portion of each composite solution was transferred to a 100-mL beaker for pH and conductivity measurements. -

2.5.4 Lactuca sativa The exposure vessels were 13-cm tall, with a top diameter of 13 cm and a bottom

diameter of 9 cm, polypropylene pots (Kord Products Ltd.). Three replicate pots were maintained for each leachate treatment tested. An 8.3-cm diameter unbleached paper filter was placed in the bottom of each pot to retain the support medium and allow for uptake of the nutrient solution by subimgation. The filter paper was moistened with reagent grade water, and then each pot was filled to a depth of 10 cm with approximately 1.5 kg of support medium (diameter at surface of the support medium = 11.5 cm; surface area = 104 cm2). The support medium consisted of washed, 20- to 40-mesh silica sand (Wedron Silica Co.) which had a pH of 7.8 and containing 0.21 % organic matter. Each pot was placed in a polypropylene saucer (Kord Products Ltd.), which served as a subimgation reservoir for the nutrient solution. The nutrient solution contained the necessary minerals and trace elements for plant growth (Table 4).

To initiate the test, the sand in each pot was moistened with 200 mL of the appropriate test solution or nutrient solution (control). Then, the sand in each pot was leveled, and ten seeds were impartially selected and planted at a depth of approximately 1 cm in each pot (30 seeds per treatment and the control). The seeds were placed in a circular pattern around the inside perimeter of the pot. Control replicates were planted first, followed by the treatment levels. One hundred milliliters of nutrient solution was added to each saucer (subirrigation) daily.

The study was conducted in an environmental growth chamber, which maintained an air temperature of 25 * 5 OC, a relative humidity of > 60% (with the exception of day 5, the humidity fell to 58%), a photoperiod of 16 hours lighU8 hours dark, and a light intensity of 900 to 1000 footcandles. Air temperature was controlled using a thermostatically-regulated heaterlair conditioner system. Air temperature and ambient carbon dioxide were monitored daily. Humidity was maintained through evaporation of water from the nutrient solution, plant transpiration, and by a humidifying system in the environmental growth chamber.

Each pot was observed on day 14 (test termination) to determine percent emergence, mortality and the morphological abnormalities (e.g., chlorosis of leaves) of the emerged seedlings. Shoot length of each surviving seedling was also measured at test termination.

2.5.5 Eisenia foetida The test was conducted in triplicate with a dilution water control and five leachate

treatments (6.3, 13, 25, 50 and 100%). For each treatment and control. 214 mL of the appropriate test solution or control were added to 600 g (dry weight) of artificial soil medium, which provided a water holding capacity (WHC) of 75%. Each test medium was mixed in a mechanical mixer. Test vessels were 400 mL polypropylene beakers, containing 200 g of soil (dry weight) and 10 mature earthworms, which were randomly distributed.


Report No. 95-1 0-6161 Page 13 of 58

The temperature was measured continuously in one test vessel using a minimum- maximum thermometer. The exposure was conducted at 20 2 2'C with the exception of test days 4 and 5, the minimum temperature fell to 17 'C. A 24 hour light photoperiod was provided with a light intensity at the soil surface ranging from 57 - 100 footcandles (613 - 1080 lux). Earthworms were not fed during the test.

On days 7 and 14, earthworm mortality and health assessments were performed. Mortality was assessed by emptying the test medium onto a tray, sorting the earthworms from the medium and testing their reaction to a mechanical stimulus at the anterior end. Mortality was defined as a lack of visible movement after gentle mechanical stimulation was applied. The general health of the earthworms was assessed and recorded by observing color changes, lethargy, softness, coiling, shortening, lengthening, lesions and the presence of cocoons. Soil moisture content, pH and temperature were measured in one replicate vessel per treatment and control at the initiation and termination.

2.5.6 n-OctonalNater Partition Coefficients The analysis was conducted at room temperature (21 "C). Separate solutions o f each

reference substance listed below were prepared by weighing approximately 50-mg (adjusted for purity) of each and diluting to 50 mL with methanol, to obtain a reference substance solution concentration of approximately 1.00 mg/rnL. All solvents were HPLC grade.

The following compounds were used as analytical standards in the preparation of the P, calibration curve:

Acetanilide, Lot No. 18389 (Thomas Scientific), purity of 100 %. Aniline, Lot No. 10126MG (Aldrich), purity of 99.5 %. Toluene, Lot No. 05041U (AldrichlSigma), purity of 99.8 %. Naphthalene, Lot No. 05902CZ (Aldrich), purity of 99 %. 1.2,4-Trichlorobenzene, Lot No. 80-1048 (Chem Service). purity of 99 %. Fluoranthene, Lot No. 08112HW (Aldrich), purity of 98 %.

Formamide, Lot No. 15231AN (Aldrich), with a purity of 99.5 %, was used to determine the dead time of the HPLC system. DDT, Lot No. HW04029MM (Aldrich), with a purity of 99+ % was used to determine the retention time (P,) upper limit.

Chipped tires and wood leachales, reference and control blank samples were analyzed in duplicate by high performance liquid chromatography with ultraviolet detection (HPLC-UV). HPLC instrumentation consisted of a Waters Model 510 pump, a Waters Model 7108 autosampler, equipped with an Applied Biosystems Model 759A variable wavelength UV detector and Hewlett-Packard Model 33968 integrator were used.

The HPLC conditions were as follows: Column: Metachem lnertsii. ODs-2, 5 gm, 250 mm (length) x

4.6 mm (inner diameter) Flow Rate: 1.0 mUrninute Injection Volume: 5.0, 100 and 200 pL (reference standards, wood chips

leachate and chipped tires leachale)

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Wavelength: 210,220 nm (210 nm for leachate samples and 220 nm for reference standards)

Range: 0.1 AUFS Mobile Phases: A: NANOpure water -

0: acetonitrile

Gradient Program

Initial 20.0 39.0 40.0

Run time: 40 minutes Equilibration delay: 15 minutes

The measured capacity factor (k) for each reference substance was calcu1ate.d using the formula:

k . ( t R - to)

to

where: tR = the mean retention time for the reference substance 1, = the dead time for the system, determined by injection a non-retained compound, tR(forrnamide) = 3.008 minutes

A calibration curve was constructed by plotting the log of the mean measured capacity factor (k) of each reference substance against the log of its n-octanol/water partition coefficient ( P ) . A linear regression analysis was performed for these log-transformed results, and the coefficient of determination, slope and y-intercept of the regression were calculated.

2.6 Statistics and Data Evaluation For the daphnid, fathead minnows, lettuce and earthworm studies, the concentration-

response relationships observed were characterized by the median lethal concentration ( ~ ~ 5 0 ) ' which is the concentration (treatment) that is calculated to be lethal to 50 percent of the organisms at the stated time interval. LC50 values were empirically estimated as being greater than the highest treatment level tested when no treatment level caused 50% or more mortality. If at least one treatment level caused mortality of greater than 50% of the test population, then a computer program (C. Stephan, U.S. EPA, personal communication, 1982) was used to estimate the LC50 value. Three statistical methods were available in the computer program: probit analysis, moving average angle method and binomial probability. Two additional computer programs estimated LC50 values using the Trimmed Spearman-Karber, Spearman-Karber and probit methods. The method selected was based on the number of treatment levels with partial mortalities (mortality greater than 0 but less than 100 percent) and on the 95 percent confidence intervals. Generally, to choose the best estimate of the LC50 value for a particular data set, the following approach was used:

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1. If the data set contained no partial mortality, then the binomial probability method was considered.

2. If the data set contained one partial mortality, then the moving average angle method. binomial probability, Spearman-Karber and Trimmed Spearman-Karber methods were considered.

3. If the data set contained two or more partial mortalities, then the probit analysis, moving average angle method, binomial probability, Spearman-Karber and Trimmed Spearman- Karber methods were considered.

4. The appropriate method with the smallest confidence interval is selected.

Survival, growth, and reproduction data were analyzed to determine the no observed effect concentration (NOEC). If the mean percent survival of a treatment was less+han or equal to the mean percent survival of the control, survival data were analyzed. Survival data were analyzed prior to growth and reproduction data. Treatment levels which caused significant survival effects were excluded from the subsequent analyses.

