Snperfand RecoWls Center SITE; V^/^^ S'M^^?— BREAK: R«^ SDMS DOCID 464937 OTHER: H WM DRAFT

STANDARD OPERATING PROCEDURES FOR THE EARTHWORM, EISENIA FOETIDA ANDREI (ANNELIDA: OLIGOCHAETA: LUMBRICIDAE), ARTIFICIAL SOIL, ACUTE TOXICITY BIOASSAY

David C. Wilborn

ManTech Environmental Technology, Inc, Ecological Site Assessment Program Environmental Research Laboratory 200 S.W. 3 5th. Street Corvallis, OR 97333

March, 1992

[Mention of trade names does not constitute endorsement or recommendation for use] TABLE OF CONTENTS

I. SCOPE AND APPLICATION 4

II. INTERFERENCES/LIMITATIONS 8 A. PH/HARDNESS ADJUSTMENT 8 B. DIRECT VERSUS INDIRECT TESTING 9 III. SAMPLE PRESERVATION, CONTAINERS, HANDLING, AND STORAGE . 10 A. SAMPLE PACKAGING MATERIALS 10 B. AQUEOUS HAZARDOUS SUBSTANCES/AQUEOUS SAMPLES. ... 11 C. SOLID HAZARDOUS SUBSTANCES 12 D. LABELING REQUIREMENTS 13 E. SAMPLE HANDLING AND PRESERVATION 14 F. SHIPPING 14 G. TOXIC MATERIALS STORAGE 17 IV. SAMPLE PREPARATION 17 A. INTRODUCTION 17 B. SAMPLE LOG-IN 17 C. SOIL AND SEDIMENT PREPARATION 18 1. Materials and Equipment 18 2. Procedure 18 V. GLASSWARE PREPARATION 20 A. DISCARD HAZARDOUS MATERIAL 20 B. PRE-SCRUB 20 C. MACHINE WASH 20 D. ACID WASH AND RO WATER DIP 21 E. ACETONE RINSE ., 21 F. RO RINSE 21 G. BAKE 22 H. EXCEPTIONS 22 I. NEW GLASSWARE 22 J. GLASSWARE STORAGE 22 VI. BIOASSAY PREPARATION PROCEDURES 22 A. FILLING OUT DATA SHEETS 23 1. Test Code 23 2. Sample Tracking Number 23 3. Dilution Scheme 23 a. Dose vs. Dilution Tests 24 4. Number of Test Containers 24 B. MARKING THE CONTAINERS 24 VII.EARTHWORM TEST PROCEDURE 25 A. METHOD SUMMARY 25 B. EQUIPMENT/APPARATUS 26 C. REAGENTS * 26 A. CONDITIONS 26 B. TIME SCHEDULE 27 C. PRELIMINARY PROCEDURES 28 1. Preparation of Artificial Soil 28 2. Screening and Mixing of Site Soil 29 3. Measurement of Soil pH 29 4. Moisture Content of Site Soil 29 5. Water Holding Capacity of Site Soil 30 D. SETUP 31 1. Test Containers and Labeling 31 2. Cover Sheet 31 3. Calculation Sheet 32 E. TEST PROCEDURES 34 1. Day 1 34 a. Weighing Out Artificial Soil 34 b. Mixing Artificial and Site Soil 34 c. Hydration of Test Soil 35 2. Day 2 35 a. Initial pH Values 35 b. Placing Test Soil Into Jars 35 c. Placing Worms Into Test Soil 36 d. Placing Test Jars in the Test Chamber . . 36 3. 7-Day Count and Observations (Optional) ... 37 4. 14-Day Count and Observations 38 IX. CALCULATIONS 39 X. QUALITY ASSURANCE/QUALITY CONTROL , 39 XI. DATA REDUCTION, VALIDATION, AND STATISTICS 39 XII. WASTE DISPOSAL 41 i\^ XI11. EARTHWORM CULTURE 45 A. PARAMETERS 45 B. WORM CULTURE CHAMBER 46 C. PREPARATION OF CULTURE TRAYS 46 . D. CARRYING CAPACITY OF CULTURE TRAYS 46 E. PREPARATION OF WORM FOOD 47 F. FEEDING AND CARE 47 G. OVERCROWDING 48 H. BEDDING CHANGES 48 I. PEST PROBLEMS 49 XIV. GLOSSARY 50

XV. REFERENCES 57

XVI. ATTACHMENTS 60 I. SCOPE AND APPLICATION The Ecological Site Assessment Program (ESAP), a section of the Ecotoxicology Branch at the Environmental Research Laboratory (ERL-C), United States Environmental Protection Agency (USEPA), has developed this procedure as part of the process of site assessment (Figure 1) to ascertain the chemical, biological, and ecological impacts of anthropogenic disturbances on natural ecosystems, to be performed as part of a battery of bioassays (Figure 2) that cover a broad range of trophic levels and physiological processes. Developing protocols and Standard Operating Procedures (SOPs) for the use of specific bioassays, as well as providing technical assistance to the public, testing labs, and other governmental agencies in the use of these bioassays, is an important parameter of the ESAP mission.

Although some of these bioassays are standardized and the ESAP is evaluating their effectiveness for hazardous waste site assessment, some are developed by the ESAP at ERL-C, and most have been modified during the evaluation process. The ESAP often receives actual site samples from Superfund sites which are used to test these bioassays, and in the process generates useful information pertaining to site remediation.

Many of the tests are of laboratory origin, and in addition to using tests in that fashion, the ESAP is particularly involved in modifying laboratory tests so they may be conducted on-site, or even in situ, to make the tests more relevant and less costly and to provide the much needed verification of accuracy in laboratory testing. Although the procedures outlined are for laboratory applications, several of the tests are currently being modified SITE ASSESSMENT

c -s ENVIRONMENTAL RESEARCH LABORATORY - CORVALLIS BIOASSAY BATTERY FLOW CHART

Ground Water & Surface Water

Microbial Aquatic Terrestrial Terrestrial Test Test Test Test Species Species SpecieI s Species Plants Invertebrates Vertebrates Plants Plants Invertebrates I 1 i I I I SOS Toxi- Amphibian Root Seed Algae Chromolcst Chromotest Cladoceran FETAX Elongation Germination Earthworm

Sclcnastrum Daphnia Xenopus L. sativa L. sativa Eiscnia E. Coli E. Coli capricornulum magna laevis et. al. ct. al. focfida -5 Aaitc/ Critical Critical ft) Chrcinic C.iMidloxic Acute CHronic Chronic Life Stage Life Stage Acute for use on-site. As those methods coalesce, protocols and standard operating procedures will be promulgated. In addition, impacts on sample integrity during sampling, transport, and test matrix preparation are being studied by the ESAP. Part of that information is presented here as portions of this SOP. Factors that are not addressed in great detail are which tests to use in what situation, the number of different tests needed, the test design (i.e., number and spacing of toxicant concentrations), as well as available statistical analysis techniques, test limitations, and data interpretation, although a thorough understanding of each of these variables is paramount to correctly using the techniques herein. Many factors affect the use of bioassays as site assessment tools. The procedures outlined in this document are merely that, procedures, the results of which may be interpreted in a myriad of different ways. The application of these procedures to specific situations, and the manner in which the data is interpreted and presented, are variables not accounted for in this document. Proper use of the data derived from using these procedures is the sole responsibility of the testing organization. Site assessment conclusions should not be attempted without the consultation of a professional environmental toxicologist using the data obtained with the described procedures. This Standard Operating Procedure addresses a specific bioassay procedure and must be used in conjunction with other appropriate documents which address Health and Safety and Quality Assurance/Quality Control (QA/QC) Protocols. This test is designed to be a laboratory assessment of the acute toxicity of soils from potentially hazardous sites using a representative soil dwelling organism. Earthworms are an appropriate tool for this assessment because they ingest soil compounds, occupy the initial level of many terrestrial food webs, are important in recycling nutrients, have wide natural dispersal, and have a close relationship with other biomasses, e.g., litter, microorganisms, roots, and other soil organisms (Bouche, 1988; Marquenie et al., 1987). Soils are screened and mixed to achieve homogeneity and then blended with an artificial soil (AS) matrix to achieve a dilution series. Although mixing, handling, and hydrating soils can alter physical, chemical, and biological conditions that affect toxicity, these alterations are considered necessary and relatively minor. Sediments have been tested with this bioassay, but the bioassay is probably not suitable for sediments. It is recommended that sediment dwelling organisms be used to assess the toxicity of sediments. Wetland soils that experience seasonal dry periods and that could be expected to support earthworm populations during the dry phases could be evaluated legitimately with this test. Modifications of this laboratory test to an on-site version are in the late stages of validation.

II. INTERFERENCES/LIMITATIONS Toxic substances may be introduced by contaminants in water, glassware, sample hardware, artificial soil, and testing equipment. Improper hazardous waste sampling and handling may adversely affect test results. Pathogenic organisms in test materials may affect test organism survival and also confound test results. Several topics pertaining to the ESAP bioassay procedures are still under discussion, and are briefly discussed below. Due to the fact that the final decision on how to best deal with each of these factors may influence the procedures outlined in this document, portions of the described procedures on which these factors have an impact are tentative. In addition to the specific issues of concern discussed below, many other factors affect the use of bioassays as site assessment tools, and the procedures outlined in this document are merely that, procedures, the results of which may be interpreted a myriad of different ways. The application of these procedures to specific situations, and the manner in which the resultant data is interpreted and presented, are variables not accounted for herein. Proper use of data derived from using these procedures is the sole responsibility of the testing organization, and resultant conclusions should not be attempted without the consultation of a professional environmental toxicologist.

A. PH/HARDNESS ADJUSTMENT Adjusting the pH and/or hardness of soil, surface or ground water, or soil eluates might alter the toxicity of the site sample to a test organism. The toxicants may become more or less available to the organism due to the form or association those toxicants are in at different pH levels. The question of whether the pH and hardness are environmental parameters (like temperature or light intensity) and should be normalized, or whether they are toxic constituents of the sample, is as yet unanswered. Arguments can be made for either school of thought. Historically, the philosophy of ESAP has been that an assessment on a pH and/or hardness adjusted sample is better than no assessment at all. Without adjustment, no characterization as to the cause of the toxicity can be made; it could be a pH toxicity, or it could be something else. With adjustment, the end user has another piece of data: the sample is toxic due to pH, and 8 the sample is or is not toxic due to something else. This seems reasonable, given that all involved parties are completely cognizant that the adjustment has had an impact on sample integrity. Certainly, pH- and hardness-tolerant organisms could be used, but the increased complexity of adding tests to the battery that are redundant except for their ability to negate the necessity of these adjustments presents added costs that must be evaluated against the data quality objectives (DQOs) and the resources available. Procedures for pH adjustment of eluates and other aqueous samples are described in the SOPs. For soils, the Seed Germination SOP describes an adjustment procedure; in the Earthworm SOP, adjustment is cautioned against. The discrepancy is due to the unresolved nature of this issue. Since it is a virtually impossible procedure, adjusting sediment or soil pH may be avoided due to difficulty and cost, even if it is considered valid. B. DIRECT VERSUS INDIRECT TESTING Direct tests are those conducted on the sample (soil, sediment, or water) without derivation. Indirect tests are those performed on a derivative of the sample (e.g., eluate, elutriate). Aquatic, microbial, and root elongation tests performed on groundwater or surface water samples are considered direct tests (see Figure 2). Tests performed on eluates of soils or sediments are considered indirect. Earthworm and seed germination tests performed on soils are direct, on eluates they are indirect. In many instances, direct tests are preferred over indirect tests because the exposure to the test organism more closely simulates environmental conditions. Determining which bioassays (or mix of bioassays) provide the best estimate of site effects, regardless of the matrix or organism, is a prerequisite to a scientifically defensible stance on what is the best assessment method. The most comprehensive assessment of a site as a whole may be with a battery of tests of as wide-ranging exposure parameters as is feasible: soil contact, soil ingestion, interstitial water contact, uptake, ingestion, etc. In accordance with DQOs, tests are selected to provide relevant information from which management decisions can be made. It is important to know from the start what assessment questions are being posed in order to select the proper tests to perform. There may be little linkage between soil toxicity and the performance of algal or daphnid tests, whereas earthworm or seed germination tests may be invaluable, depending on the type of toxicant and the soil constituents. Non-water soluble constituents bound or tightly adhered to soil particles will not be contained in an eluate, whereas unbound water soluble constituents would. If the principal question relates to surface water or groundwater runoff into streams, lakes or wetlands, or groundwater flow off site (e.g., off-site well water contamination), it may be most appropriate to test derivatives of soils.

Because the definition of the limitations of most toxicity tests is poorly developed, there will be an interim period where tests may not match the questions as perfectly as one might hope. For this reason, we present the operating procedures for direct and indirect tests with few caveats about their application. As the field progresses, limitations will be defined more precisely and this issue will evolve into a more scientifically defensible stance.

III. SAMPLE PRESERVATION, CONTAINERS, HANDLING, AND STORAGE Proper collection, packaging, handling, and shipping of hazardous site samples is critical. Proper packaging and shipping ensures sample integrity, safety in handling, an adequate amount for sample processing and a data base for future sampling requirements.

A. SAMPLE PACKAGING MATERIALS. 1. 80 Quart Ice Chest with latch closure. Coleman Model # 5256, or equivalent. The Coleman Company, Wichita, Kansas­ 1-800-835-3278.

2. 5-gal steel paint cans with 0-ring fitted crimp lids. Freund Can Company #1260-4450 or equivalent. 1-800-621-2808.