Data were subjected to Dunnett's Test or Bonferroni's 1-Test (Dunnett, 1955, 1964; EPA, 1989) to determine the (NOEC). Dunnett's or Bonferroni's &Test were preceded by Shapiro- Wilks' and Bartlett's tests which test for normality and homogeneity of the data set. If either Shapiro-$Vilks' or Bartlett's tests failed, Steel's Many-One Rank Test or Wilcoxon Rank Sum Test with Bonferroni Adjustment, a non-parametric procedure, was used to establish the NOEC. All comparisons are made at 95% level of certainty (P s 0.05) except Shapiro Wilks' and Bartlett's Tests, where 99% level of certainty was applied (P s 0.01).

For the algae study, the cell density of each culture from the treatment and control samples at 96-hours was calculated by dividing the number of cells counted by the number of fields examined. Means and standard deviations for cell density were calculated for each treatment level and the control from individual replicate values.

The EC50 values (the concentration (treatment level) of leachate which reduced cell densities by 50%) were calculated based on cell densities after 96 hours of exposure. The EC50 values were empirically estimated as being less than the lowest treatment tested when the lowest treatment caused >50% reduction of cell densities.

Retention times were analyzed to the determine the P,, for isolated peaks in the leachates. If the retention time of the resolved peaks were within the range of the reference substance retention times, the resolved peak's mean measured capacity factor (k,,, calculated in the manner described above) was interpolated by the calibration curve. The partition coefficient of each resolved peak from each leachate (log(P,),,) was interpolated from the log-transformed calibration curve using the following formula:



where: P,, = the log-transformed partition coefficient (log(P,)) of the peak k,, = the mean measured capacity factor of the peak

b = the Y-intercept of the linear regression analysis of the calibration curve m = the slope of the linear regression analysis of the calibration cuwe

For peaks with a retention time outside the range of the reference substance, the log(P,) was expressed as a limit. If the compound eluted more rapidly than the fastest-eluting reference substance its partition coefficient was estimated to be less than the partition coefficient of that reference substance. If the compound eluted more slowly than the slowest- eluting reference substance its partition coefficient was estimated to be greater than the partition coefficient of that reference substance.

3.0 RESULTS -

3.1 Leachate Preparation Differences between the two leachates were already measurable on the day the test

materials and water were combined. There were considerable differences ih the dissolved oxygen and pH between the two leachates. The dissolved oxygen in the chipped tire leachate remained relatively stable for the first 4 days ranging between 8 and 10 mglL (Figure 1). The dissolved oxygen dropped dramatically between days 4 and 5 to approximately 2 mgIL, and remained between 1 and 2 mglL for the remaining leaching period. The dissolved oxygen demand in the wood chips leachate was much greater than in the chipped tire leachate (Figure 1). The dissolved oxygen in the wood chips leachate dropped from 9 mg/L on day 0 to 0.7 mglL on day 1 and generally remained below 1 mglL for the remainder of the leaching period.

The leachates were not aerated until they were siphoned from the tanks following the 7- , day leaching period. At that time 2.8 L volumes of the chipped tire leachate were aerated at a rate of approximately ~ ~ b . u b b l , e s p.e_r,rni,ou!_e. After 30 minutes of aeration the dissolved oxygen had risen to 6.0 mglL, and after 1 hour of aeration the dissolved oxygen was 6.4 mglL. The aeration rate was increased to approximately 500 bubbles per minute for another 30 minutes ,. . . .

.;, : and the dissolved oxygen concentration remained at 6.4 mglL. The chipped tire leachate . ,.. . % *

. . dilutions were prepared at that time. I; '

The dissolved oxygen recovery in the wood chips leachate was slower and not as effective. Following the same aeration procedure for the chipped tire leachate, the dissolved oxygen in the wood chips leachate was 2.0 mglL after 30 minutes, 2.5 mglL after 1 hour (-100 bubbleslmin) and 3.0 mglL afler 30 minutes a1 500 bubbles pet minute. Aeration continued at 500 bubbles per minute for another hour anddissolved oxygen had risen to 4.9 mglL. The wood chips leachate dilutions were prepared at that time.

The pH of the chipped tire leachate decreased slightly over the 7 day leaching period beginning at 6.9 and dropping to 6.4 (Figure 1). The pH of the wood chips leachate decreased steadily for the first 2 or 3 day to approximately 5 and then remained relatively stable ranging between 4.7 and 4.8 (Figure 1).



The conductivity of the two leachates were similar and increased slightly over the 7 day period from 150 to 170 pmhoslcm to 210 to 220 vmhoslcm. Hardness and alkalinity also increased in both leachates. Hardness in the chipped tire leachate, wood chips leachate and dilution water was 68, 56, and 36 mglL as CaCO,, respectively (Table 5). Alkalinity in-the chipped tire leachate, wood chips leachate and dilution water was 40, 24, and 22 mglL as CaCO,, respectively (Table 5). Ammonia concentrations in the chipped tire and wood chips leachates were similar, 0.5 and 0.8 mgfL as N, respectively (Table 5). Both leachates were also colored, the chipped tire leachate was slightly yellow, and the wood chips leachate was brown.

The two leachates and the dilution water were analyzed for baselneutral extractables (EPA Method 625) and selected metals (Table 6). No baselneutral extractables were detected in the dilution water. lsophorone (2 pglL) and 2,6-dinitrotoluene (45 vglL) were the only base neutral extractables detected in the chipped tire leachate. Di-n-butylphthalate (4 pglL), dimethyl phthalate (14 vglL), phenol'(l10 pglL), 2-methyl phenol (34 pglL), and 4-methyl phenol (31 vglL) were measured in the wood chips leachate. Barium (0.0040 mglL) was the only selected metal detected in the dilution water. Barium (0.0367 mglL), lead (0.0031 mglL), and zinc (2.95 mglL) were measured in the chipped tire leachate, as well as trace levels of chromium, mercury, and silver. Barium (0.0528 mg1L)~Iead (0.0020 mglL): and zinc (0.284 mg/L$vere also measured in the wood chips leachate, along with trace levels of chromium and silver.

3.2 Toxicological Results 3,2.1 Ceriodaphnia dubia Table 7 summarized the ranges of dissolved oxygen, pH, temperature and conductivity

in the test solutions for both leachates over the 7-day testing period. The dissolved oxygen, pH temperature, and conductivity of the test solutions from the chipped tire leachates were adequate to support C. dubia. Low dissolved oxyoen concentrations and pH in the 50%3_and_ 100% w~_chips-leachaiec~entra~s~owl6ha~e~b~e~n~~sppn~ible forra,aazome of the -- ., toxicity observed in the wood chips test solutions. Aeration at 100 bubbles per minute was ~ o v i d e d to the C. dubia test solutions for the wood chips leachate at the initiation of the study. Even with small tes!y$umm~jl5 mL), the 100 bubbles p.er.minuteae~ationratecogl_d_n~ maintain dissolved o ~ c o n . c c e ~ t ~ i o n s above .... 4 mglL ._ . . in the 100% wood chips leachate-and - - aijsolved oxyg.e.o.co~.cen!~a~n~~b_~!~e~6~mgIL.in.!h.e~5.0%.w.oodch1~s Ie-achate. The aeration was however able to increase the pH in the 100% and 50% test concentrations.

3- o z - All of the daphnids in the 13% to 100% chipped tire leachates and half of the daphnids in

the 6.3% chipped tire leachate died within the first 48 hours of the study. The 48-hr LC50 for the chipped tire leachate was 6.3% (Table 8). A second study with C. dubia was initiated 24 hours afler the initial study. The second study had a control, and 1.6%, 3.1%, and 6.3% chipped tire leachate concentrations. Survival afler 48 hours in the 6.3% leachate was 40%, while survival in the remaining concentrations was 100% (Table 9). Survival in the 6.3% chipped tire leachate was 0% at the end of the first 7-day study, compared to 90% in the control (Table 10). Survival in the 6.3% chipped tire leachate was 10% at the end of the second 7-day study (Table 11). Survival in the 3.1%, 1.6%, and the control was 100% at the end of the second study (Table 11). The mean number of offspring produced in the 6.3%, 3.1%, and 1.6% concentrations were 0. 9, i

1 . and 11, respectively (Table 11). The mean number of offspring produced in all three concentrations were significantly less than the control. The NOEC of the chipped tire leachate L:- was 3.1% for survival and <1.6% for reproduction.

Springborn Laboratories, inc.


All of the daphnids in the 50% and 100% wood chips leachates died within the first 48 hours of the study (Table 8). Survival in the remaining wood chips leachate concentrations was 90% and in the control 100% (Table 8). The 48-hr LC50 for the wood chips leachate was 34%. Survival decreased some by the end of the 7-day exposure, with 50, 80, and 80% survival in the 25%. 13%, and 6.3% wood chips leachates, respectively (Table 10). The mean number of offspring produced in the 25%, 13%, and 6.3% wood chips leachates were 6, 15, and 18, respectively (Table 12). The NOEC for survival in the wood chips leachate was 25%, even though there was only 50% survival. The NOEC for reproduction in the wood chips leachate was 13%.