3. Plasti cBags, 18 x 24", 4 mil polyethylene. Consolidated Plastics Company, Part # 90452HA or equivalent. 1-800-321-1980.

4. Nalgene HDPE or HDPP container, leak-proof screw cap. Consolidated Plastics Company, Part # 37087AJ (30 ml), 37089AJ (60 ml), 37091AJ (125 ml), 37093AJ (250 ml), 37095AJ (500 ml), 37096AJ (1000 ml) series; 38087AJ (1 L) , 38089AJ (2 L) , 38091AJ (4 L) series; 35713AJ (6 L) , 35715AJ (10 L) series; or equivalent. 1-800-321-1980. 5. High Density Polyethylene (HDPE) Plastic Pails with covers. Consolidated Plastics Company, Part # 33400AJ (1.0 Gal), 33404AJ (3.5 Gal) or equivalent. 1-800-321-1980. 6. ORM-E Labels. 10 Labelmaster, Chicago, IL 60646. (312) 478-0900. 7. Re-useable cold-packs, "Blue Ice" or equivalent. 8. Black PVC (electrician's) tape; Duct Tape. B. AQUEOUS HAZARDOUS SUBSTANCES/AQUEOUS SAMPLES. Throughout the sampling operations, great care should be exercised to keep the shipping containers and packaging materials as uncontaminated with sample residue as possible.

If any correlative analytical chemistry is required, consult the EPA document "Methods For Chemical Analysis Of Water and Wastes", since possible sample splitting and analyte specific sample preservation techniques may be required.

Chain-of-custody procedures must always be followed when handling hazardous wastes from Superfund sites when the data may be used for enforcement purposes. Refer to the EPA document SW 846 "Test Methods For Evaluating Solid Waste Physical/Chemical Methods" for specific procedures.

1. Using Figure 3, determine the volume of sample required to perform the number of bioassays requested for each sample. 2. Choose the sample container size (#4, Sample Packaging Materials, above) that will contain, with as little excess as possible, the volume of sample required. 3. All sample containers should be rinsed with sample water before being filled with sample. 4. Fill the container to the brim with the sample, screw the cap on TIGHT, seal the cap in place with black PVC (electrician's) tape, stretching the tape around the cap junction to form a complete seal.

5. Place the container in a 4 mil plastic bag (#3, Sample Packaging Materials, above), twist, and seal with PVC tape. Place that plastic bag, along with a plastic identification tag made out with indelible ink, into a second 4 mil bag, then seal the second bag with PVC tape. The identification tag should have the sampling site, date, and any applicable tracking codes legibly written. 6. Place the sealed sample(s) upright into an ice chest (#1, Sample Packaging Materials, above), and fit frozen cold packs amongst the sample(s). An equal volume of cold packs to sample volume is optimum to keep the samples cool. Fill any empty space in the ice chest with an absorbent (vermiculite, floor-dry, etc.). The absorbent acts both as insulation and to stabilize the sample

11 containers. Close the lid and latch it. Duct tape should then be wrapped around the ice chest to secure the lid. For most sample volumes, more than one sample may be placed in the same ice chest. Careful identification is critical in this case. As an alternative, samples may be placed in steel paint cans without cold packs (although still packed with absorbent), but it is strongly recommended that samples are sent cooled to preserve sample integrity. 7. Copies of Sample Data Sheets (Attachment #1) for all samples in an ice chest should be placed in a waterproof document protector or Ziploc bag and enclosed in the ice chest with the samples. This is an addition to the originals sent under separate cover (see "Shipping", below). 8. Label the ice chest properly (see "Labelling Requirements", below), identifying the sampler, date, and any sample tracking codes required. All packages that contain liquids must be marked or labeled "THIS END UP" and have arrows indicating proper orientation. NOTE: Watery sediment samples should be packaged as liquids.

C. SOLID HAZARDOUS SUBSTANCES Throughout the sampling operations, great care should be exercised to keep the shipping containers and packaging materials as uncontaminated with sample residue as possible. If any correlative chemistry is required, consult the EPA document SW 846 "Test Methods For Evaluating Solid Waste Physical/Chemical Methods" since possible sample splitting and analyte specific sample preservation techniques may be required. Chain-of-custody procedures must always be followed when handling hazardous wastes. Refer to the EPA document SW 846 "Test Methods For Evaluating Solid Waste Physical/Chemical Methods" for specific procedures.

1. Using Figure 3, determine the amount of sample required to perform the number of bioassays requested for each sample. Notice this is dry weighty more sample will be needed to account for moisture in the sample. 2. Use the size pail (#5, Sample Packaging Materials, above) that will contain, with as little excess as possible, the volume of sample required. If more than one pail is necessary for the sample, great care must be taken to insure the sample aliquots are identical. 12 3. Line the plastic pail with two 4 mil plastic bags (#3, Sample Packaging Materials, above). Fill the inner bag with sample material to a level about three inches from the top of the pail. Seal the inner bag with PVC tape. Place the proper identification on a plastic tag using indelible ink inside the second bag, on top of the inner bag, then seal the second bag with PVC tape. 4. Secure the lid on the pail, insert the pail into an ice chest and fit frozen cold packs (#1, Sample Packaging Materials, above), around the pail(s). An equal volume of cold packs to sample volume is optimum to keep the samples cool. Fill any empty space in the ice chest with an absorbent (vermiculite, floor-dry, etc.), close the lid and latch it. The absorbent acts both as insulation and to stabilize the sample containers. Duct tape should then be wrapped around the ice chest to secure the lid. If sample volume is small, more than one sample may be placed in the same ice chest. Two 3.5 gal pails fit nicely inside the ice chest specified above. Careful identification is critical in this case. As an alternative, pails may be placed in steel paint cans without cold packs (although still packed with absorbent), but it is strongly recommended that samples are sent cooled to preserve sample integrity. 5. Copies of Sample Data Sheets (Attachment #1) for all samples in an ice chest should be placed in a waterproof document protector or Ziploc bag and enclosed in the ice chest with the samples. This is in addition to the originals sent under separate cover (see "Shipping", below). 6. Label the ice chest properly (see "Labelling Requirements", below), identifying the sampler, date, and any sample tracking codes required.

D. LABELING REQUIREMENTS All containers must be identified according to the labeling requirements discussed below. A sample data sheet. Attachment #1, must be filled out for each sample with as much detail as possible. U.S. Department of Transportation (DOT) regulations require that environmental samples collected from hazardous materials disposal sites are identified as Miscellaneous (Class 9), Environmentally Hazardous Substance, solid (or liquid), N.O.S., UN3077. Identification should take the form of pre-printed "misc." labels (Attachment #2), with the other identification marked directly on the shipping container with paint or an indelible marker. This marking and label must be applied to two opposite sides of the shipping container of all collected environmental samples. The 13 designation identifies the sample as being potentially hazardous, flammable, corrosive, poisonous, etc., but containing less than a reportable quantity of the sample. All such marks or labels should be clearly identifiable, (light on dark or vice-versa) and permanently affixed. Labels should be filled out with permanent ink.

If sample contents are known, or if reportable quantities of various substances (poison, corrosive, etc.) are contained or anticipated in the sample, then labeling must comply with DOT CFR­ 49 specifications. These specifications are found in section 172.101 of the DOT Hazardous Materials Shipping and Handling Regulations. These regulations can be found at the office of any carrier authorized to haul hazardous materials.

Labels should be filled out as in Attachment #2. The terminology "Hazardous Substance" should be employed for environmental samples with potential or known toxicity. "N.O.S." means "Not Otherwise Specified". The label must state whether the sample is in a liquid or solid matrix. The designation "UN3077" should be written along with "Environmentally Hazardous Substance" and "N.O.S." directly on the shipping container.

E. SAMPLE HANDLING AND PRESERVATION The time elapsed between collection of a sample and the initiation of a toxicity test should be kept at an absolute minimum. For aqueous samples, this elapsed time should not exceed 72 hours. All samples should be shipped by an overnight delivery carrier. All samples should be shipped cooled. Planning and coordination between the sampling team and the testing laboratory are absolutely critical to minimize holding time. Numbers of samples and sampling rate will vary according to the bioassays performed and the testing lab capacity, and a sampling and shipping schedule should be agreed upon before sampling commences.

F. SHIPPING Samples should be shipped by overnight carrier (Federal Express or equivalent). Sample Data sheets (Attachment #1) should be mailed under separate cover concurrent with sample shipping. Contact a Federal Express shipping office for information on how to complete the necessary Federal Express forms for the specific material being shipped.

14 Figure 3

QUANTITIES OF AQUEOUS HAZARDOUS SUBSTANCE, SOLID HAZARDOUS SUBSTANCE, AND/OR ELUATE REQUIRED TO PERFORM TOXICITY TESTS AND ROUTINE CHEMICAL ANALYSES

Aqueous or Solid Hazardous Substance

Test Aqueous Substance Sample Required or Eluate Required (g, dry weight) (mL)

Daphnia 600 150

Algae 600 150

Root Elongation 240 60 (per species)

Earthworm N/A 3630

Seed Germination N/A 1200 (per species)

Microbial Tests 100 25

Chemical Analyses 1000 250

Note: The above figures are per test, a test being a three replicate 100% screen and a three replicate, six concentration definitive test.

15 G. TOXIC MATERIALS STORAGE Upon receipt, samples will be moved by a designated staff member to a hazardous materials storage environmental chamber kept at 4°C within one hour of notification of receipt by shipping and receiving. For custody purposes, that chamber should remain locked.

Storage of potentially toxic samples in the laboratory should be either in a vented refrigerator or in a hood. If site samples are outside a hood in the lab for any reason, they must be properly sealed and packaged.

IV. SAMPLE PREPARATION A. INTRODUCTION This section covers all those aspects of sample handling from receipt of samples to the point where samples are suitable for testing. Sample handling specific to particular bioassays is covered in the bioassay procedures themselves. Preparation of hazardous waste samples must be extensively covered in appropriate Health and Safety Protocols, as this activity involves the highest hazard when working with potentially toxic samples.

B. SAMPLE LOG-IN Upon receipt, samples will be moved by a designated staff member to a hazardous materials storage environmental chamber kept at 4^0 within one hour of notification of receipt by shipping and receiving. For custody purposes, that chamber should remain locked.

An eight-character (easily used as a file name for most computer software) alphanumeric sample tracking number can be assigned to each sample and entered into a log book to keep track of sample receipt and code number assignment, for example, DCN36000. The first two digits depict the site, compound, or test species (in this example, DC represents Drake Chemical). The next digit can be one of four letters: P, H, B, or N (P = pH adjustment, H = hardness adjustment, B = both, and N = neither). The next two digits can represent the week of the current year, (i.e., 01 to 52), and the last three digits (001, 002, 003, 004, etc.) can stand for the consecutive sample tracking number (e.g., DCN36000, DCN36001, DCN36002, etc.).

The sample numbers can be assigned in consecutive order as follows: Soil 000-099 16 Sediment 100-199 Surface Waters 200-299 Ground Waters 300-399 Extracts (TCLP, etc.) 400-499 Standards (Cu, Zn, etc.) 500-599 Miscellaneous 600-699 QA/QC 700-799 Media (AS, etc.) 800-899 Fits no category above 900-999 The first two digits on the Test Code and the sample tracking numbers should coincide for each run. The sample tracking originate when the samples are received on-site and entered into the logbook. The logbook may also be used as a cross reference. The sample tracking numbers are recorded for future referencing and tracking. With 676 possible origin codes, it is highly unlikely that two samples in two different years having the same week and sample number codes would, in addition, possess the same character origin code.

C. SOIL AND SEDIMENT PREPARATION 1. Materials and Equipment Rubbermaid cart; air tanks; personal protective equipment; tyvek clothing; steel-toed rubber boots; respirators; 1/4-inch mesh stainless steel screen; metal paint can or Hobart mixing bowl; plastic bags; sink; spatulas; rubber scrapers; Hobart Mixer Agitator; pastry knife; crystallizing dish; balance; moisture/elution notebook; vented oven; elution notebook; twist- tie closure.

2. Procedure The waste site soil and/or sediment samples, still in their original shipping containers, are transported on a gray Rubbermaid cart from the storage area to the sample preparation area. Initial site soil preparation is most easily and efficient done in a containment room which can be hosed down at the end of each work day, which has a large stainless steel sink below hip level which can easily accommodate two persons working with bulky personal protective equipment and sample buckets and shipping containers, which has a high velocity ventilation system, and which is equipped with full-face, positive pressure respirator support equipment. In the sample preparation area at least three size "K" breathable air tanks should be on-line for use by a two-person crew intending to work on bottled air for approximately 4 hours. If only elution is to be done, each size "K" air tank is turned on and checked to see that there is at least 1000 psi left in one tank. If screening is to be done, all three empty tanks should be changed. If the tanks need to be changed, follow appropriate procedures. Two

17 technicians must work together whenever bottled air is used. The door should be locked when the room is vacated. Personal protective equipment is worn according to the appropriate Health and Safety Protocol. Steel-toed rubber boots should be used when handling air tanks. The samples are screened through a 1/4-inch mesh stainless steel screen placed on a plastic-bag-lined (4 ml) metal paint can or Hobart mixing bowl in Room 8-21. Oversize material such as rocks, sticks, and debris from the samples are placed into plastic bags, labeled with the sample tracking number, and placed in storage in the original container until determination of toxicity by bioassay has been made. At the completion of bioassays, the debris is discarded according to toxicity of the sample. No significant amount (>g or ml) of uncharacterized material, liquid or solid, should be disposed of in the dump, down the sink, or into garbage dumpsters. However, as much as 100 g per sample are washed unavoidably down the sink when the equipment is cleaned after each sample is processed. Great care should be exercised to reclaim as much soil as possible from the equipment using spatulas and rubber scrapers before washing. Small amounts (gram or milliliter quantities) spilled during sample preparation may be placed in the garbage can to be placed into a dumpster.

After screening, the sample is mixed in a Hobart Mixer Agitator for two 15-minute periods at speed 1 using the pastry knife. Scrape the sides of the mixing bowl with a large spatula at the end of each 15-minute mixing period. A 125 g subsample is taken from the homogenized sample and placed into a weighed crystallizing dish using a balance. The initial weight of the dish + 125 g wet soil is recorded in the moisture/elution notebook. Then it is dried at 100 ± 5°C for at least 16 hours in a vented oven. The final dry weight of the dish + soil is recorded in the elution notebook (Attachment # 3). When returning to weigh the dried sample, bottled air should he used, but the full suit is not required unless another technician is working with open raw sample in the room. The moisture content is determined according to the following formula: (q initial weight dish + wet soil) - (a dry weight of dish + soil) = m 125 g = initial wet weight of soil only 250-225 = 0.2 125 Then, m = fraction moisture (0.2 in the above example). The value m is used to determine the amount of wet soil and water for elution. The dried subsample is placed in a plastic bag, labeled with 18 sample ID and chemical number, taped closed, and stored until disposal of the entire sample residuals. The original sample is transferred to its original plastic pail lined with a 4-ml plastic bag. The bag is closed with a twist-tie closure. Any visible sample residue on the exterior of the original plastic pail will be cleaned off in the containment room. The plastic pail is placed in the original shipping container. The sample, contained in original shipping containers, is then transported back to refrigerated storage.

All equipment, utensils, carts, etc. which have come into contact with the sample are cleaned by personnel prior to removal from the containment room. The floors are wet-broomed at the end of each work day. V. GLASSWARE PREPARATION The purpose of the glassware preparation procedure is to remove all solid material, organic contaminants, and metals so that nothing remains on the glassware to chemically or biologically contaminate the next bioassay.

A. DISCARD HAZARDOUS MATERIAL After a test result is calculated, check the EC50s of that sample on all the other tests run. If the EC50 for any test is less than 20%, treat that whole sample and its dilutions as hazardous liquid, and dispose of the liquid into a 2-liter rigid- sided, polyethylene-propylene container (refer to the Waste Disposal section). Remember to put the name as well as the tracking number of the sample, the sample volume in liters, and your full last name and first initial on the container. Do not use abbreviations.

B. PRE-SCRUB

All glassware should be scrubbed with a brush to remove solid material like algal cells, daphnia skins, or soil. Glassware may be easier to clean if they are allowed to first soak in the sink with hot water and detergent. Index cards may be placed on a group of dishes indicating the next step to be performed in the washing process. C. MACHINE WASH

Check dish washing machine for debris on the screen and remove as per equipment requirements. The usual machine settings are: MFR on, auto, 1st rinse on, drain off, 3 minutes extended wash. However, the extended wash cycle can be extended to more than 3 minutes and the MFR (mineral-free rinse) is omitted for non-pyrex glassware. Phosphorous-free detergent may be used.

19 Put the glassware on the spindles of the rack, and put the rack into the dishwasher. Put the shelf over the dishes so they will not fly off the spindles. Shut the door and turn on the machine by holding down the start button for about 10 seconds. The machine will go through a pre-rinse cycle and then stop. Open the door, throw in about a spoonful of VWR non-phosphate detergent powder kept next to the dishwasher, close the door, and press start again. Now the machine will go through its cycle. Put the "acid wash" index card on the front of the dishwasher. D. ACID WASH AND RO WATER DIP Set up two plastic (34 x 28 x 14 cm) tubs, one for 10% v/v HCl, and one for RO water. Normal makeup procedure for the 10% HCl is as follows: take a 2-liter jug marked 10% HCl and put some RO water in it. Using a graduated cylinder, measure out 200 ml of concentrated 12 N HCl under the hood and pour it into the jug. Make up to the 2-liter mark with RO water. Dip each piece of glassware successively into the HCl solution and the RO water tubs. This does not have to be done under a hood, however the HCl mist may be somewhat irritating to the respiratory tract and some workers prefer to wear a dust and mist mask. Neutralize the HCl with sodium carbonate before discarding down the drain. E. ACETONE RINSE Dishes that are not baked, such as buret stopcocks, other plastic ware, filtration apparatus, as well as glassware contaminated by sites with oil and grease must be acetone-washed to remove organics. Both acetone and pesticide baking serve the same function to remove organics (generally, only one is necessary). The acetone must be used under the hood. Set up four tubs in the hood; although reagents (except acetone) do not need to be under the hood, it is more convenient to have them all side by side. The tubs should contain left to right: acetone, tap water, 10% HCl, and RO water. Dip each piece of glassware 'successively into the acetone, tap water, HCl, and RO water baths. When finished for the day, place the acetone in the acetone collection container, and neutralize the HCl with sodium carbonate before discarding down the drain.

F. RO RINSE Thoroughly rinse everything in RO water. RO water can be easily transported around the lab in a 5-galIon carboy. Using a rubber stopper and tubing mechanism, the carboy can be pressurized by attaching to a laboratory air valve for ease of rinsing glassware. Tape the rubber stopper down using strapping tape. Hook up tubing to the house air inlet. Be sure the pressure relief valve is open. Turn on the air so that the regulator reads about 20 6-8 psi. Put the clean dishes in a round metal basket for baking. Do not bake plastic. For acetone-rinsed glassware that will not be baked, it is more convenient to actively RO-rinse and dry in the lab. G. BAKE Dishes may be baked overnight (16 hour cycle) in an oven that reaches 350° C (Blue-M or equivalent). If the oven is still on in the morning, turn chamber power OFF before you open the door (DO NOT BAKE PLASTIC). H. EXCEPTIONS 1. Flint glass jars must be machine rinsed with MFR rinse OFF, and are not baked, so they must be acetone-rinsed as above. They are finally dried in the drying oven in Room 200. 2. There are special racks for Petri dishes that fit on top of the flat dishwasher rack. 3. When biological contamination may be a concern, glassware can be autoclaved after baking. 4. Burettes should be cleaned as follows: Take apart and scrub well with detergent. Acetone and 10% HCl-rinse the plastic parts; 10% HCl-rinse the glass part. Rinse well with RO water. Pesticide-bake everything but the plastic stopcocks. 5. Contaminate items like filtration equipment for sample processing, that do not go into the dishwasher, are washed by hand in a ventilated sink or fume hood. The same steps are repeated, except for the dishwasher and bake steps. Squirt-bottles are used for acetone, HCl, and RO water.

I. NEW GLASSWARE Begin with the 10% HCl rinse and continue through the remainder of the cycle.

J. GLASSWARE STORAGE After the glassware is baked, openings are covered with foil before putting away in the cupboards. Glassware stored upside down in drawers is not covered with foil. Broken glass goes into the appropriate containers.

VI. BIOASSAY PREPARATION PROCEDURES

Each test can be divided into three parts: preparation, initiation, and termination of the test. Preparing the test refers to all pre-exposure activities necessary to perform each test. 21 Initiating the test refers to beginning or starting the actual exposure. Terminating the test refers to all post-exposure activities.

Preparing, initiating, and/or terminating a test may be performed by a different technician each time, which requires consistency and conformity so that the necessary information and equipment is available when needed. The "responsible technician" is the person who conducts the test. However, the responsible person who sets up the test will also need to initial the paper­ work.

A. FILLING OUT DATA SHEETS 1. Test Code A cover sheet (Attachment #4) is given a Test Code, for example UC093088A. The first two letters in every Test Code depict the site, compound, or test species (in this example, UC refers to United Chrome). The following six digits represent the date (e.g., 093088 is September 30, 1988. The Test Code is comprised of either eight or nine characters. The ninth character is represented by a letter (A, B, C, D, etc.) which indicates that more than one test was run with the same Test Code (e.g., UC093088A, UC093088B, UC093088C, etc.).

2. Sample Tracking Number See section on Sample Log-in and Storage, above. 3. Dilution Scheme The dilution scheme should be calculated for the test. A logarithmic series of at least five concentrations should be employed (e.g., 100-10-1-0.1-0.01%) for a range-finding test. For a definitive test, at least 5 concentrations in a geometric series related by a 0.5 multiplier should be used. A standard dilution scheme might be 100-50-25-12.5-6.25-3.125% of toxicant by dry weight. Another scheme might be 64, 32, 16, 8, 4, 2, 1, 0.5 ppm.

Test volume is 200 g dry weight, and the percent toxicant in decimal form is multiplied by 200 as is the percent diluent, for example, 0.25 x 200 = 50 g, for a 25% site soil concentration. If 25% toxicant is available, 75% diluent is needed (0.75 x 200 = 150 g). Thus, to make a 25% dilution, 50 g of site soil and 150 g of diluent (artificial soil) are needed on a dry weight basis. a. Dose vs. Dilution Tests

A dose test is a mixture of toxicant and media (200 g of artificial soil an earthworm assay) in which less than 1 percent 22 of a toxicant is added, and a constant amount of media is used for all concentrations. A dilution test is a mixture of toxicant and media (diluent) in which more than 1 percent of a toxicant is added to the test vessel for some or all concentrations, and an appropriate reduced percentage of diluent is used for those concentrations. The difference is the media: it is either "dosed" or diluted. Dose tests are normally done with toxicants that can be manipulated in concentration (e.g., chemical compounds) to maintain the constant amount of media (and less than 1 ml toxicant spike). Reference toxicants (or standards) are usually tested in this manner. Units for dose tests are usually in mg/liter of a substance; dilution tests are usually in percent of total test volume. Almost all environmental samples are completed as dilution tests. The only possible exception might be an eluate so toxic that the EC/LC50 was approximately 0.5%, so that the highest concentration tested was no more than 1% (e.g. 0.5 ml in 50). This, by definition, could be termed a dose test, although normally that label is reserved for toxicants in which the active ingredient is known and can be expressed as mg/liter. 4. Number of Test Containers The number of containers to use is figured by adding the number of concentrations used for a test, multiplying by 3 (because each concentration is tested in triplicate), adding 3 more containers for the negative control, and three for the positive control. This calculation should be written on the back of the data sheet if it is not already there. For a standard 11 concen­ tration test, the number of beakers would be (11 x 3) + 3 -i- 3 = 39 containers total. Once all the blanks have been filled in, the dilution scheme has been determined, and the units filled in on the front of the sheet, the paperwork is nearly complete. At this point, it is advised that another technician check the dilution scheme. B. MARKING THE CONTAINERS After the dilution scheme has been figured out, the test containers (beakers, flasks, petri dishes, jars, etc.) are marked. When more than 1 sample (test) is being initiated in a day, the containers for each test are marked with a different color. Containers for each test are numbered (e.g., l through 33) and the first of each concentration marked with the concentration and the chemistry number in addition to the container number (e.g., container #4 would be marked 80%, UCN36000; #5 and #6 would not). Container number 1 should have the Test Code (e.g., UC093088B) and the test water description (e.g.. United Chrome Bench Treatability 23 Cell F) in addition to the container number (1), the chem number (e.g., UCN36000) and the concentration (e.g., 100%). The negative and positive controls are marked as such, dated, and marked 1 through 3.

VII.EARTHWORM TEST PROCEDURE

A. METHOD SUMMARY The procedures in this document are a modification of the methods described by Edwards (1984) and Callahan et al. (1985). It is a detailed description for culturing earthworms, preparing a test, setting up a test, putting on a test, and performing data analysis. Similar general procedures were published in Greene et al. (1989). Working with hazardous soils suggests the potential for exposure of workers and this issue should be addressed before testing, but this document does not include a Health and Safety Protocol. Some level of familiarity with routine laboratory procedures and equipment is required for the successful completion of this test.

The earthworms are exposed (in triplicate) in a test chamber at 22 ± 2°C to a negative control of AS and to various concentrations of site soil (SS) mixed with AS for a period of 14 days. Mortality and sublethal endpoints (behavioral and pathological observations) are checked at 14 days and optionally at 7 days and median lethal concentration (LC50) values are calcualted. Each replicates is hydrated with reagent water to create a moist environment for the worms. The replicates consist of 200 g (dry weight) of test soil (TS) to which 10 worms are added. Test soil refers to a hydrated mixture of AS and SS or the hydrated negative control.

Some uncertainty exists as to the taxonomic status of the test organism being utilized. Eisenia foetida (Savigny) is a well known species that is used extensively in toxicity testing. Bouche (1972) reported this species as consisting of two subspecies, Eisenia foetida foetida and Eisenia foetida andrei. Oien and Stenersen (1984) and Jaenike (1982) did electrophoretic work that led them to consider these as two separate species. Fender (1985) also reported the existence of two separate species, but indicated that literature references have not always made a distinction between the two forms. However, differences between their sensitivities to toxicological testing may not be significant (Edwards, 1984). Until this issue is resolved, it was decided to accept Eisenia foetida as the scientific name of the organism and to utilize the subspecies Eisenia foetida andrei.