3.2.2 Pimephales promelas Table 13 summarized the ranges of dissolved oxygen, pH, temperature and conductivity

in the test solutions for both leachates over the 7-day testing period. The dissolved oxygen, pH temperature, and conductivity of the test solutions from the chipped tire leachates were adequate to support the fathead minnows. Low dissolved oxygen concentrations and pH in the

' 25%. 50%, and 100% wood chips --. leachate .-... concentrations could have been respon~ible..f~iat i7 .O.

_.__._,_i . ... - least some of the !~xity_obse-~~jjnj_h.e~~o_o.d_ch~s~Je~! solutions. The low dissolved oxygen in the wood chips leachate occurred even though aeration was-proded at 100 bubble per minute at the initiation of the study (USEPA, 1989). Figure 2 shows the depressed dissolved oxygen concentrations in the wood chips leachate, even at the lowest concentrations, afler 24 hours with aeration. Aeration was increased to 200 bubbles per minute in the solutions supporting fish afier the first 24 hours, which helped maintain dissolved oxygen above 40% of saturation for the remainder of the study period. \ l'i

/ \\I * ,.. :,?, l All of the fish in the 13% to 100% chipped tire ieachates died within the first 24 hours of J , % I/.. 3, '.

the study. The 48-hr LC50 for the chipped tire leachate was 8.9% (Table 14). Survival in the - . 6.3% chipped tire leachate was 89% at the end of the 7-day study, compared to 96% in the control (Table 15). The mean weights of the fathead minnows at study termination in the 6.3% chipped tire leachate and the control were nearly equal, 0.224 mg and 0.223 mg, respectively (Table 16). The NOEC of the chipped tire leachate was 6:3%, for both survival and growth.

. . All of the fish in the loo%, 50%, and one replicate of the 25% wood chips leachate also died within the first 24 hours of the study. The 48-hr LC50 for the wood chips leachate was 25% (Table 14). Survival in the 25%, 13% and 6.3% wood chips leachate were 38%, 87%, and 80%, respectively at the end of the 7-day study (Table 15). Growth of the surviving fish in the wood chips leachate was good, with the mean weights of the fathead minnows in all of the wood chips leachates (6.3% - 25%) exceeding the control mean weight (Table 16). The NOEC of the wood chips leachate was 13% for both survival and growth. - ., , . :, . . .-

The replicate with 100% mortality in the 25% wood chips leachate was the replicate with the lowest dissolved oxygen concentration (3.0 mglL). Excluding this replicate from the LC50 and NOEC calculations produced only minor changes. The 48-hr LC50 increases from 25% to 30% and the NOEC for growth and survival remain the same.

3.2.3 Selenastrum capricomutum The p m o $ tests in~re~_e_d~behyeep-experi~ment~ini~a!bon~,and termination. The pH of

the chipped tire leachate solutions ranged between 7.1 and 7.4 at test initiatiori'andranged



between 7.9 and 9.6 at test termination (Table 17). The pH of the wood chips leachate solutions ranged between 6.5 and 7.4 at test initiation and ranged between 8.0 and 9.6 at termination (Table 18). The pH in both experiments decreased with increasing concentrations of leachate. '.

Conductivity either decreased or remained the same between experiment initiation and I . I . . . . termination in both tests. The conductivity of the both the chipped tire and wood chips leachate solutions ranged between 120 and 300 pmhoslcm at initiation and 90 and 300 pmhoslcm at . . 9.

I , . termination (Tables 17 and 18). The conductivity of each solution generally increased with increasing leachate concentration.

Algal growth was 100% inhibited in all of the chipped tire leachate test concentrations, with no growth in the 50% and 100% solutions (Table 19). Algal growth in the wood chips leachate was 100% inhibited in the 50% and 100% solutions and 64% to 71% inhibited in the 6.3%, 13%, and 25% solutions (Table 19). The lack of a dose response in wood chips leachates between 6.3%.and.25% suggests that.alga!..gr*h inh.ibi!/on.m.ay have-been-a.cesult.ef competition or predation in the leachate solutions. Predation or competition interactions may -/- -, . .. . . . . . . . . . . .. . h c b e . e n introduced because-tke leachates . . -- were .. .. . not s~e.rilized.pri~o~to use .... . in the experiments. - .

The EC50 concentrations for both leachates were ~6 .3%. A t-test comparing the two leachates at the same leachate concentrations showed that the algae cell densities in the 6.3%. 13%, and 25% chipped tire leachates were significantly less than the respective algae cell densities in the wood chips leachates.

3.2.4 Lactuca sativa Lettuce seedling emergence was not effected by either of the leachates. Mean percent

emergence for the chipped tire leachate ranged between 83% and 97% (Table 20). Mean percent emergence for the wood chips leachate ranged between 93% and 97% (Table 20).

The lea~ha~s~did,h~~e.v_er,~_m~uI~~~l~tt_~~~_e~.g~~wt~~ Mean shoot length increased by 1 ~ 3 3 % -. to . - 25% compared to the control in the chipped tire leachales (Table 21). The increased

shoot length was significant in all test concentrations except for the 13% chipped tire leachate. Mean shoot length increased by 5% to 22% in the wood chips leachate (Table 21). A significant shoot length increase was measured in the 50% wood chips leachate.

3.2.5 Eisenia foetida Soil pH in the two experiments were similar and remained relatively constant throughout

the 14-day exposure. The pH in both the chipped tire leachate dosed soils and the wood chips leachate dosed soils ranged between 6.6 and 7.1 (Table 22).

Earthworm survival was not effected in any of the treated soils during the 14-day exposure. One earthworm died in one of the control replicates and one earth worm was missing in one of the 50% chipped tire leachate replicates (Tables 23 and 24). All other earthworms were recovered alive at the end of the exposure period. The EC50 concentrations for both leachates were >loo%.

3.3 n-OctonalNJater Partition Coefficients Retention time, capacity factors (k), log k, and the log P, of the six reference substance

for the calibration curve are present in Table 25. Retention time, capacity factors (k ) , log k.



calculated log P, and calculated P, results from the chipped tire and wood chips leachates are presented in Table 26. T-vee* were resolved in the chipped tire leachate. Six of the peaks were strongly polar compounds, with a mean calculated P, less than 7.9. The peak with -,

/-- the greatest retention had a mean calculated P, of 210, which is less than the P, fo~toluene. 1 i

-None of these constituents would be expected to bioaccumulate to any significant degree. An .- .. .

example chromatogram of the chipped tires leachate is presented in Figure 3.

T~e~.w~c42_.pegk_s resol_ved.in the wood chips leacha!e, including four peaks which were only isolated in one of the two analyses. There were 25 peaks which had retention times less than aniline and a calculated P, less than 7.9. Eleven of the remaining peaks had a calculated P, in a range similar to the chipped tires leachate, ranging between 7.6 and 186. Five of the remaining six peaks had a calculated P, behveen 438 and 20,800. Two of these peaks were only isolated in one of the two analyses. There was one peak with a calculated P, greater than 1,580,000. An example chromatogram of the wood chips leachate is presented in Figure 4.

-

4.0 DISCUSSION

Both leachates were toxic to the aquatic organisms and non-toxic to the terrestrial organisms, with the chipped tire leachate being more toxic than the wood chips leachate. l he- -- toxicity to aquatic organisms was strongly correlated with the zinc concentrations in the two leachates. The zinc concentration of the 100% chipped tire and wood chips leachates were 2.95 5, - mglL and 0.284 mglL, respectively. Acidlbase extractable organics and the other metals

- analyzed were found at concentrations at least an order of magnitude below LC50 concentrations. There were no constituents in the tire leachate which showed a potential to bioaccumulate. The highest calculated P, in the chipped tire leachate was 210. There were three constituents in the wood chips leachate which had a calculated P, greater than 10,000.