B. EQUIPMENT/APPARATUS The equipment and apparatus include: 1) earthworm culture chamber, 2) earthworm test chamber, 3) test containers—one-pint, 24 glass canning jars with lids and rings, 4) reagent water system or source, 5) analytical balance, 6) benchtop balance—heavy duty, toploading, 1 g readability, 9,000 g capacity, 7) benchtop balance­ -toploading, 0.01 g readability, 400-600 g capacity, 8) temperature recording device—portable, 7-day, circular chart, e.g., Dickson and Tempscribe, 9) cement mixer, 10) sieve—stainless steel, 1/4 inch mesh, 18 in. diameter, 11) laboratory (food) mixer—stainless steel, 5 gal. capacity bowl, e.g., Hobart, 12) drying oven—up to 110°''' 13) pH meter, 14) earthworm sorting/counting tray, 15) vented laboratory hood, 16) 3 2 gal. plastic garbage cans, 17) large flat tray, 18) laboratory cart, 19) 5 gal. plastic pails, 20) plastic culture trays, 21) computer, 22) mechanical pipettes, 23) magnetic stir plate with magnetic stir bars, 24) shelf system to hold worm culture trays, 25) black plastic, 26) plywood boards—30 x 38 cm, 27) routine laboratory glassware—funnels, Erlenmeyer flasks, graduated cylinders, volumetric flasks, and beakers, and 28) routine laboratory materials—scratch pads, spatulas, scoops, filter paper, disposable petri plates, parafilm, marking pens, disposable rubber gloves, notebooks, plastic bags, and Ziplock bags-

C. REAGENTS The reagents include: 1) silica sand—grade 70, 97.1% particle size of 0.053-0.3 mm, 2) Canadian sphagnum peat moss (Sphagnum)—that portion passing through a 2.36 mm sieve, 3) kaolin clay—97% kaolinite with particle size under 40 microns, 4) calcium carbonate—99% purity, 5) dry alfalfa (Medicago sativa) pellets— minimum 15% protein, maximum 30% fiber, maximum 12% ash, minimum 1% fat, and maximum 12% moisture, 6) reagent water—deionized, distilled or better, 7) dish soap, and 8) pH buffers—4, 7, and 10.

VIII.PROCEDURE A. CONDITIONS

1- Temperature : 2 2 ± 2°C 2. Light Quality: incandescent or fluorescent 3. Lighting Intensity: minimum 400 lux 4. Photoperiod: continuous illumination 5. Test containers: 1-pint, glass canning jars with rings and lids; 1-2 mm vent holes in lids 6. Weight of TS: 200 g (dry weight) per container 7. Hydration of SS: 75% of WHC 8. Hydration of AS: 45% of dry weight of components 9. AS (% dry weight): 10% Canadian sphagnum (Sphagnum) peat moss, 20% kaolin clay, 70% silica sand; pH adjusted with calcium carbonate (99% purity) 10. Test organism: Eisenia foetida andrei 11. Life stage of test organism: clitellate adults 12. Organisms per container: 10 25 13. Replicates: 3 14. Test duration: 14 days 15. Effect measured: mortality and sublethal effects (behavioral and pathological) 16. Feeding during test: none 17. pH measurement: initial and at 14 days or 100% mortality 18. Temperature measurement: continuous B. TIME SCHEDULE The following illustration of a possible time schedule follows the examples utilized in this manual. There can be some variation in the schedule. The various procedures scheduled prior to Wednesday can be performed at any time. The procedures scheduled for Wednesday and Thursday can be performed in one day, if time permits.

Prior to Monday Prepare batch of AS. Measure pH of AS. Monday (3 days before test is put on) Screen and mix SS. Begin moisture content measurement of SS. Begin WHC measurement of SS. Measure pH of SS. Tuesday (2 days before test is put on) Conclude moisture content measurement of SS. Conclude WHC measurement of SS. Prepare paperwork and do calculations. Label test jars. Wednesday (day before test is put on) Mix AS and SS. Hydrate TS. Thursday (day test is put on) Obtain initial pH values. Place TS into test containers. Introduce worms to TS. Place test containers into test chamber. Thursday (7 days of exposure) (optional) Do 7-day count of worms. Make 7-day observations. Take final pH values if 100% mortality. Thursday (14 days of exposure) Do 14-day count of worms. Make 14-day observations. Take final pH values. Calculate LC50.

C. PRELIMINARY PROCEDURES The following procedures are undertaken before the worm test 26 is set up: preparation of AS; screening and mixing of the SS; moisture content measurement of SS; pH measurement of AS and SS; and WHC measurement of SS. These procedures are outlined below. 1. Preparation of Artificial Soil The AS used in this test was developed with the advice of pedologists to overcome the variability between different soil types and has an adsorptive capacity resembling typical loam soils (Edwards, 1984). The following constituents are mixed together on a dry weight basis: 10% Canadian sphagnum (Sphagnum) peat moss (that portion passing through a 2.36 mm screen); 20% kaolin clay (97% kaolinite with particle size under 40 microns); and 70% silica sand (grade 70, 97.1% particle size of 0.053-0.3 mm) (Edwards, 1984). After these materials are mixed together, an amount of calcium carbonate (99% purity) equal to about 0.4% of their total weight is added to the mixture to adjust the pH to 7.0 ± 0.5. The exact amount of calcium carbonate used will depend upon the pH of the peat moss utilized. For example, 50 kg of AS would have 200 g of calcium carbonate added to it. Kaolin clay can be purchased from EPK, a division of the Feldspar Corporation. Granusil brand of silica sand, produced by Unimin Corporation, can be utilized. The materials and source of the materials need to be standardized as much as possible.

A large batch of AS, e.g., 50 kg, can be prepared for use in many tests or a smaller batch can be prepared for each specific test. The following directions are for the preparation of a 50 kg batch of AS needing 0.4% calcium carbonate for pH adjustment. First, a quantity of peat moss is screened through a 2.36 mm (Tyler equivalent 8 mesh) screen to yield 5 kg of screened peat. Over­ sized material is discarded. A fourth, or 1,250 g, of the screen­ ed peat is placed into each of four plastic bags. Next, 5,000 g of clay is weighed into each of two plastic bags and 8,750 g of sand into each of four plastic bags. Into each of two 400 ml beakers is weighed 100.0 g of calcium carbonate. All of the materials just weighed, along with a plastic garbage can, are moved to the mixer. The mixer should be clean and dry. Two bags of peat and one bag of clay are poured into the mixer and mixed for 5 minutes. Two bags of sand are added and mixed for another 5 minutes. The 100.0 g of calcium carbonate is added and mixed for another 5 minutes. This mixture is poured into the garbage can. The procedure is repeated with the remaining materials. When the second load is done, it is left in the mixer and the first load (now in the garbage can) is dumped back into the mixer and the combined batches are mixed for 10 minutes. This final mixture (50,000 g of AS plus 200 g of calcium carbonate) is poured back into the garbage can and moved to storage. If the proper amount of calcium carbonate to use is not known, it is best to start with less than 0.4% and check a subsample of the AS for pH after the first load has been mixed mixed. If needed, more calcium carbonate can be added, the materials can be mixed for another 5 minutes and 27 the pH can be rechecked. The amount of calcium carbonate can then adjusted for the second load of AS in the mixer. Each new batch of AS is given a number corresponding to the day it was mixed, e.g., batch # 062392, and its pH is measured. A record book containing pertinent data such as amount of AS prepared, pH, batch number and brand of peat and other constituents, etc. can be maintained. 2. Screening and Mixing of Site Soil All site soils are homogenized by being screened through a 1/4-inch stainless steel screen and then mixed for 1/2 hour in a stainless steel mixer, e.g., Hobart. See Sample Preparation section for details of screening and mixing. 3. Measurement of Soil pH Soil pH is measured using a modification of method 9045 of the EPA/OSW (1986). A Slurry of soil and reagent water is prepared at a ratio of 1 g soil:2.5 ml water (20 g of soil and 50 ml water in a 100 ml glass beaker is convenient). The slurry is mixed with a magnetic stir bar for 5 minutes and allowed to settle for a minimum of 30 minutes. A glass, combination pH electrode is inserted into the supernatant. The pH meter reading is allowed to stabilize (a maximum of 10 minutes) before the pH is recorded. Site soil samples are checked for pH after they are screened and mixed. Site soils are not normally pH adjusted. If the pH values of a SS are outside of the range 5-9 and an adjustment is to be made, one option would be to adjust the soil to the nearest value within that range. 4. Moisture Content of Site Soil Moisture content of SS is needed to determine the amounts of AS and SS to mix together on a dry weight/dry weight basis. A 100­ 125 g subsample of the screened and mixed SS is weighed, dried at 100 + 5°C for 24 hours or until a constant weight is determined and then re-weighed to gravimetrically determine the moisture content. Moisture content can be expressed as a percent or as a decimal moisture fraction (MF). The moisture fraction can be determined on a dry weight basis (MF^) or a wet weight basis (MF„). Values from either determination can be used in performing this bioassay, but differing sets of calculations are required depending upon the selection of a dry or wet weight basis. The calculations in this document are designed to utilize wet weight basis moisture content values. Details are discussed in General Laboratory Procedures; Sample Preparation.

28 5. Water Holding Capacity of Site Soil WHC values can be used in determining the hydration of a SS. The procedures are as follows. A circle of styrofoam (ca. 16 mm thickness) is cut to a diameter (e.g., 224 mm) so as to fit inside of and float in a bucket (e.g., 3 gal.) of water. In the center of the styrofoam, an 8 mm diameter, beveled hole is made. A circle of 185 mm diameter, coarse-porosity, qualitative, crepe filter paper (VWR grade 617) is folded into quarters and placed in a 100 mm (top inside diameter) glass funnel with a stem of approximately 96 mm. Using mechanical pipettes, 9 ml of reagent water are slowly added to the filter paper to uniformly wet the entire surface. The weight of the funnel and wet filter paper is measured and recorded (e.g., 163.9 g). After the soil has been screened and mixed, a subsample of approximately 50 g (wet weight) is removed and added to the funnel with wet filter paper and the weight of this whole "unit" is measured and recorded as the initial weight (e.g., 213.9 g). The styrofoam is placed in the bucket of water, and then the funnel with soil is placed in the center hole and allowed to float freely in an upright position. An appropriate amount of water needs to be in the bucket so that a lid can be placed on the bucket and so that stem of the funnel is about 100 mm off the bottom. The lid is placed on the bucket and the soil is allowed to hydrate by capillary action for 24 hours after which a final weight of the "unit" is obtained (e.g., 247.6 g). The initial weight is subtracted from the final weight to obtain the amount of water held by the soil (1 ml = 1 g for water). Utilizing the initial and final weight examples from immediately above, the amount of water held by the soil is 33.7 g (ml).

The amount of moisture (water) present in the original 50 g of wet soil before it was hydrated is calculated below. The example uses a wet weight moisture fraction (MF^) of 0.20. water present in soil = (wt. wet soil) (MF„) = (50 g) (0.20) = 10 g (ml) The amount of dry soil in the original 50 g of wet soil is calculated as follows: dry soil present in wet soil = (wet soil) - (water present) = (50 g) - (10 g) = 40 g WHC is equal to the total amount of water (amount of water originally present plus the amount added through capillary hydration) that the dry soil can hold as follows: WHC = water originally present -i- water added by hydration 29 amt. of dry soil = 10 ml + 33.7 ml = 43.7 ml = 1.093 ml/g 40 g 40 g Therefore, the WHC of this specific soil is 1.093 ml water/g dry soil and the moisture content on a wet weight basis is 20%. Both of these values are used for calculations that follow. A percentage of the WHC is used as a hydration standard for this document. Other methods of hydration could be utilized, e.g., hydration to a percent of the dry weight of the soil. What is important is that the earthworms have a moist environment, but that the soil is not hydrated so that standing water is present. D. SETUP At this point, all information needed for the bioassay has been collected: WHC for the SS, pH values for the SS and AS, and moisture content of the SS. Attachments #4 and #5 are examples of a cover sheet and a calculation sheet used with each test. The setup procedures are done 2 days before the test begins and consist of filling out the two sheets mentioned above and marking the test jars. 1. Test Containers and Labeling Standard one-pint, glass canning jars with lids and screw rings are used as test containers. The lids have a small (2 mm) hole drilled or punched in their center to allow air exchange. New lids and rings are used for each test. All test concentrations are made in triplicate. The side of each jar is labeled with the concentration and the replicate number, e.g., 40%, #1. Negative controls are labeled "neg. C". All of the jars needed for the test are organized, by concentration, on a Rubbermaid laboratory cart. The first replicate of each concentration, in addition to the above information, is labeled with the test code and chemistry number, e.g., 40%, #1, PH110787, PHN45002. Felt-tip permanent markers (not black) are used for labeling. 2. Cover Sheet Next, the cover sheet is filled out. The test code consists of a two-letter designation for the site or the chemical followed by the date the test is put on. Directions concerning these codes are given in the General Laboratory Procedures. The name of the person performing the test is placed after the word "inve­ stigator". The name of the site where the soil was collected is placed on the line for site soil. In addition, each site sample usually has a number or letter designation which is placed after its name, e.g., 40-22A. The time at which the test is started is placed on the cover sheet. The WHC value for the site soil, 30 expressed in terras of 100 g of dry soil, is placed on the cover sheet. The batch number of the AS being used in the test is noted. Hydration of the SS is expressed in terms of percent of WHC. The SS portion of the TS is hydrated at 75% of its WHC value. The pH values of the SS (after it is screened and mixed) and the AS are recorded. The AS portion of the TS is hydrated at 45% of the dry weight of its components (Hague and Ebing, 1983). Moisture content of the site soil is reported as percent or as a fraction. The test uses three replicates per concentration, 10 worms per replicate, and therefore 30 worms per concentration. Site soils are tested as percent of site soil (dry weight) per concentration. A standard test has a minimum of five concentrations (Edwards, 1984; van Gestel and van Dis, 1988) of SS and a negative control. A test utilizing 5 concentrations of SS, a high concentration of 80% and a dilution factor of 0.5, for example, would set up the following series of concentrations: 80%, 40%, 20%, 10%, and 5%. The concentrations are recorded in the proper column on the lower portion of the cover sheet. The rest of the cover sheet is filled out when the test is started and when the worms are counted at 7 (optionally) and 14 days.

3. Calculation Sheet The calculation sheet is completed using a number of simple formulas and the information on the cover sheet. Percent site soil is required in column 1 on the left. These are the test concentrations recorded on the cover sheet. A 40% concentration is a mixture of 40% SS and 60% AS mixed on a dry weight/dry weight basis. The weight of dry SS used in producing the concentration in column 1 is required in column 2. The 40% concentration from the calculation sheet is used as an example in working through the calculations. As noted previously, 200 g (dry weight) of the TS is added to each replicate. Therefore, 600 g (dry weight) of test soil is needed for each concentration. An additional 60 g (dry weight) are added to account for the loss of TS due to removal of a subsample for initial pH value, adhesion to container walls, etc. Each concentration is made up in a batch of 660 g (dry weight) and then divided into its three replicates to insure that the repli­ cates are as homogeneous as possible. The calculations for this column are done in the following manner (rounded to whole number): weight dry SS = (660 g) (test concentration fraction) = (660 g) (0.40) = 264 g Since a certain amount of water (1 ml of water = 1 g) is contained in the SS, the amount of wet SS needed to yield the proper amount of dry SS is entered in column three. These cal­ culations are rounded to a whole number in the following manner: weight wet SS = dry weight SS 1-moisture fraction = 264 g = 264 g = 330 g

31 1-0.20 0.80 The amount of hydration water needed for the site soil is entered in column four. As the cover sheet indicates, the value is 75% of the WHC and in this example equals 33.8 ml/100 g (roun­ ded to nearest tenth). These calculations are done in the fol­ lowing manner (rounded to a whole number): water for dry SS = (dry weight SS) (hydration value) = (264 g) (33.8 ml/100 g) = 89 ml The amount of AS that is mixed with the SS to produce the desired concentration in column 1 is entered in column 5. AS, as prepared, is dry. The calculations are rounded to a whole number and done in the following manner: weight dry AS = (660 g) - (weight dry SS) = (660 g) - (264 g) = 396 g The amount of hydration water needed for the AS portion of the TS is entered in column 6. This amount is based on 45% of the dry weight of the components of the AS. Since the AS is dry as prepared, the calculations are as follows (rounded to a whole number): water for dry AS = (weight dry AS) (45%) = (396 g) (0.45) = 178 ml The total volume of water needed for the TS is entered in column 7. The calculations for this column are done in the following manner: water for TS = (water for dry SS) -t- (water for dry AS) = (89 ml) + (178 ml) = 267 ml The amount of water in the wet SS is entered in column 8. This amount is based on the moisture content of the SS. In the example, a 20% moisture content is used. Therefore, 20 ml of water exists in every 100 g of wet soil. The calculations for this column are rounded to a whole number in the following manner: water existing in wet SS = (weight wet SS) (moisture content) = (330 g) (20 ml/100 g) = 66 ml The total amount of water that is actually added to the test soil is entered in column nine. The calculations for this column are done in the following manner: water added to TS = (water for TS) - (water in wet SS) = (267 ml) - (66 ml) = 201 ml The wet weight of TS that is added to each replicate of a particular concentration is entered in column 10. The calculations for this column are done using the following equation (rounded to a whole number) where X equals the wet weight of TS per replicate (1 ml water = 1 g): dry weight per cone. = dry weight per replicate dry weight per cone. + water for TS X 32 660 g = 200 g 660 g + 267 g X 660 g = 200 g 927 g X X = 281 g

E. TEST PROCEDURES Test procedures consist of initiating the test and counting and observing the worms after 7 (optionally) and 14 days of exposure. Initiating the test requires the performance of procedures during two successive days. During day one (the day before the test is put on) the the AS and SS are mixed together and the test soils are hydrated. During day 2 (the day the test is put on), the initial pH values are taken, the test soils are placed in the test jars, worms are introduced to the test soils, and jars are placed in the testing chamber. If necessary and time permits, day 1 and 2 duties can be combined into a single day.

1. Day 1 a. Weighing Out Artificial Soil AS is weighed for each concentration and the controls and is placed in 30 x 30 cm Ziplock bags. A lightweight, plastic tray (approximately 19 cm square and 6 cm deep) is used to support the Ziplock bags. The tray and bag are tared on a top-loading balance. Dust is kept to a minimum if the AS is weighed in a hood. Using the information in column 5 of the calculation sheet, the proper amount of AS for each concentration and the controls is weighed and placed .into Ziplock bags. Each bag is marked with the concentra­ tion using a permanent marking pen. Using the 40% concentration as an example, 396 g of AS is placed into a Ziplock bag. For each control, 660 g of AS is needed. b. Mixing Artificial and Site Soil

The appropriate amount of SS is added to each Ziplock bag. This step is performed in the hood. The SAT Health and Safety Protocol prohibits one from bringing an entire SS into the labor­ atory, so an aliquot is used. The aliquot is removed while the worker is in the soil shed. The aliquot is placed into a plastic bag that is placed into a "diaper pail" that is then placed into a 5-gallon metal bucket. The aliquot is then transported to the laboratory using a Rubbermaid cart and placed in a hood. One of the plastic bags containing AS is placed in the plastic tray noted above and tared on the top loading balance. The correct amount of wet SS is added to the AS (see values in column 3 of the calculation sheet). A stainless-steel scoop or spatula is used to

33 dispense the site soil. In the example, 330 g of wet SS is added to 396 g of AS. After dispensing the SS, each bag of SS and AS is mixed by hand to ensure that the two constituents are well mixed. After mixing, the material is known as test soil. c. Hydration of Test Soil Hydration of the test soil is also performed in the hood. The needed values are in column 9 of the calculation sheet. The correct amounts of water are added to the test soils using a 100­ ml graduated cylinder. In the example, 201 ml of water is added to the 40% concentration. The hydrated TS is mixed by kneading the plastic bag by hand to ensure a uniform distribution of moisture. The negative control is hydrated with 297 ml of RO water. All of the mixed and hydrated test soils are left in the hood overnight for equilibration unless the test is to be put on in a single day. 2. Day 2 a. Initial pH Values Initial pH values of the high and low concentrations and the negative control are measured and this information is recorded on the cover sheet. To obtain the pH values, a 20-g sample is taken from the appropriate Ziplock bag (before the replicates are distributed) with a stainless steel spatula and mixed with RO water (see section above on measurement of pH). These measurements are done in the hood. b. Placing Test Soil Into Jars The test soils are placed in the test containers (in the hood) using a spatula or scoop. The values from column 10 of the calculation sheet are used to determine the appropriate amount in each jar. The test jar is tared on the top-loading balance in a hood. The test soil is often consolidated into a mass in the Ziplock bag. This mass is separated with the spatula or scoop before the TS is placed in the jar. If large interstitial spaces occur after placing the TS into the jars, the TS is pressed down to remove these spaces. The TS is not pressed unless these spaces occur. In the example, 281 g of wet TS is required for each replicate of the 40% concentration. c. Placing Worms Into Test Soil The worms are now weighed and placed into the test jars (still in the hood). A culture tray of worms is brought into the lab. The worms used in testing are adult, clitellate worms with a wet weight range of 300-600 mg each (Inglesfield, 1984; Edwards, 1984). 34 The worms are introduced into the test jars in any sequence. The bottom half of a plastic disposable petri dish (140 mm in diameter by 10 mm in height) is tared on the Sartorius top-loading balance. Ten worms (one at a time) are rmoved from the bedding, "dunked" in room temperature RO water to remove bedding, placed on a paper towel for very quick absorption of excess water, placed into the petri dish and weighed as a group of ten. The weight of the 10 worms is recorded in grams on the cover sheet for the container in which they are to be placed. The petri dish with worms is gently emptied onto the surface of the TS in the selected replicate. The worms are allowed to burrow into the TS. It is important not to damage the worms in this process. Worms are not used if they appear to be injured. Each replicate is to have a biomass of 3.0 to 6.0 g. In the example, the 40% concentration has a biomass of 4.11 g for replicate number 3. Once the worms are placed in the test jar, the lid (with the rubber gasket down) and screw ring are secured. d. Placing Test Jars in the Test Chamber The jars are now placed onto a Rubbermaid cart and taken to Room 8-1 of Greenhouse 8 where the test chamber is located (Attachment #6). Four chambers are located along the northeast wall. Chamber number one is utilized by the SAT. The test chamber is a modified refrigerator that produces a temperature controlled environment. The tests are run at 22 + 2°C. A temperature recording chart (Tempscribe) is kept inside the chamber during the test- Care of this temperature chart is discussed in General Laboratory Procedures and Quality Assurance and Quality Control. The worms are exposed to continuous lighting (Edwards, 1984), which promotes burrowing. The lighting is to be at a minimum intensity of 400 lux. A shallow pan of water, approximately 28 X 19 X 6 cm, is placed in the bottom of the test chamber to maintain humidity. The jars are randomly placed on the shelves. Plywood racks designed to hold seven test jars can be used to facilitate handling of the jars. These racks are stored in laboratory Room 8-4 of Greenhouse 8 (Attachment #6). The starting time for the test is recorded on the cover sheet.

3. 7-Day Count and Observations (Optional) The worms are counted, mortality is noted and behavioral and pathological observations are made. The test jars are removed from the test chamber and transported to the laboratory with a Rubbermaid cart. A polycarbonate sorting tray, stored in Room 8­ 21 of Building 8-20 or in Room 8-4 of Greenhouse 8, is used to count the worms. The tray is 18 cm in height at one end and tapers to 13 cm at the other end. The low end has a circular hole under which the test jars are placed after they have been emptied onto the top of the sorting tray.

35 Before the jars are emptied onto the sorting tray, the worms are evaluated for non-burrowing which is noted on the observation sheet for 7 days (Attachment #7). As the count is made, the following observations are also made: ulceration, coiling, "balling" together, contraction, rigidity, elongation, mid- segmental swelling, segmental constriction, and segmental loss. These and any other observations are entered on the observation sheet for 7 days. Each replicate is counted and the number of mortalities is entered on the cover sheet. A worm is considered dead if it does not respond to a gentle mechanical stimulus to its anterior end (Edwards, 1984). If no mortality occurs, a zero is placed on the cover sheet. The sorting tray is placed in a hood and one repli­ cate of the negative control is emptied onto it. The lid is set nearby for holding worms. The emptied jar is placed under the low end of the sorting tray. The soil is gently sorted with gloved fingers or with the aid of a flat, stainless-steel spatula. As the worms are located, they are evaluated and placed in the lid, held by the ring. Ten worms are accounted for in each replicate. Mortality seldom occurs in the negative controls. After the worms are accounted for, the soil is pushed off the sorting tray back into the test jar. The worms are placed back into the jar on the surface (Edwards, 1984) and allowed to burrow into the soil again. The other replicates of the negative control are counted and the jars placed back on the cart. The tray is wiped off with paper towels.

Dead worms are removed from the soil and discarded. If any replicate of a concentration has 100% mortality, the TS and the dead worms are placed into double plastic bags, labeled as outlined in General Laboratory Procedures; Waste Disposal and held until the end of the bioassay. Worms decay rapidly in the moist testing environment and, if 10 worms are not accounted for, they are considered to have died and completely decomposed. In addition, worms can lose a number of segments and still be able to move. This "half" of a worm is still considered alive. The sorting tray is washed with soap and water and then dried with paper towels after the last replicate of the positive control is counted. Next, the lowest test concentration is counted. If the test jars are counted from the lowest to the highest concentration, the tray is not washed between concentrations and is just wiped off with paper towels. The procedures outlined above are continued until the last replicate of the high concentration is counted, the worms are placed back into the jars, and the jars are placed back onto the Rubbermaid cart. If 100% mortality, i.e., 30 dead worms, occurs in the high or low concentrations or controls on the 7-day count, the final pH for that concentration is taken. A 20-g aliquot is removed from one of the replicates after it has been emptied onto the sorting tray. An aliquot containing dead worms 36 is not taken. The pH values are placed on the cover sheet and day 7 is noted in parentheses. The sorting tray is washed with soap and water after the final jar is counted. The tray of water for maintenance of humidity is checked to see if it needs more water. The final step for the 7-day count is the calculation of the percent effect (percent mortality) for each concentration. This information is placed on the cover sheet under the column labeled "percent E." The calculations are made by dividing the total number of dead worms in a concentration by 30 (the total number of worms initially) and converting this fraction to a percent value. The test jars are now taken back to the test chamber and randomly placed inside. 4. 14-Day Count and Observations The procedures for this day are similar to those for the 7­ day count. The chart from the Tempscribe is removed and checked to see if the temperature has remained constant. This chart is kept in a file. A photocopy of this chart can be attached to the paperwork for the test. The jars are removed from the test chamber and taken to the laboratory with a Rubbermaid cart. Each replicate is counted as per the procedures for the 7-day count. Observations are made as at 7 days and entered on the 14-day observation sheet. If survival does not change between 7 and 14 days, the mortality counts and the percent effect values are carried forward from the 7-day count. In the example, the 40% concentration, as shown on the cover sheet, had 100% effect at 14 days as opposed to 96.7% at 7 days. This is the final count and the sorting tray does not need to be cleaned until all jars are evaluated. The TS is not placed back into the jars. It, along with the dead and living worms, is placed in double plastic bags and held as potentially hazardous material until toxicity information is obtained from all of the other bioassays (see General Laboratory Procedures; Waste Disposal). Used rings and lids are discarded. The final pH values are taken from the highest concentration remaining, the lowest concentration and the negative control (day 14 is noted in parentheses). The procedures are followed as outlined during the 7-day count. The percent effect values are also calculated for the 14 day counts and noted on the cover sheet.