Figure 5 shows a comparison of zinc toxicity from the literature with Ceriodaphnia reticulata, algae (S. capricomutum), and fathead minnows to the toxicity results from the two leachates, expressed in mg/L of zinc. Mount and Norberg (1984) reported zinc 48-hour LC50 concentrations for several Cladoceran species; Ceriodaphnia reticulata 0.076 mgIL; Daphnia magna 0.068 mglL; and D. pulex 0.107 rnglL. The C. dubia 48-hour LC50 concentrations were 6.3% and 34% for the chipped tire and wood chips leachates, respectively. These dilutions result in zinc concentrations of 0.186 and 0.097 rnglL for the tires and wood chips leachates, respectively. The NOEC for survival and reproduction of C. dubia, expressed as zinc, in the chipped tire leachate were 0.091 mglL (3.1%) and <0.047 mglL (<1.6%), respectively. compared to 0.071 mglL (25%) and 0.037 mg/L (13%), respectively in the wood chips leachate

Zinc 96-hour LC50 concentrations for fathead minnows ranged between 0.238 to 2.54 mglL (Norberg and Mount, 1984, and Hobson and Birge, 1989), depending on water hardness, and fish age. Norberg and Mount (1984) used <24 hr old fish and sofl water for their studies and reported a zinc LC50 of 0.238 mglL. The fathead minnow 48-hour LC50 for the chipped tires and wood chips leachates were 0.263 rnglL (8.9%) and 0.071 rnglL (25%), respectively. Norberg and Mount (1984) reported the fathead minnow NOEC for growth at 0.184 mglL. The NOEC for both growth and surrival in the chipped tire leachate was 0.186 mglL (6.3%). The NOEC for both growth and survival in the wood chips leachate was 0.037 rnglL (13%).

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Both the chipped tires leachate and the wood chips leachate significantly suppressed algae growth at all concentrations tested. Blaise, et al., (1986) reported a zinc 96-hour EC50 of 0.0447 mglL for S. capricomufum. There was 100% inhibition when zinc concentrations in the leachates exceeded 0.142 mglL; all of the chipped tire leachate concentrations and the 100% and 5Q% concentrations of thewnod chips leachale. The zin~cmcentrations in the 6.3% to 25% wood chips leachate ranged between 0.018 and 0.071 mglL. Algae grovjlh was approximately 67% inhibited at all three of these concentrations. The lack of a dose response at these three concentrations may suggest that there is competition or predation by another organism when zinc concentrations dropped below an certain threshold.

Neither the earlhworms nor the lettuce were negatively affected by the leachates. Lettuce growth was actually stimulated by the Ieachates. One reason for the lack of toxicity is the dilution of the toxic fractions of the leachate which takes place by combining a volume of leachate to a mass of soil. Assuming the toxic components of the leachates are all available, the 100% leachate for the earthworms was diluted to 36% when mixed with the soil (214 mL leachatel600 g soil). This would result in a maximum zinc concentration in the chipped tire leachates of 1.7 mglkg and 0.102 mglkg in the wood chips leachates. The 100% leachafes for the lettuce exposures were diluted to 13% when mixed with soil (200 mL leachate11,500 g soil), resulting in 0.384 mglkg and 0.037 mglkg zinc in the chipped tire and wood chips leachates, respectively. Zinc is also much less toxic to terrestrial species. Zinc NOEC for plants ranges between 11 mglL and 800 mglL (Gorsuch, el a/. , 1990, and Gorsuch, eta/., 1994).

The results from this five to compare the relative toxicity of two leachates, to an array of and animals. The results also met a secondary objective, which w uses andlor mechanisms of toxicity. The

elative safety or risk associated with using shing process or procedure

ntration of the wash or s a function of pH and water

tires or wood ch areas with well buffer soils and water would programs could be focused around

The results also demonstrated the importance o f a matrix approach to evaluating complex produd mixtures. Algae may have been the most sensitive species tested for both leachates, assuming that the majorit were toxicant related and not a result of competition or predation lettuce growth was stimulated with increasing volumes of leachate. C. sensitive than the fathead minnows in the chipped tire leachate and the more sensitive than the C. dubia in the wood chips leachate. by either leachate. The earthworms were exposed to the observed effect (theoretical soil concentration study plan identified the probable

that the chipped tires did not The analyses also showed that

of metals, as well as possibly



Both the chipped tires leachate and the wood chips leachate significantly suppressed algae growth at all concentrations tested. Blaise, etal., (1986) reported a zinc 96-hour EC50 of 0.0447 mglL for S. capncomutum. There was 100% inhibition when zinc concentrations in the leachates exceeded 0.142 mglL; all of the chipped tire leachate concentrations and the 100% and 50% concentrations of the wood chips leachate. The zinc concentrations in the 6.3% to 25% wood chips leachate ranged between 0.018 and 0.071 mglL. Algae growth was approximately 67% inhibited at all three of these concentrations. The lack of a dose response at these three concentrations may suggest that there is competition or predation by another organism when zinc concentrations dropped below an certain threshold.

Neither the earthworms nor the lettuce were negatively affected by the leachates. Lettuce growth was actually stimulated by the leachates. One reason for the lack of toxicity is the dilution of the toxic fractions of the leachate which takes place by combining a volume of leachate to a mass of soil, Assuming the toxic components of the leachates are all available, the 100% leachate for the earthworms was diluted to 36% when mixed with the soil (244 mL leachate1600 g soil). This would result in a maximum zinc concentration in the chipped tire leachates of 1.1 mglkg and 0.102 mglkg in the wood chips Ieachates. The 100% leachates for the lettuce exposures were diluted to 13% when mixed with soil (200 mL leachate11,500 g soil), resulting in 0.384 mglkg and 0.037 mglkg zinc in the chipped tire and wood chips leachates, respectively. Zinc is also much less toxic to terrestrial species. Zinc NOEC for plants ranges between 11 mglL and 800 mglL (Gorsuch, etal., 1990, and Gorsuch, et a/., 1994).

The results from this study met their primary objective to compare the relative toxicity of two leachates, to an array of aquatic and terrestrial plants and animals. The results also met a secondary objective, which was to identify potential causes andlor mechanisms of toxicity. The results from this study will be useful in identifying the relative safety or risk associated with using chipped tires as a road bed material. For example, any washing process or procedure performed on chipped tires can be evaluated measuring zinc concentration of the wash or leachates before and after washing. ZincJoxicity in water is a function of pH and _water__. hardness, thus using chipped tires or wood chips in areas with well buffer soils and water would rr&e risks to aquatic organisms. Field monitoring of pilot programs could be f ~ e e d d a ~ o u n d m_o-oing zinc concentrations in surface waters and ground water.

The results also demonstrated the importance of a matrix approach to evaluating complex produd mixtures. Algae may have been the most sensitive species tested for both leachates, assuming that the majority of effects seen in the algae were toxicant related and not a result of competition or predation by organism. In contrast, lettuce growth was stimulated with increasing volumes of leachate. C. dubia were more sensitive than the fathead minnows in the chipped tire leachate and the fathead minnows were more sensitive than the C. dubia in the wood chips leachate. Earthworms were not effected by either leachate. The earthworms were exposed to the highest concentrations of zinc with no observed effect (theoretical soil concentration of 1.1 mglkg). The analytical phase of the study plan identified the probable cause of toxicity to the aquatic organisms and demonstrated that the chipped tires did not leachate chemicals with a high potentia!for bioaccumulation. The analyses also showed that the wood chips leachate has a low pH allowing the leaching of metals, as well as. p.ossit!!y. leaching three chemicals with a potential for bioaccumulation.

. ... , ,

, . ._, 1

Sprinobom Laboratories. Inc.

~ i ~ o r t NO. 95-10-6161 Page 22 or 58

REFERENCES

Blaise, C., R. Legault, N. Bermingham, R. Van Coillie, and P. Vasse. 1986. A Simple Microplate Algal Assay Technique for Aquatic Toxicity Assessment. Toxicity Assessments-l:261- 281.

Norberg, T.J. and D.I. Mount. 1985. A new Fathead Minnow (Pirnephales promelas) Toxicity Test. Environmental Toxicology and Chemistry, 4(5):711-718.

Green, J.C., C.L. Bartels, W.J. Warren-Hicks, B.R. Parkhurst. G.L. Linder, S.A. Peterson and W.E. Miller. 1989. Protocols for the Short Term Toxicity Screening of Hazardous Waste Sites. EPA 60013-881029.

Gorsuch, J.W., R.O. Kringle, and K.A. Robillard. 1990. Chemical Effects on the germination and Early Growth of Terrestrial Plants. Plants for Toxicity Assessment, ASTM S i P 1091, Philadelphia, PA. pp. 49-58.

Gorsuch, J.W., M. Ritter, and E.R. Anderson. 1994. Comparative Toxicity of Six Heavy Metals, Using Root Elongation and Shoot Growth of Three Plant Species. Environmental Toxicology and Risk Assessment - Third Volume, ASTM STP 1218, Philadelphia, PA.