IX. CALCULATIONS The percent effect values are calculated at the end of the 7 and 14-day counts and entered on the cover sheet. Mortality in the negative control must not exceed 10% (Edwards, 1984). A median lethal concentration (LC50) with corresponding 95% confidence intervals (Edwards, 1984) is statistically determined for the site soil for both 7 and 14 days using the trimmed Spearman-Karber method. Personal computers are available for determining LC50 values in Rooms 289 and 250. The computer program selection menus have a Spearman-Karber option. Attachment #8 is 37 a printout of the questions asked by the program with the appropriate answers, following the example in this manual. Capital letters Y and N are used when answering questions yes and no. Plot and summary files are created. The name of the plot and summary file is the test code from the cover sheet. Enter the date the test was started. Test number is the chemical number from the cover sheet. LC50 is estimated; use capital L. The species name is Eisenia foetida. Chemical name is the name of the site (Poison Hollow). The units for concentration are percent. The number of concentrations does not include the controls; there are 5 concentrations in the example. The concentrations are entered from low to high. The number of individuals at each concentration is equal. The number of individuals at each concentration is 30. The unit for the duration of the experiment is days. The duration of the test is 7 or 14 days. The number of mortalities at each concentration is the total of the three replicates- The automatic trim calculation is selected and the LC50 value with confidence intervals appears on the computer screen. A copy is sent to the printer (Attachment #9). The results are saved in the plot/summary file, and the program does not continue unless more LC50 values are to be calculated.

X. QUALITY ASSURANCE/QUALITY CONTROL The acceptability of the test is based on the percent germination of the negative control. At least 90% of the organisms in negative controls must survive in order for the test to be acceptable (27 out of 30 worms). XI. DATA REDUCTION, VALIDATION, AND STATISTICS Due to the transient nature of bioassay statistics, this suggested procedure may change significantly as better ways to handle data reduction are learned. Data is either processed on the Spearman-Karber program or a specific program design that can transform data into log form. Response is expressed as percentage effect of a toxicant concentration relative to the control for "continuously distributed" data and as percent mortality for "quantal" data. The log of the toxicant concentration can be plotted against the log of the response measured using the Statgraphics program. With this program, an EC50 is determined, and 95% confidence intervals are graphically obtained. The EC50 value and confidence intervals are then manually transformed back from log notation. The Spearman-Karber program uses this log transformation, and transforms the results back before producing an output. The Spearman-Karber program is used for all tests that are measured as LC50s, that is, they do not compare the toxicant response to the control yield but to the number of organisms 38 originally exposed, always a constant ("quantal" response tests). In tests of this nature, the control is used to determine if the test is valid, that is, if a certain percentage of the control organisms survive. For tests that are measured as EC50s ("continuously distributed" data, that is, toxicant concentration yield relative to control yield), the Spearman-Karber program is not applicable; the log transformation and subsequent Statgraphics EC50 and confidence interval deterTtiination must be done. Confidence intervals are graphically obtained from the Statgraphics printout, unlike the Spearman-Karber that figures them mathematically. The tests of this type include the Root Elongation test. Of the bioassays calculated as EC50s, each has a program specific to its design for the log transformation and data reduction, and these numbers are all run through the Statgraphics program.

An alternative procedure for continuously distributed data is to use not only the linear model on log transformed data in the Statgraphics program, but also the linear, multiplicative, and exponential models on the untransformed data and choose the model that has the highest r-squared value for determining the EC50 and 95% confidence intervals. This method minimizes the number of tests that need to be re-run due to lack of satisfactory confidence intervals.

All data from bioassays in final form are either in a Statgraphics or Spearman-Karber format, have an EC or LC50 determined with confidence intervals if possible, and if not are rerun or have explanations offered for why no such determinations were possible. Each technician terminating a test is responsible for entering the data into the Spearman-Karber program, if that is applicable, and turning in that output along with a copy of the original data to the team coordinator. If the Spearman-Karber program is not applicable, the technician terminating the test is responsible for reducing the data using the data reduction program specific for that bioassay, and submitting the output from that program plus a copy of the original data to the team coordinator. Since the Spearman-Karber program truncates data objectively, and some subjectivity is inherent with the Statgraphics program, only one technician uses the Statgraphics program. If only one technician makes this subjective decision, it is assumed a higher level of continuity and comparability between bioassays will be obtained. For each site investigated, a bioassay result sheet is assembled that includes the results (EC/LC50s, 95% confidence intervals, etc.) of all bioassays done on that site. That sheet, along with a similar document for chemical analysis, form the basis of the report to the principal investigator, who draws conclusions from this data and submits a subsequent report to the party requesting the investigation. The raw data is kept on file; copies

39 may be submitted to the party requesting the investigation at the discretion of the principal investigator. XII. WASTE DISPOSAL Return of Test residuals to the site of origination or to a waste disposal facility Waste can be unused site soil contained in its original shipping container that is no longer needed and has been determined to be toxic; unused eluates that are stored in a container in which they were put when generated, are no longer needed and have been determined to be toxic; material contaminated with soil or eluate or standards in the preparation and bioassay process which can include contaminated equipment, disposable testing materials, the eluate-diluent mixtures, the soil-diluent mixtures, the organisms, and any other contaminated materials; and chemical standards. Raw site samples and eluates are stored at 4oC are stored either in the hazardous materials storage area or in a vented refrigerator in the laboratory. The bioassay process can produce up to approximately a 4:1 increase in contaminated materials, and raw soil is often accessed a number of times, thus making it unworkable to store spent sample and accumulated wastes in the original shipping containers. Leftover toxic eluates should be moved from the refrigerator in the testing lab to cold storage as well as wastes generated from completed bioassays, and kept with unused toxic raw samples when analyses have been completed and toxicity has been assessed. Solid and liquid wastes produced in the lab are stored at that location or moved to await toxicity determination ("pending" material). Material deemed non-toxic is disposed of as such and toxic material is either moved or remains at that location. Remaining soil and eluates are stored in a vented refrigerator in the lab until toxicity data for that particular sample determines its disposal as non-toxic or it to await disposal as toxic waste upon completion of that specific site (see attached waste flow chart. Attachment #12). Eluates are stored in high-density polyethylene/propylene containers (HDPE/P) upon completion of the processing part of sample preparation (for processing detail, see Processing SOP). Eluates remain in this container until they are deemed non-toxic and discarded in the sink or turned over to the TSCO for disposal. Solid wastes generated from testing are double-bagged in 4 mil plastic bags with the inner bag opening sealed inside the outer bag to reduce possible leakage. A tag filled out with the identifying information for that sample should be enclosed in a small Ziploc bag, sealed, and 40 inserted between the two containment bags with the information facing outward so that if the tag on the outside was torn off, waterlogged, or the information somehow obliterated that the material could be identified. This tag should not be labeled "Solid Hazardous Substance" since this label is put in place before toxicity determination and unlike the tag on the outside cannot simply be torn off if the material is deemed non-toxic and disposed of as refuse. This tag is for emergency identification only, and must be accompanied by a tag on the outside of the package for toxic samples. On the outside of the second bag the closure is twisted tightly, folded over, and sealed with electrical or other suitable tape (duct tape or strapping tape), and a plastic tag tied around the closure with wire. The tag is marked with the generic label "Solid Hazardous Substance"; this should appear on all outer tags. The sample tracking number must be present and the concentration either in percent raw sample or percent eluate (mg/liter or mg/kg toxicant if not a site sample); the actual description of the sample is also pertinent information, as well as type of waste (worm test, RE test, SG test, processing waste etc.), and the weight (solid) or volume (liquid). Packaging, tagging, and weighing of wastes should occur when the material becomes waste. When a test is terminated or samples are processed, the contaminated material should be packaged, tagged, and weighed and placed in the North hood in the lab which is purged weekly. In addition, at the time of packaging as waste, a "Hazardous Materials Disposal Log" sheet should be filled out for that particular package of waste if it is known toxic. This log should contain the date, the name of the material or site as complete as possible with no abbreviations, the chemistry number, the amount (in kilograms or liters), concentration range (1-100%, 5-12 mg/L, etc.), and the name of the worker filling out the form. At the top of the form it should clearly be stated if the material is liquid or solid, and if more than one chemistry number or sample waste is contained in the same package the NUMBER (the date: e.g., bottle # 102488) of that package (this is only applicable to wastes where spent sample from a number of different tests is grouped together in one container. This is common practice for spent positive controls, standards, and residuals of multiple tests from the same site sample).

Packaged solid wastes, leftover eluates, and leftover soils do not need separate container numbers and can be identified by their tags. They do, however, need to be entered on disposal log forms, and more than one container can go on the same form provided it is the same material or from the same site. Liquid wastes of different chemistry number stored in the same container are the only packages that have disposal log sheets filled out for them while in the lab, all other packages are logged when they are deemed toxic). When the raw samples or eluates themselves are to 41 be discarded (testing for that site is complete and the PI has authorized removal) they are taken from the lab refrigerator and are labeled, tagged, and weighed (or volume determined). Soil samples in original shipping containers should be removed from the HPDE bucket, but must remain in the plastic inner bags when placed in larger vessels (e.g. 55 gal drums) for disposal. Although when the sample is originally screened it should be placed in 2 bags 4 mils thick if not sent in that fashion, that is not always the case. Upon determination as toxic if the raw sample is removed from the bucket to be disposed of, in addition to being weighed and tagged it should be correctly bagged and labeled with felt pen on the bag itself since there is no inner tag. If the sample remains in the HDPE bucket that bucket must be properly labeled. If the sample remains in the bucket because of some compromise of the inner containment, the entire bucket should be placed in a 4 mil plastic bag and sealed and tagged as above, or the sample must be re-packaged under bottled air (see Health and Safety Protocol).

Eluates should be labeled directly on the HDPE/P container, and the screw cap tightened securely and taped with electrician tape (for this purpose no substitute should be used). A tag filled out as above should be attached to the neck of the container, or optimally the entire container placed in a 4 mil plastic bag and sealed and tagged as above. At the time raw samples and unused eluates are packaged as waste they must be weighed (volume determined) and that information entered on the tag and all information concerning that sample entered onto a disposal log sheet (see example). Wastes are stored under refrigeration until they are sorted according to the TSCO's directive. This usually involves placing similar materials together in 55 gallon drums. For wastes which are shipping back to the site of origin (this should be the bulk of the material tested) it must be placed in drums or proper shipping containers and send it out according to DOT regulations. For liquids, absorbent ;Ls placed in the bottom of outer containers to soak up any possible leakage. ALL material in the hazardous materials storage area should be properly labeled so the only additional steps necessary are to either discard the pending material as refuse if it is deemed non-toxic, log pending onto the disposal log forms if toxic, or to place toxic materials in shipping containers for disposal as wastes or for shipment back to the site of origin. All wastes should be disposed of as soon as possible. Laboratory Pretreatment of Test Residuals to Reduce Waste Volume Due to the high cost of waste disposal, the volume of liquids wastes containing organic toxicants can be reduced by percolation 42 through charcoal to remove the toxicants and allow disposal of the resultant solution down the sink (after it has been tested to determine if all toxicants were successfully removed). The toxicant-laden charcoal can then be incinerated. The large reduction in weight of material requiring incineration is the driving force behind implementation of this procedure. A preliminary test can be completed to estimate the amount of organic material that a known amount of charcoal will bind. A known concentration of azosulfamide solution, a bright red dye, can be percolated over a known amount of charcoal and, when the filtrate begins to come through colored, the amount of solution percolated through can be recorded and the milligrams azosulfamide per gram charcoal can be figured. This value will differ depending upon the type of charcoal used. The charcoal should be incinerated when it is estimated to be at 2/3 capacity; this margin of error can be used to account for variation in the affinity for organic materials to bind to the charcoal. Again, the resultant solution should be tested for toxicants before it is disposed of, and re- filtered if necessary. In order to estimate the point at which the charcoal filtration material has been 2/3 saturated with organics, and to meet requirements for incineration of the charcoal, accurate records of the actual amounts of toxicants lodged on the charcoal will be required. To do this, a program should be developed that calculates the total amount of toxicant from the highest concentration, the number of dilutions, and the "factor." The Federal Register and other sources have advocated designing tests with a concentration series that incorporates a "multiplier" (factor), a number that is multiplied by each successive descending concentration to get the next concentration for testing. The Federal Register advocates using a 0.1 multiplier (100, 10, 1, 0.1, 0.01 is an example descending concentration series) for range- finding tests and a 0.5 multiplier (100, 50, 25, 12.5, 6.25) or 0.3 multiplier (100, 30, 9, 2.7, 0.81) for definitive tests. Ascending concentration series can also be generated using the "multiplier" by dividing each successive concentration by the "multiplier." An ascending example using a 0.5 "multiplier" or "factor" would be 2, 4, 8, 16, 32, 64. Of course, if the order of concentrations were reversed, one would multiply each concentration by the factor; it is simply a matter of where one wants to start. There are factors and beginning concentrations that work well together (providing series that have a minimum of decimal places for dilution). There are an infinite number of concentration series possible using this method.

For this in-house waste remediation program to work efficiently, restricting concentration series used in testing to those incorporating single multipliers is optimal. Figuring the total toxicant in a mixed concentration waste sample when other concentration series have been used can be difficult, especially 43 if not done by the tester. If single multipliers are not used, it will be up to the tester to calculate the total toxicant in a waste. If possible, use single multipliers in test designs, and complete an entry on a hazardous waste disposal log sheet (Attachment #10) that includes all the required information at the time the waste is generated (at the conclusion of a test if no further analysis is scheduled). Only like materials should be placed in any one single waste receptacle, but multiple test wastes of the same material may be combined. At the present time, only organic wastes are being reclaimed, but it is anticipated that a system for metals wastes and mixed wastes (such as eluates) will be developed in the future. Only aqueous matrix wastes can be disposed of using this system; solid wastes must still be sent off site.