Hobson;J.F. and W.J. Birge. 1989. Acclimation-Induced Changes in Toxicity and Induction of Metallothionein-Like Proteins in the Fathead Minnow Following Sublethal Exposure to Zinc. Environmental Toxicology and Chelmsitry, 8(2):157-169.

Mount, 0.1. and T.J. Norberg. 1984. A seven-Day Life-Cycle Cladoceran Toxicity Test. Environmental Toxicology and Chelmsitry, 3(3):425-434.

OECD, Pans 1989. Guidelines for Testing of Chemicals. Physical Chemical Properties: Guideline 117;"Partition Coefficient (n-octanollwater), HPLC Method", adopted by the Council on 30 March 1989.

Stephan, Charles. 1982. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal Communication to Dr. Lowell Bahner, chairman ASTM Task Group on Calculating LC50's.

U. S. Environmental Protection Agency. 1989. Short-Tern Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. EPA/600/4- 89/001.



TABLES



Table 1. A summary of the toxicity test results performed with dilutions of leachate from chipped tires and wood chips.

Test Type and Enpoint % Leachate

Chipped Tires Wood Chips

Water Flea (Ceriodaphnia dubia)

6.3 34 3.1 25 < I .6 13

I '

48-hr LC50 NOEC Survival NOEC Reproduction

Fathead minnow (Pin; 48-hr LC50

NOEC Survival NOEC Growth

s promelas) 8.9 25

6.3 13

6.3 13

Green algae (Seienastrum capricomufum) 96-hr EC50 Cell Density <6.3 ~ 6 . 3 ~ NOEC Cell Density ~ 6 . 3 <6.3a

Lettuce (Lactuca sativa) 7-day LC50 >lo0 >lo0

14-day LC50 >I00 >lo0

NOEC Growth >lOOb >I00

Earthworm (Eisenia foetida) 7-day LC50 >lo0 >I00

14-day LC50 >I00 > lo0

a Cell densities for the 25%, 13% and 6.3% were similar, but significantly less than the sterile control sample (approximately 67% reduction compared to >98% reduction in all chipped tire dilutions). The reductions in these dilutions may have been a result of competion or predation by microorganisms rather than a toxicological response.

Lettuce growth increased with increasing concentrations of chipped tire leachate.



Table 2. Composit ion of algal growth medium (AAP medium) used in this study.

Compound Concentration

NaNO, 25.5 mg A.lL

MgC126H20 12.16 mg A.lA

CaC12.2H20 4.41 mg A. lL

MgS0..7H20 14.7 mg A.lA

Additional nutrient required, personal communication. Dr. R.R.L. Guillard, June 1991

Source: Miller. W.E.. J.C. Greene and T. Shiroyama. 1978. The Selenastrum capricomutum Printr algal assay bottle test. EPA 60019-78-018, U.S. Environmental Protection Agency. Corvallis, OR.

Springborn Laboratories, lnc.


Table 3. Monitoring equipment and methods used during the conduct of the toxicity tests.

EquiprnenffMethod Parameter

Specific Conductivity YSI Model #33 Salinity-Conductivity-Temperature meter and probe

Jenco Model 601A pH meter LaMotte Model HA pH meter -

Dissolved Oxygen YSI Model # 33 Dissolved Oxygen Meter

Daily Temperature Fisher Alcohol Thermometers

Continuous Temperature Fisher Minimum-Maximum thermometers

Light Intensity GE Type 217 Light Meter IL 1350 Radiometer/Photometer

Humidity Hanna Thermohygrometer

Ambient Carbon Dioxide Beckman Ind. Corp. Model #880 Carbon Dioxide Gas Analyzer

Hardness 130.2 (Titrimetric, EDTA)

Alkalinity 310.1 (Titrimetric, pH 4.5)

Total Residual Chlorine Hach Test 330.1 (Amperomertic)

Ammonia as N 4500-NH,G



Table 4. Stock solutions used to formulate the nutrient solution for lettuce.

Stock Solution # Chemical Formula glLa

1 Potassium dihydrogen phosphate KH2P0, 131.1

2 Potassium nitrate KNO, 101.1

3 Calcium nitrate Ca(NO,), 4H,O 236.2 -

4 Magnesium sulfate MgSO, 7H20 246.5

5 Iron Chelated with EDTA C,,H,,N20,FeNa 10.0

6 Trace Elements:

Boric acid 0- -

Zinc chloride

Cuprous chloride

Molybdenum oxide

Manganese chloride

7 Sodium hydroxideb

a All stock solutions are prepared in distilled water.

Sodium hydroxide is uiilized to adjust the pH of the final nutrient solution (Table 2)

Afler formulation, each stock solution is placed in a screw-capped amber 1 - 4 L glass bottle Unused stock solutions can be stored for up to six months.



Table 5. Results of the characterization of the leachate samples and dilution water.

Sample I.D.: Chipped Tires Wood Chips Dilution Leachate Leachate Water'

Parameter Measured . .

Alkalinity (mg/L as CaCO,) 40 24 22

Hardness (mglL as CaCO,)

Specific Conductivity (~~mhoslcm)

Temperature (OC)

Dissolved Oxygen ( m W

Salinity (ppt)

Total Residual Chlorine (mglL) <0.01

Total Ammonia as N (mglL) 0.5

4.7 7.1

~ 0 . 0 1 <0.01

0.8 Not Analyzed - --

a Dilution water used for the daphnid, fathead minnow, lettuce and earthworm studies.



Table 6. Chemical analyses performed on the chipped tires and wood chips leachates and the control water.

Sample I.D. Chipped Tires Wood Chips Control Detection Leachate Leachate Limit

PARAMETER ( ~ g w -- Arsenic ND ND ND 2

Lead 3.1 2.0 ND 0.8 Mercury

- Selenium Silver

-t. Barium Chromium. Total Chromium, Hexavalent

+Zinc -Cadmium

Acenaphthene Acenaphthylene Anthracene Benzo(a)anthracene ~enzo(b)fluoranthene Benzo(k)fluoranthene Enzo(a)pyrene Benzo(g,h,i)perylene bis(2-Ch1oroethyl)ether

--bis(2-Chloroethoxy) 4is-(2-Ethylhexyi)phthalate - bis(2-chloroisopropyl)ether

4Bromophenyl Phenyl Ether - 2-Chloronaphthaiene --4-Chlorophenyl Phenyl Ether Xhrysene

- Dibenzo(a,h)Anthracene Di-n-butylphthalate 1,3-Dichlorobenzene 1,2-Dichlorobenzene I ,4-Dichlorobenzene Diethyl Phthalate Dimethyl Phthalate

0.4 ND 0.3

36.7 2

ND 2950 ND ND- ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND N D N D ND ND

Springbom Laboratories, inc.


Table 6. (continued) Chemical analyses performed on the chipped tires and wood chips leachates and the control water.

Sample I.D. Chipped Tires Wood Chips Control Detection Leachate Leachate Limit

PARAMETER (uglL) 2.4-Dinitrotoluene ND ND ND 1 Di-n-octyl phthalate ND ND ND 10 Fluoranthene ND N D N D 1 Fluorene N D ND ND 3 Hexachlorobenzene ND ND. N D 2 Hexachlorobutadiene ND N D N D 3 Hexachloroethane ND ND ND - 5 Indeno(l,2,3-cd)Pyrene ND ND. ND 1 lsophorone 2 N D ND 2 Naphthalene ND ND N D 2 Nitrobenzene ND ND ND 2 Phenanthrene ND ND ND 1 Pyrene ND ND ND 3 1,2,4-Trichlorobenzene ND ND ND 2 4-Chloro-3-methylphenol ND ND ND 2 2-Chlorophenol ND N D N D 5 2,4-Dichlorophenol ND ND ND 2 2&DimethyIphenol ND ND N D 3 2,4-Dinitrophenol ND ND ND 3 2-Methyl-4,6-dinitrophenol ND ND ND 2 2-Nitrophenol ND ND ND 2 4-Nitrophenol ND ND ND 2 Pentachlorophenol N D N D N D 1 Phenol ND 110 ND 7 2,4,6-Trichlorophenol ND ND N D 1 Benzidine ND N D ND 5 Hexachlorocyclopentadiene ND ND N D 1 N-Nitrosodiphenylamine ND ND ND 3 Butyl Benzyl Phthalate ND ND ND 1 Nnitrosodi-n-propylamine N D N D ND 3 3,YDichlorobenzidine ND ND ND 1 2,6-Dinitrotoluene 45 N D N D 1 2-Methyl Phenol N D 34 ND 3 4-Methyl Phenol ND 3 1 ND 3 N-Nitrosodirnethylamine ND N D ND 10



Table 7. The water quality ranges measured during the short-term static-renewal exposure of Ceriodaphnia dubia to chipped tires and wood chip leachates.

Chipped Tires Leachate Treatment Dissolved PH Temperature Conductivity

Level Oxygen ("c) (pmhoslcrn) (% Leachate) (rnglL)

Controla 7.4 - 8.3 6.8 - 7.5 25 160

Wood Chips Leachate Treatment Dissolved PH Temperature Conductivity

Level Oxygen ("c) (pmhoslcm) (% Leachate) (mglL)

1 OOa 3.1 - 4.1 4.9 - 6.9 24 - 25 260 a At test initiation, oil-free aeration at approximately 100 bubbles per minute was initiated in each test vessel.

Springborn Laboratories. Inc.

Report No. 95-1 0-6161 Page 32 of 58

Table 8. The number of organisms surviving at 48-hours during the short-term static-renewal exposure of Ceriodaphnia dubia to chipped tires and wood chip leachates.

Chipped Tires Leachate Test A B C D E F G H l J 48-hour % %

%Solution Number of Organisms SurvivinglReplicate Survival (SD)' Inhibition (% Leachate) 48-hours

Control I 1 1 1 1 1 1 1 1 1 1 OO(0) 6.3 1 1 1 1 0 0 0 0 0 1 50(53) 13 0 0 0 0 0 0 0 0 0 0 o(0) 25 0 0 0 0 0 0 0 0 0 0 o(0) 50 0 0 0 0 0 0 0 0 0 0 o(0) 100 0 0 0 0 0 0 0 0 0 0 O(0)

Wood Chips Leachate

Control 1 1 1 1 1 1 1 1 1 1 lOO(0) N A 6.3 1 1 1 1 1 1 1 0 1 1 90(32) 10 13 1 1 1 0 1 1 1 1 1 7 90(32) 7 0 25 1 1 1 0 1 1 1 1 1 1 90(32) 10 50 0 0 0 0 0 0 0 0 0 0 o(0) 100 100 0 0 0 0 0 0 0 0 0 0 O(0) 100

SD = Standard Deviation "A = Not Applicable



Table 9. The number of organisms surviving at test termination during the short- term static-renewal exposure of Ceriodaphnia dubia to chipped tires and wood chip leachates.

Chipped Tires Leachate Test A B C D E F G H I J Day 7 % %

%Solution Number of Organisms SurvivingIReplicate Survival (SD)' inhibition (% Leachate) Day 7

Control 1 1 1 0 1 1 1 1 1 1 90(32) NAa 6.3 0 0 0 0 0 0 0 0 0 0 cWc 100 13 0 0 0 0 0 0 0 0 0 0 WC 100 25 0 0 0 0 0 0 0 0 0 0 o(o)= - 7 00 50 0 0 0 0 0 0 0 0 0 0 'Wc 100 100 0 0 0 0 0 0 0 0 0 0 o(o)c 100

Wood Chips Leachate

Control 1 1 1 0 1 1 1 1 7 7 90(32) N A 6.3 1 1 1 1 1 1 1 0 1 0 80(42) 11 13 1 0 1 0 1 1 1 1 1 1 80(42) 11 25 0 0 0 0 1 1 1 1 1 0 50(53) 44 50 0 0 0 0 0 0 0 0 0 0 we 100 100 0 0 0 0 0 0 0 0 0 0 WC 100

' SD = Standard Deviation NA = Not Applicable Statistically different compared to the control, based on Fisher's Exact Test.



Table 10. The total number o f offspring produced at test termination o f the short-term static-renewal exposure of Ceriodaphnia dubia to chipped tires leachate.

Chipped Tires Leachate Test A B C D E F G H I J M.ean %

%Solution Number o f ORspringlReplicate % Survival(SD)' Inhibition _(% Leachate)

Control 20 18 21 0 22 24 21 21 18 30 20(7.7) 6.3 0 0 0 0 0 0 0 0 0 0 o(0) 13 0 0 0 0 0 0 0 0 0 0 ow) 25 0 0 0 0 0 0 0 0 0 0 o(0) 50 0 0 0 0 0 0 0 0 0 0 o m 100 0 0 0 0 0 0 0 0 0 0 O(0)

Wood Chips Leachate

Control 20 18 21 0 22 24 21 21 18 30 20(7.7) N A 6.3 28 20 13 21 26 22 18 0 28 2 18(10) 9 13 12 3 21 0 19 22 19 8 21 20 15(8.2) 26 25. 2 3 0 0 15 10 8 12 5 0 6(5.5)' 72 50 0 0 0 0 0 0 0 0 0 0 o(0) 100 100 0 0 0 0 0 0 0 0 0 0 O(0) 100

a SD = Standard Deviation NA = Not Applicable Statistically different compared to the control, based on Steel's Many-One Rank Test.



Table 11. The number of organisms surviving at 48-hours during the short-term static-renewal exposure of Ceriodaphnia dubia to chipped tires leachate.

Chipped Tires Leachate -

Test A B C D E F G H I J 48-Hours% % %Solution Number of Organisms SurvivinglReplicate Survival (SD)' Inhibition

. 1% Leachate) 48-Hours

Control 1 1 1 1 1 1 1 1 1 1 lOO(0) NAB 1.6 1 1 1 1 1 1 1 1 1 1 lOO(0) 0

i 3.1 1 1 1 1 1 1 1 1 1 1 lOO(0) 0 6.3 1 1 0 0 1 1 1 1 0 0 60(52) 40

SD = Standard Deviation NA = Not Applicable



Table 12. The number of organisms surviving and offspring produced during the short-term static-renewal exposure of Ceriodaphnia dubia to chipped tires leachate.

Chipped Tires Leachate Test A B C D E F G H I J Dav 7 % % - -

%Solution Number of Organisms SurvivinglReplicate survivai (SD)' Inhibition (Oh Leachate) Day 7

Control 1 1 1 1 1 1 1 7 1 1 1 OO(0) NA3 1.6 1 1 1 1 1 1 1 1 1 1 lOO(0) 0 3.1 1 1 1 1 1 1 1 1 1 1 lOO(0) 0 6.3 1 0 0 0 0 0 0 0 0 0 IO(32)' 90

Offspring Produced

Control 21 9 I5 10 18 20 25 24 27 13 le(6.3) N A 1.6 14 0 9 6 6 4 22 20 16 14 Il(7.2)' 39 3.1 5 6 14 13 4 3 11 I1 11 8 9(3.9)$ 53 6.3 0 0 0 0 0 0 0 0 0 0 o(0) 100

' SD = Standard Deviation a NA = Not Applicable

Statistically different compared to the control, based on Fisher's Exact Test Statistically different compared to the control, based on Dunnett's Test.



Table 13. The water quality ranges measured during the short-term static-renewal exposure of Pimephales promelas to chipped tires and wood chip leachates.

- Chipped Tires Leachate

Treatment Dissolved PH Temperature Conductivity Level Oxygen ("(2 (pmhoslcm)

(% Leachate) (mglL)

Wood Chips Leachate Treatment Dissolved PH Temperature Conductivity

Level Oxygen ("C) (gmhoslcm) 1% Leachate) (mglL)

a At test initiation, oil-free aerafion at approximately 100 bubbles per minute was initiated in each test vessel. Although aeration was functioning properly, on day 1, the dissolved oxygen was below 40% saturation and aeration was increased to approximately 200 bubbles per minute.

Springborn Laboralones, Inc.


Table 14. Percent survival o f fathead minnows (Pimephales promelas) at 48-hours exposed t o chipped tires and wood chip leachate during the short-term static-renewal test.

Chipped Tires Leachate

Treatment Percent Survival Level 48-hour Percent

(% Leachate) A-Rep B-Rep C-Rep Mean (SD)' Inhibition

Control 100 100 100 1 OO(0) NAB 6.3 100 87 100 96'7.7) 4 13 0 0 0 o(0) 100 25 0 0 0 o(0) 100 50 0 0 0 O(0) - 100 100 0 0 0 O(0) 100

Wood Chips Leachate

Treatment Percent Survival Level 48-hour Percent

(% Leachate) A-Rep B-Rep C-Rep Mean (SD J' Inhibition

Control 100 100 100 1 OO(0) NAB 6.3 100 80 93 91 (1 0) 9 13 100 100 100 1 OO(0) 0 25 0 80 73 51 (44) 49 50 0 0 0 o(0) 100 100 0 0 0 O(0) 100



~ e p o r t No. 95-10-6161 Page 39 of 58

Table 15. Percent survival of fathead minnows (Pimephales promelas) at test termination (day 7) exposed t o chipped tires and wood chip leachate during the short-term static-renewal test.