XIII.EARTHWORM CULTURE A. PARAMETERS Population density, type and availability of food, pH and moisture content of substrate, temperature and consistency of temperature all contribute to fecunciity, growth rate and age of sexual maturity in Eisenia foetida (Hartenstein, 1982; Hartenstein et al., 1979; Neuhauser et al., 1980; Reinecke and Kriel, 1981; Reinecke and Venter, 1987; Reinecke and Viljoen, 1990; Tomlin and Miller, 1980). Tomlin and Miller (1980) reported maturation periods, from cocoon to fully clitellate worm, for Eisenia foetida ranging from 48 days to 117 days at 25 and 13° C, respectively. Tsukamoto and Watanabe (1977) reported hatchability and the number of young worms emerging from a single cocoon to decrease with a rise in temperature from 10 to 25° C for Eisenia foetida while the incubation time of cocoons decreased with the rise in temperature.

B. WORM CULTURE CHAMBER The culture chamber is maintained at a constant temperature of 22 ± 2° C (Neuhauser et al., 1986b). Hartenstein (1982) concluded that Eisenia foetida could be produced optimally over the range 18-25° C. The culture trays are maintained on a shelf system (rack), e.g., 2X4s and plywood. The chamber is kept under continuous lighting (Edwards, 1984) to induce the worms to remain in their trays. A record book tracking activities, e.g., preparation of food and bedding, of the culture is maintained.

C. PREPARATION OF CULTURE TRAYS

The cultures are grown in plastic Rubbermaid trays measuring about 34 X 28 X 14 cm. Bedding consists of Canadian sphagnum peat moss (Sphagnum) adjusted to a pH of 7 with calcium carbonate (99%

44 purity) (Edwards, 1984). The worms can be cultured at a pH range of 5-9, but a pH of 7 is optimum (Kaplan et al., 1980). A toploading balance with 1 g readability is used to weigh 700 g of unscreened peat moss (with large debris removed) which is placed into a large plastic bag. A toploading balance with 0.01­ 0.1 readability is used to weigh approximately 20 g of calcium carbonate which is added to the bag of peat moss. The proper amount of calcium carbonate will vary with the pH of the peat moss utilized. The peat moss and carbonate are mixed by shaking and inverting the plastic bag. The adjusted bedding is placed in a clean culture tray. Trays are cleaned by washing with soap, tap water and a scratch pad, rinsing with tap water, and finally rinsing with reagent water. Approximately 2,300 ml of reagent water are added to the bedding, which is mixed by hand until it is uniformly wet. The exact amount of water added will depend upon the type of peat moss and its ambient moisture content. The goal is to produce a suitable environment for the worms with no standing water on the bottom of the tray. The trays contain a volume of about 9,000 cm^ with a surface area of about 900 cm^ when the wet bedding is added. The materials and source of the materials used for the bedding should be standardized. Trays are covered with a piece of black plastic to maintain moisture and a piece of plywood (30 x 38 cm) is used to keep the plastic in place. Worms are placed on the surface of the bedding and allowed to burrow. Those worms not burrowing within about 5 minutes should be discarded.

D. CARRYING CAPACITY OF CULTURE TRAYS Neuhauser et al. (1980) calculated carrying capacities for Eisenia foetida. in a volume of 300 cm^ with a surface area of 78 cm^, to range from about 6 to greater than 23 g of worm depending upon the type of food source/substrate. This is about 0.02-0.08 g of worm per cm' of substrate. The number of worms that a tray holds is a function of the size and age of the worms. Adult worms have distinct, fully developed clitella and weigh a minimum of about 300 mg. Sub-adult worms have visible, but not fully developed, clitella and are about 150-300 mg in weight. Juvenile (young) worms do not have clitella and are usually less than 150 mg in weight. For optimal reproduction, it is recommended that the trays containing 9,000 cm' of bedding hold a maximum of 245 g of worm, i.e., 0.03 g/cm'. For example, 350 adult worms weighing 700 mg each would be equal to 0.03 g/cm\

E. PREPARATION OF WORM FOOD Fermented alfalfa (Medicago sativa) pellets are used for food. Dry pellets (protein, minimum of 15%; fiber, maximum of 30%; ash,' maximum of 12%; fat, minimum of 1%; and moisture, maximum of 12%) can be purchased at most feed stores. The quality of alfalfa pellets and their source should be standardized. Worm food is

45 prepared in 3 gallon plastic pails with lids. Eighteen hundred g of pellets and 3,600 ml of reagent water are added to each pail and mixed by hand. Lids are placed on the pails which are stored in the incubation chamber. Each pail is marked with the date of preparation of the food. The food is allowed to ferment for two weeks before use. F. FEEDING AND CARE Feeding and examination of the worm cultures is done twice a week, Tuesday A.M. and Friday P.M. is convenient, unless the population of a tray is low, e.g., 100 adults, when only once a week is sufficient. If high growth and reproduction rates are to be achieved, regular feeding of the worms is required (Reinecke and Viljoen, 1990). A Feeding Data Sheet (Attachment #11) is kept for each tray which contains data on food, moisture and other pertinent information. Disposable rubber gloves are used when handling the worms and are changed between each tray to reduce the possibility of the spread of disease. If worms crawl or fall out of the trays due to handling, they are discarded. Food should be consistently available, but it is important not to add too much food to a tray at one time. Mortality has been observed in some trays with excess food due possibly to the production of toxic gases from decay of the alfalfa. If, for example, a tray of 350 adults can consume a volume of 800 ml in a week, then they should be given 400 ml in two feedings. On Tuedsay, care of the cultures begins by removing and discarding any remaining food. Accumulations or pockets of mites and enchytraeids along with a small amount of the surrounding bedding are also removed by hand. Removing the excess food helps to reduce these pests. If a large amount of food is left, too much was added during the prior feeding and a reduced amount is needed. Food is now spread evenly over the surface of the bedding. The proper amount of food is a function of the number and age of worms in a tray. Recent feeding records give an idea of how much food to add to a particular tray. Care of the cultures on Friday begins with the removal of excess food and any mites or other pests as on Tuesday. The plastic cover should be examined for the presence of large numbers of mites or enchytraeids and replaced if necessary. Next, determine if the tray needs more moisture by turning the bedding over for examination. This procedure also prevents the bedding at the bottom of the tray from becoming too compressed. If it appears as if the bedding is dryer than when it was first prepared, or if the surface of the bedding is dry, then reagent water needs to be added. How much water to add is a subjective decision. The bedding needs to be moist, but with no standing water. If a handful of bedding is squeezed, it should be moist enough so that water runs out. Water is added by pouring or spraying it evenly over the surface of the bedding. The bedding is then turned over by hand again to distribute the moisture. Finally, food is added

46 as on Tuesday. G. OVERCROWDING Once a tray exceeds the carrying capacity of adult worms as described in section D above, the tray needs to be split into two trays for optimum growth of the worms. A tray with less than the carrying capacity of adults, but with a large number (300-800) of young worms also needs to be split. Failure to split trays when needed leads to more frequent changes of bedding as described in section H below. Experience allows one to be able to estimate numbers of worms in a tray. First, a new tray of bedding is prepared. One-half of this new bedding is removed and placed on a clean surface, e.g. , a piece of plastic. One-half of the bedding and worms from the crowded tray is removed and placed into the new tray. The new bedding on the clean surface is then placed in the old or previously crowded tray. Both trays are mixed by hand to create a uniform mixture of the old and new bedding. Records of split trays can be recorded on the Feeding Data Sheet (Attachment #11). Overcrowding can also be reduced by removing adult worms from crowded trays and introducing them directly into a bioassay or by placing them in a new tray of bedding.

H. BEDDING CHANGES The bedding tray needs to be changed at a minimum of every six months. Trays usually need to be changed due to excessive moisture and the potential build up of anaerobic conditions. Frequency of change is a reflection of the age and number of worms in a tray. Trays, especially crowded ones, become excessively wet over time even if they are not over-watered. This may be due to the moisture added in the fermented feed and/or to water as a respiration by-product. First, a new tray of bedding is made as described above. Bedding from the old tray is spread out evenly on the top of the new bedding- The tray is then allowed to sit under room lighting for a day or two. After this time, the old bedding, which is generally darker in color, is removed and discarded. Most of the worms will have burrowed into the new bedding. This procedure, however, does not recover 100% of the worms nor any of the cocoons and consequently should not be utilized in a culture with only a few number of trays. An alternative method is to thinly spread the contents of the old tray onto a large, flat surface and allow it to be exposed to room lighting for three to four hours. Cocoons and worms can then be removed by hand and placed in the new bedding (cocoons under the surface). This is, however, very time consuming and can result in injury to young worms and cocoons. Records of bedding changes can be noted on the Feeding Data Sheet (Attachment #11).

47 I. PEST PROBLEMS Some of the pests associated with the culture of worms are fungus gnats, soil mites, Collembola and enchytraeids. None of these pests in low numbers appear to be a problem for the culture of healthy worms. Gnats are seasonal and are mostly a nuisance for the caretaker of the worms. Large numbers of mites and enchytraeids appear to compete for food with the worms and mites have been observed on dead or dying worms. Biocides are not used for the control of pests because of their potential effect on earthworm health or testing sensitivity. Control of pests consists of removal by hand as described in section F above or by disposal of infected trays. Different geographical regions may have their own distinct types of pests.

48 XIV. GLOSSARY Acute: A short-term, severe reaction to a stimulus. In this test, the response or endpoint measured is mortality, but other endpoints can be measured in an acute test.

Aliquot: A subsample or portion of a whole.

Aqueous matrix: A fluid water-based environment, substrate, or carrier within which something develops, exists, or proceeds. AS: Artificial soil. A synthetic soil prepared in-house with a specific formulation. AS is used as the diluent medium and also as the medium for the positive and negative controls. Aspirator bottle: A specific piece of glassware used for aspira­ tion that has a flat or slightly domed bottom, straight sides, normal neck, and a tubulature or spout protruding from the side flush with the bottom. Usually made of heavy glass. ASTM: American Society for Testing and Materials. ATCC: American Type Culture Collection. Brood cycle time: The time interval between a given birth event and the next birth event, usually in reference to a female organism. Buffer: Any substance in a solution that tends to stabilize the concentration of another constituent in that solution or tends to stabilize the difference between antagonistic forces. Sample Tracking Number: The tracking number for a chemical solution, site sample, or other toxicant. The number contains as much information about the material as possible. Usually eight characters or less.

Clitellum: The fleshy "ring" or "saddle" of glandular tissue found on certain mid-body segments of many oligochaetes. It is the most visible feature of an adult earthworm and secretes the cocoon into which eggs and sperm are deposited.

Data sheet: A sheet accompanying a test on which data, test parameters, and other pertinent information is recorded. Diluent: Material (fluid or solid) used to dilute or make less concentrated some other material. Dilution scheme: The series of dilutions necessary to create the series of toxicant concentrations desired in a toxicity test.

Direct (terrestrial, aquatic) test: 49 Dissolved oxygen (DO): Oxygen (Oj) that is dissolved in aqueous solution. Quantified in mg/1. DO: Dissolved oxygen.

Dose test: A test in which the toxicant is less than or equal to 2% of the total volume or weight of the exposure matrix. The "diluent" or carrier volume is constant for different concentrations in a dose test, and the concentration of the "spike" or "dose" is varied [again, the spike or dose is always less than or equal to 2% (1 part in 50) by weight or volume of the exposure matrix],

DQO: Data quality objective. EC50: Effective concentration, 50%. The concentration of a toxicant that effects the test organism by 50% based on the specific endpoint of a test. For example, if root lengths are being measured, the EC50 would fall at the point where the root lengths were reduced from the control by 50%, or half. If the control average were 10 mm, the EC50 would be calculated for where the average toxicant exposure length would be 5 mm.

EDTA: (Ethylenedinitrilo) tetraacetic acid, disodium salt. A chelator which is one of the ingredients of AAM (algal assay media) and Woods Hole MBL media. Eluant or eluent: A solvent used in eluting. Eluate: The solution that results from eluting.

Elute: To wash out: to remove (absorbed material) by use of a solvent. Specifically to mix a site soil with water to put water- soluble soil constituents into solution.

Endpoint: The definitive qualitative juncture in a procedure or process upon which or from which conclusions can be made. The final measured quality; culmination. ERL-C: Environmental Research Laboratory—Corvallis.

Eyeballing: A non-quantitative subjective analysis that relies on experience, intuition, or luck for its accuracy. From the practice of looking at something and estimating a level, quality, or quantity.

Filter-sterilized: Passed through a filter of a pore size small enough to exclude microbial contaminants.