Treatment Percent Survival Level Day 7 Percent


Control 93 93 100 96(3.8) NAb 6.3 87 80 100 89($0) 7 13 0 0 0 o(0) 100 25 0 0 0 o(0) 100 50 0 0 0 O(0) - 100 100 0 0 0 O(0) 100

Wood Chips Leachate



Control 93 9 3- 100 9613.8) N Ab 6.3 100 67 73 80(18) 16 13 100 73 87 87(13) 9 25 0 47 67 38(34) 60 50 0 0 0 o m 100 100 0 0 0 O(0) 100

a SD = Standard Deviation a NA = Not Applicable



Table 16. Mean weight of fathead minnows (Pimephales promelas) exposed t o chipped tires and wood chip leachate during the short-term static-renewal test. -


Treatment Weioht fmo) Level Day 7 Percent


Control 0.251 0.169 0.248 0.223(0.046) NA' 6.3 0.222 0.268 0.181 0.224(0.044) 0

- Wood Chips Leachate

Weight (ma) Treatment Level Day 7 Percent


Control 0.251 0.169 0.248 0.223(0.046) NAB 6.3 0.249 0.426. 0.343 0.339(0.089) -52 13 0.400 0.394 0.299 0.364(0.056) 6 3 25 - 0.301 0.319 0.310(0.012) -39




Table 17. Conductivity, pH, temperature and light intensity measured during the 96- hour exposure of Selenastrum capricornutum t o chipped tires leachate.

- Treatment Conductivity

Level pH lumhcslcml (% Leachate) 0 Hour 96 Hours 0 Hour 96 Hours

Control 7.4 9.6 120 90

MinimumlMaximum Temperature (DC)

0 - 24 Hours 24 - 48 Hours 48 - 72 Hours 72 - 96 Hours

24/24 24/24 24/24 24/24

Light Intensity (footcandles)

0 Hour 24 Hours 48 Hours 72 Hours 96 Hours

300 - 400 300 - 400 300 - 400 300 - 400 300 - 400 a Sample was not nutrified with AAP medium.


Reporl No. 95-10-6161 Page 42 of 58

Table 18. Conductivity, pH, temperature and light intensity measured during the 96- hour exposure o f Selenastrum capricomutum to wood chips leachate.

Treatment Conductivity Level pH -

(% Leachate) 0 Hour 96 Hours 0 Hour 96 Hours

Control 7.4 9.6 120 90

MinimumlMaxirnum Temperature ("C)

0 - 24 Hours 24 - 48 Hours 48 - 72 Hours 72 - 96 Hours

24/24 24/24 24/24 24/24

Light Intensity (footcandles)

0 Hour 24 Hours 48 Hours 72 Hours 96 Hours

320 - 440 320 - 440 320 - 440 320 - 440 320 - 440 ' Sample was not nutriiied with AAP medium.



Table 19. Cell densities (x l o 4 celislmL) o f Selenastrum capricomutum after 96 hours o f exposure t o chipped tires and wood chips leachate.


Treatment Cell Density ( x 10' cellslmLI Level 96 Hour Percent


Control 150 150 260 190(65) NA" 6.3' 1 .O 4.5 2.3 2.6(1.8) 100 13' 1 .O 0.25 0.50 0.58(0.38) 100 25' 0.25 0.50 0 0.25(0.25) 100 50' 0 0 0 0 100 100' 0 0 0 0 - 100

Wood Chips Leachate

Treatment Cell Densitv f x 10' cellslmLI Level 96 Hour Percent

(% Leachate) A-Rep 8-Rep C-Rep Mean (SD)' Inhibition

Control 150 150 260 190(65) NAB 6.3' 64 65 66 65(1 .I) 66 13' 52 50 63 55(7.0) 71 25' 70 57 75 67(9.6) 64 50' 0.75 0.50 1.5 0.92(0.52) 100 100' 0 0 0 O(0t 100 1 OOW 0 0 0 O(0) 7 00

' Mean and sandard deviation (SD) were calculated from original raw data. no1 from the rounded MIUS presented in this table. Sample was not nuirified with AAP medium. NA = Nol applicable. Blcated cells and cell fragments were obsewed. Cell fragments were obsewed. Cell fragments and micrwrganisms were observed



Table 20. Percent emergence of lettuce (Lactuca sativa) seeds exposed to chipped tires and wood chip leachate during the seedling emergence test.


Treatment Percent Emeroence Level Day 14 Percent

1% Leachate) A-Rep B-Rep C-Rep Mean (SD). Inhibition

Control 100 90 100 97(5.8) NA" 6.3 100 80 100 93(12) 4 13 100 90 90 93(5.8) 4 25 90 80 80 83(5.8) 14 50 80 100 100 93(12) 4 100 100 70 100 90(17) - 7

Wood Chips Leachate

Treatment Percent Erneroence Level Day 14 Percent


Control 100 90 100 97(5.8) NAa 6.3 90 100 100 97(5.8) 0 13 100 90 90 93(5.8) 4 25 100 100 90 97(5.8) 0 50 100 90 90 93(5.8) 4 100 90 90 100 93(5.8) 4

SD = Standard Deviation a NA = Not Applicable



Table 21. Shoot length of Lettuce (Lactuca sativa) seeds exposed t o chipped tires and wood chips leachate during the seedling emergence test.


Treatment Mean Shoot Lenath Level Day 14 Percent

1% Leachate) A-Rep &Rep C-Rep Mean (SD)' Inhibition8

Control 7.8 7.4 7.6 7.6(0.20) N A' 6.3 9.4 8.5 8.5 8.8(0.50)" -1 6 13 8.6 8.8 8.3 8.6(0.30) -1 3 25 9.9 8.4 9.8 9.4(0.80)' -24 50 9.6 9.8 9.2 9.5(0.30)' -25 100 8.9 9.2 10.1 9.4(0.6)' -24

Wood Chips Leachate

~reatment Mean Shoot Lenath Level Day 14 Percent

(% Leachate) A-Rep B-Rep C-Rep Mean (SD)' Inhibition'

a SD = Standard Deviation Negatjve numbers represent an increase in mean shoot length as compared to the control. NA = Not Applicable Significantly increased as compared to the control, based on Dunnett's Test.


Repori No. 95-10-6161 Page 46 of 58

Table 22. Soil pH and temperature measured during the 14-day exposure of Eisenia foetida to chipped tires and wood chips leachate.


Treatment Temperature Level pH I°CI

(% Leachate) 0 Hour Day 14 0 Hour Day 14

Control 7.0 7.1 19 20 6 3 6.9 6.6 79 20 13 6.7 6 7 19 20 25 6 6 6.7 19 20 50 6.6 6 7 19 20 100 6 6 6.7 19 20

Liqht Intensity (footcandles)

0 -Hour Day 7 Day I4

57 - 85 60 - 80 80- 100

Wood Chips Leachate

Treatment Temperature Level pH PC)

(% Leachate) 0 Hour Day 14 0 Hour Day 14

Control 7.0 7.1 19 20 6.3 6.7 7.0 20 19 13 6.7 6.6 20 20 25 6.6 6.8 20 20 50 6.6 7.1 19 20 100 6.6 7.0 19 20

Liqht Intensity (footcandles)

0 - Hour Day 7 Day I4

57 - 85 60 - 80 80 - 100



Table 23. Percent survival observed on day 7 of the toxicity test exposing earthworms (Eisenia foetida) to chipped tires and wood chips leachate.



(% Leachate) A-Rep &Rep C-Rep Mean (SD)' Inhibition

Control 90 100 100 97(5.8) NAB 6.3 100 100 100 1 OO(0) -4 13 100 100 100 1 OO(0) -4 25 100 100 100 1 OO(0) -4 50 90 100 100 97(5.8) 0 100 100 100 100 lOO(0) - -4

Wood Chips Leachate



Control 90 100 100 97(5.8) NA" 6.3 100 100 100 1 OO(0) -4 13 100 100 100 1 OO(0) -4 25 100 100 100 1 OO(0) -4 50 100 100 1 OOc 1 OO(0) -4 100 100 100 100 I OO(0) -4

a SD = Standard Deviation ' NA = Not Applicable



Table 24. Percent survival observed on day 14 of the toxicity test exposing .--

earthworms (Eisenia foet idsr fo chipped tires and wood chips leachate.



(% Leachate) A-Rep B-Rep C-Rep Mean (SDp Inhibition

Control 90 100 100 97(5.8) NA' 6.3 100 100 100 1 OO(0) -4 13 100 100 100 1 OO(0) -4 25 100 100 100 1 OO(0) -4 50 90 100 100 97(5.8) 0 100 100 100 100 1 OO(0) -4 -

Wood Chips Leachate



Control 90 100 100 97(5.8) NA' 6.3 100 100 100 1 OO(0) 4 13 100 100 100 1 OO(0) -4 25 100 100 100 1 OO(0) -4 50 100 100 100 1 OO(0) -4 100 100 TOO 100 lOO(0) 4




Table 25. HPLC analyt ical resul ts fo r the reference substances used to prepare the calibration curve.a

Reference Replicate Retention kb log k log PDIL Substance Time (min)

Acetanilide 1 18.246 5.067 0.705 1.0

Acetanilide

Aniline

Aniline

- Toluene

- Toluene

Naphthalene

Naphthalene

1,2,4-Trichlorobenzene

1,2,4-Trichlorobenzene

Fluoranthene

Fluoranthene

' All values presented in this table were calculated based on unrounded experimental resub, not the rounded values above. Apparent minor discrepancies in the resub presented may be anributed to rounding.

' The capacity factor (k) for each reference substance was calculated as k = (tR - b)&, where tR is the mean retention time for the reference substance and t, is the dead time for the system, determined by injecting a non- retained compound. On this HPLC system, = lR(forrnamide) = 3.008 minutes. Log(P,) values listed for reference substances are from Table 1, Recommended Reference Compounds, in OECD Guideline 117.


Repori No. 95-10-6161 Page 50 of 58

Table 26. Calculated n-OctonalNJater Coefficients for chipped tires and wood chips leachate.

RT k log k calc'd Pow Mean .

log Pow Pow Chipped Tires

4.886 0.62 -0.204 0.90 < 7.9433 < 7.9433 12.713 3.23 0.509 < 0.90 < 7.9433 < 7.9433 14.012 3.66 0.563 < 0.90 < 7.9433 < 7.9433 15.174 4.05 0.607 < 0.90 < 7.9433 < 7.9433 15.916 4.29 0.633 < 0.90 < 7.9433 < 7.9433

17.945 4.97 0.696 < 0.90 < 7.9433 < 7.9433 18.440 5.13 0.710 0.903 8.0045 7.8210

18.990 5.31 0.725 1.128 13.4204 13.3403 19.982 5.64 0.752 1.514 32.6362 32.0230

20.833 5.93 0.773 1.827 67.1756 66.2155 21.146 6.03 0.780 1.939 86.8502 85.1849 22.282 6.41 0.807 2.328 212.86 210.121

Wood Chips 5.589 0.86 -0.066 < 0.90 < 7.9433 < 7.9433 7.989 1.66 0.219 < 0.90 < 7.9433 < 7.9433 12.766 3.24 0.51 1 < 0.90 < 7.9433 < 7.9433

13.046 3.34 0.523 < 0.90 < 7.9433 < 7.9433 13.520 3.50 0.544 < 0.90 < 7.9433 < 7.9433 13.826 3.60 0.556 < 0.90 < 7.9433 < 7.9433 14.114 3.69 0.567 < 0.90 < 7.9433 < 7.9433

14.252 3.74 0.573 < 0.90 < 7.9433 < 7.9433 14.781 3.91 0.593 < 0.90 < 7.9433 < 7.9433 14.967 3.98 0.600 < 0.90 < 7.9433 < 7.9433 15.156 4.04 0.606 < 0.90 c 7.9433 < 7.9433

15.379 4.1 1 0.614 < 0.90 < 7.9433 < 7.9433 15.565 4.18 0.621 < 0.90 < 7.9433 < 7.9433

15.776 4.25 0.628 < 0.90 c 7.9433 < 7.9433 15.955 4.31 0.634 < 0.90 < 7.9433 < 7.9433 16.241 4.40 0.643 < 0.90 < 7.9433 < 7.9433

16.339 4.43 0.647 < 0.90 < 7.9433 < 7.9433

16.559 4.51 0.654 < 0.90 c 7.9433 < 7.9433 16.699 4.55 0.658 < 0.90 < 7.9433 < 7.9433


Reporl No. 95-10-6161 Page 51 of 58

Table 26. (continued) Calculated n-Octonal~Water Coefficients for Chipped tires and wood chips leachate.

RT k log k calc'd Pow Mean loq Pow Pow



FIGURES



Figure 1. The dissolved oxygen and pH of the chipped tire and wood chips leachate during the 7day leaching period.

Dissolved Oxygen of the Leachates

pH of the Leachates

Chipped tire leachate

10 - - - 2 8; - E - - c - w 6 - z -

0 - n 4 - a, - > - - 0 - "l

2 2: - - -

0

\ - '4 - \

Chipped tire leachate

\ \ \ \ \ \ Wood chips leachate

\ & - - - C - ~ . - . - * - - L - ~ _ -A

I I I I I I I 1

Days

In - .- 7 ; 5 6 - r - n *

- - 5 - - -

- -


: Z \ Wood chips leadiate b, -. *--4 --.

Y--4--L, --A

4 I I I I I I I I

. O 1 2 3 4 5 6 7


Figure 2. The mean dissolved oxygen concentrations from the fathead minnow beakers of the chipped tire and wood chips leachates after the first 24 hours of exposure.

0 ! I I I I i I 1 Control 6.3% 13% 25% . 50% 100%

Treatment Levels


- Report No. 95-10-6161 Page 55 of 58

Figure 3. A representative chromatogram from the n-octanollwater coefficient analysis of the chipped tire leachate.



Figure 4. A representative chromatogram from the nsctanollwater coefficient analysis of the wood chips leachate.

Springborn Laboratories, inc.

. . ,- ,. : :y .. * ' . , , - - r ,, ,,+., , 9 ,.. ' 2 , i 2 . L ir- ,. . . . ., , ! . ;/,,-r. , . . i - - .- :. ,.

1 1.- .- 1 ,p ;;,, ,I.; ;; , ..> 9 b/[. >,A ,J ; 13 .:, ,. - ;, :., 3. .. c , <: Report No. 95-10-6161 ." 1

7 . . 0 : ~ //r r 4 & p b g e 57 of 58 ... L ! I /

Figure 5. Historic zinc toxicological responses compared to the toxicity observed in the chipped tire and wood chips leachate, expressed a s pglL zinc.

i Chipped Tires

100% leachate

50% leachate

13% leachate I

1 'Fish LC50 i 6.3% leachate i 'Ceriodaphnia dubia LC50 I 'Fish NOEC - growth & survival *Algae 100% inhibition 3.1% leachate 'Ceriodaphnia dubia NOEC - survival

1 1.6% leachate 'Cen'odaphnia dubia NOEC - reproduction 4 . 6 % leachate

Literature Wood Chips

100% leachate

Fish LC50

Fish NOEC - growth 4 I

50% leachate

'Ceriodaphnia dubia LC50 Fish NOEC - orowth 4

Zenbdaphnia rekulata LC50 f 25% leachate 'Fish LC50

i 'Ceriodaphnia dubia Algae EC5O - growth NOEC - survival

13% leachate 'Fish NOEC - growth & survival 'Ceriodaphnia dubia NOEC - reproduction

4 6.3% leachate 'Algae 67% inhibition


Report No. 95-10-6161 Paqe 58 of 58

SIGNATURES AND APPROVAL

SUBMITTED BY:

PREPARED BY:

Springbom Laboratories Inc. Environmental Sciences Division

790 Main Street Wareham, Massachusetts 02571-1075

Ann M. Askew Date Study Director

James Hoberg Study Director

Date

Debra Teixeira Study Director

Date

Ronald C. Biever Program Manager Environmental Services

Date

Marjorie E. Dix Date Principal Chemist

John Mao Study Director

Date

Nancy Garvey Study Director

Date


Documents

A COMPARATIVE STUDY OF THE TOXICITY OF … COMPARATIVE STUDY OF THE TOXICITY OF CHIPPED TIRES AND WOOD CHIPS LEACHATE Submitted To: Minnesota Department of Transportation Office of