Fish-food-yeast (FFY) solution: A nutrient solution for Daphnia 50 consisting of baker's yeast and fish food (Trout Chow PRll or equivalent) mixed in water. Gilson Pipetman automatic pipettor: An automatic pipettor made by the Rainin Company that delivers a predetermined (preset) amount of liquid repeatedly by use of a plunger. HA: Hardness-adjusted. Hardness: The amount of magnesium, calcium, and a few other minor metals contained in a given volume of solution and usually reported as mg/1 calcium carbonate (CaCO,). HWAT: Hazardous Waste Assessment Team. ICPAES: Inductively coupled plasma-atomic absorption spectroscopy. Indicator: Any of various substances used to indicate the acidity or alkalinity of a solution, the beginning or end of a chemical reaction, the presence of certain substances, etc., by changes in color. Indirect test: Initiation: Beginning; starting; putting a process in effect. Inoculum: An amount of an organism suspension or mixture in any matrix used to inoculate a volume of media or substrate. Insolubility: The level or degree to which a substance is insoluble, that is, is not soluble, cannot be dissolved. LC50: Lethal concentration, 50%. The concentration of a toxicant that causes mortality in 50% (half) of specific test organisms exposed. LC50: Median lethal concentration. The concentration of toxic substance to which test organisms are exposed that is es­ timated to be lethal to 50% of the test organisms. The LC50 is usually expressed as a time-dependent value, e.g., 96-hour or 14-day LC50; the concentration estimated to be lethal to 50% of the test organisms after 96 hours or 14 days of exposure. The LC50 value is derived by interpolation or statistical analysis. Macronutrients: Nutrients (usually inorganic compounds) that are the primary food sources or primary components used to create a food source to be used as the primary energy source for a population or culture. Meniscus: The curved upper surface of a liquid column that is concave when the containing walls are wetted by the liquid 51 and convex when not. Micronutrients: Nutrients (usually inorganic compounds) that are necessary for physiological processes of an organism, but are not primary energy sources, such as vitamins and trace metals. MilliQ reagent grade RO water (or RO water): Water from a MilliQ (Trademark of the Millipore Company) water treatment system in which the water is passed through a fiber prefilter, a reverse osmosis filter, a carbon filter, and two ion-exchange filters. This ultrapure water is monitored in purity by its resistivity, and is considered reagent grade when its resistivity is greater than 10 megohms. Mineral-free rinse: A cycle on automatic dishwashers that replaces the usual tap water with distilled or de-ionized water. This water is usually unheated, whereas the tap water rinse is heated. Applying cold mineral-free water to hot non-pyrex glassware may cause fracture. Motile: Exhibiting or capable of movement. MSA: Mine Safety Appliance. A company that manufactures and distributes safety equipment. Multiplicative factor: Any factor by which a number or series of number is multiplied by to give a certain result. Usually a condensation of an equation or series of equations. Negative control: The control, usually in three or more repli­ cates, that accompanies any test and is void of the parameter being tested. Test "exposures" that give the "normal" response. These controls may be used to validate the test or may be used as a base against which to compare the toxicant exposure response. Negative control (earthworm bioassay): A treatment in a toxicity test that duplicates all the conditions of the exposure treatments, but contains no test material. One hundred percent dilution medium or AS is used as the negative control in this test. The negative control is used to demonstrate the absence of toxicity in the basic test conditions, e.g., health of -test organisms, quality of dilution medium, etc. NIOSH: Nutrient limitation: That point at which a population can no longer increase or sustain itself due to the lack of some constituent required for growth. Organism: Any individual animal or plant having diverse organs and parts that function together as a whole to maintain life 52 and its activities. PAR: Photosynthetically active radiation. Percent effect: PI: Principal investigator. Plated cultures: Microbial or plant cultures growing on nutrient or other agar petri plates. Population densities: The number of inter-breeding organisms existing within a clearly defined area or volume and expressed as the number of individuals per unit volume or area. Positive control (earthworm test): A component of a toxicity test that duplicates all the conditions of the exposure treatments, but contains an effective concentration of a known toxicant that is added to the dilution medium (AS in this test). The positive control is used as a routine, continuing check for changes in sensitivity in the test organism due to such factors as feeding, source of worms, culturing changes, etc. Positive control: A toxicant control, usually three or more replicates, that verifies that the organism being tested is responding to a toxicant in the normal fashion. A reference toxicant is used for positive controls and the response is usually charted on a "control chart." Preparation: Readying; outfitting; assembling the necessary components for an event or for putting a process in effect. Protocol: A document outlining guidelines for procedures or processes. Not a step-by-step procedure, but general rules with which various more definitive procedures must comply. Randomization: To place ordered objects in an unordered array. Specifically, to remove any semblance of order from a group of toxicant-exposure vessels to minimize the possibility of spatial effects being perceived as effects from some tested variable. RE: Root elongation. Reconstituted water: Tap water, well water, partially treated water, or synthetic water that has had chemicals added to increase its hardnegs. Synonymous with "hardness-adjusted" water. Reference toxicant: Toxicant with a known response for a par­ ticular organism against which test data or response data is compared. 53 Regimen: A regulated process or procedure that is repeated systematically, often at regular time intervals or as prepa­ ration for an event. Rigid-sided polypropylene or polyethylene waste container: A container made of high-density polypropylene or polyethylene that maintains its own shape, does not conform to the shape of the liquid therein, and is only slightly flexible (as opposed to low-density polypropylene which is very flexible). RO water: Reverse osmosis water. In the ESAP, the term RO water refers to the reagent grade water that is produced by the MilliQ system with carbon and ion-exchange cartridges. In actuality, RO water is a laboratory grade water that goes into the MilliQ system after going through a prefilter and a reverse osmosis cartridge. It would be more accurate to call the water used by the ESAP MilliQ water or reagent grade water, but the term RO water is commonly used. RO: Reverse osmosis. Sensu stricto: Of known or exactly defined constitution. Not composed of any portion of an unknown or unquantified sub­ stance. Serial Dilution: A series in which all dilutions are reduced by the same factor. Solid matrix: An organic or inorganic non-fluid particle-based environment, substrate or carrier within which something develops, exists, or proceeds. SOP: Standard operating procedure. Detailed, step-by-step, instruction for the performance of a bioassay, laboratory process, or other scheme. Standard operating procedures are not to be confused with protocols, which are guidelines and usually do not have details specific to a particular labora­ tory. SS: Site soil. The material being tested for its toxicity. In this manual, SS refers to the soil or sediment received by the laboratory for toxicity testing. Standard: Solution of known chemical constitution against which test data or solutions are compared. Sterilized: Having any living organisms or spores killed or removed, either by heating, autoclaving, filtering, microwa­ ving, chloroforming, irradiating or any other method. Supernatant: The fluid portion of a suspension that has been 54 centrifuged to remove some or all suspended particles.

Survivorship: The number of organisms (or percent of originally exposed organisms) alive after a period of exposure to a particular level of toxicant. Terminating: Ending; stopping; bringing to a close (as in a process). Test code: The tracking code for a particular test. The code is singular so as to minimize data mixup. The code contains as much information about the test as possible. Usually 8 characters or less.

Titrant: The standard used in titration (see titration). Titration: The process of finding how much of a certain substance is contained in a known volume of a solution by measuring volumetrically how much of a standard solution is required,to produce a given reaction.

TOC: Total organic carbon. Toxicant: Material that has an expected or verified negative impact on the life processes of any organism, or of a par­ ticular organism, under certain conditions (most toxicants are concentration dependant).

TS: Test soil. A combination of AS and SS mixed together in a known concentration. In this manual, the TS is referred to in terms of percent of SS by dry weight, e.g., a 75% TS (concentration) consists of 25% AS and 75% SS on a dry weight/dry weight basis.

TSCO: Toxic Substances Contol Officer. Turbidity: The level or degree to which a solution is turbid, that is, has particulate matter or sediment suspended in it. USEPA: United States Environmental Protection Agency. WHC: Water-holding capacity. The amount of water that a soil or sediment retains, after a specific time period, following complete saturation. WHC is expressed as ml water/100 g soil. In this test, WHC is measured after a minimum of three hours and a maximum of 24 hours.

55 XV. REFERENCES

Bouche, M.B. 1972. Lombriciens de France, ecologie et systematique. Publ. 72-2. Inst. Natn. Rech. Agron., Paris, France, 671 pp.

Bouche, M.B. 1988. Earthworm toxicological tests, hazard assessment and biomonitoring. A methodological approach. In CA. Edwards and E.F. Neuhauser, eds.. Earthworms in Waste and Environmental Management. SPB Academic Publishing, The Hague, The Netherlands, pp. 315-320.

Callahan, C.A., L.K. Russell and S.A. Peterson. 1985. A comparison of three earthworm bioassay procedures for the assessment of environmental samples containing hazardous wastes. Biol. Fert. Soils 1:195-200.

Edwards, CA. 1984. Report of the second stage in development of a standardized laboratory method for assessing the toxicity of chemical substances to earthworms. Report EUR 9360 EN. Commission of the European Communities, 99 pp-

Fender, W.M. 1985. Earthworms of the western United States. Part 1. Lumbricidae. Megardrilogica 4:93-129. Greene, J.C, CL. Bartels, W.J. Warren-Hicks, B.R. Parkhurst, G.L. Linder, S.A. Peterson and W.E. Miller. 1989. Protocols for short term toxicity screening of hazardous waste sites. EPA 600/3-88/029. U.S. Environmental Protection Agency, Environmental Research Laboratory, Corvallis, OR.

Hague, A. and W. Ebing. 1983. Toxicity determination of pesticides to earthworms in the soil substrate. J. Plant Diseases and Protection 90:395-408.

Hartenstein, R. 1982. Metabolic parameters of the earthworm Eisenia foetida in relation to temperature. Biotechnol. Bioeng. 24:1803­ 1811.

Hartenstein, R., E.F. Neuhauser and D.L. Kaplan. 1979. Reproductive potential of the earthworm Eisenia foetida. Oecology (Berl.) 43:329-340.

Jaenike, J. 1982. "Eisenia foetida" is two biological species. Megadrilogica 4:6-7.

Kaplan, D.L., R. Hartenstein, E.F- Neuhauser and M.R. Malecki. 1980. Physicochemical requirements in the environment of the earthworm Eisenia foetida. Soil Biol. Biochem. 12:347-352.

Marquenie, J.M., J.W. Simmers and S.H. Kay. 1987. Preliminary 56 assessment of bioaccumulation of metals and organic contaminants at the Times Beach confined disposal site, Buffalo, N.Y. Misc. Paper EL-87-6. Dept. of the Army, U.S. Army Corps of Engineers, 67 pp.

Neuhauser, E.F., P.R. Durkin, M.R. Malecki and M. Anatra. 1986a. Comparative toxicity of ten organic chemicals to four earthworm species. Comp. Biochem. Physiol. 83C:197-200. Neuhauser, E.F., R. Hartenstein and D.L. Kaplan. 1980. Growth of the earthworm Eisenia foetida in relation to population density and food rationing. Oikos 35:93-98. Neuhauser, E.F., R.C Loehr and M.R. Malecki. 1986b. Contact and artificial soil tests using earthworms to evaluate the impact of wastes in soil. In Hazardous and Industrial Solid Waste Testing; Fourth Symposium. STP 886. American Society for Testing and Materials, Philadelphia, PA, pp. 192-203. Oien, N. and J. Stenersen. 1984. Esterases of earthworms- III. Electrophoresis reveals that Eisenia fetida (Savigny) is two species. Comp. Biochem. Physiol. 78C:277-282. Reinecke, A.J. and J.R. Kriel. The influence of constant and diurnally fluctuating temperatures on the cocoon production, hatching time, and number of hatchlings of Eisenia foetida (Lumbricidae, Oligochaeta). Proceedings, Workshop on the Role of Earthworms in the Stabilization of Organic Residues, Vol. 1, Kalamazoo, MI, April 9-12, pp.167-177.

Reinecke, A.J. and J.M. Venter. 1987. Moiasture preferences, growth and reproduction of the compost worm Eisenia fetida (Oligochaeta). Biol. Fertil. Soils 3:135-141.

Reinecke, A.J. and S.A. Viljoen. 1990. The influence of feeding patterns on growth and reproduction of the vermicomposting earthworm Eisenia fetida (Oligochaeta). Biol. Fertil. Soils 10:184­ 187.

Tomlin, A.D. and J.J. Miller. 1980. Development and fecundity of the manure worm, Eisenia foetida (Annelida: Lumbricidae), under laboratory conditions. In D.L. Dindal, ed. , Soil Biology as Related to Land Use Practices. Proc. 7th. International Soil Zool. Coll. of ISSS. EPA, Washington, DC, pp. 673-678.

Tsukamoto, J. and H. Watanabe. 1977. Influence of temperature on hatching and growth of Eisenia foetida (Oligochaeta, Lumbricidae). Pedobiologia 17:338-342. U.S. Environmental Protection Agency. 1986. Test methods for evaluating solid waste. Volume II: Field manual, physical/ chemical methods. SW-846, 3rd ed. Office of Solid Waste and Emergency 57 Response, Washington, D.C van Gestel, CA.M. and W.A. van Dis. 1988. The influence of soil characteristics on the toxicity of four chemicals to the earthworm Eisenia fetida andrei (Oligochaeta). Biol. Fertil. Soils 6:262-265.

58 XVI. ATTACHMENTS Attachment #1 SAMPLE INFORMATION SUMMARY FORM

1) Date of Collection. 2) Name of Sample Site, 3) Location of Site 4) Type of Sample Soil Groundwater Sediment Other Surface Water

5) Date Received at ERL-C 6) Chemical Constituents

Known .

Suspected

7) Collected By: Name,

Address

Phone

59 Attachment # 2

S u. t:> ^ "t ^ n c: ^ SoX±ci,. IsT.O-S­ ,. UN 3 O"?" V

60 01 r> zr 3 ft) 3

(JO Attachment # 4 Cover Sheet For Eisenia Definitive Site Soil Bioassay ample No.: Project: Species: 'ype of Test:. .Investigator:. WHC of S.S, ml/g (Dry Wt. Container Size:, pH of A.S.: pH of S.S.:. rfyd. of A.S.: % of Dry Wt. Hyd. of S.S.:. of WHC oisture Content of S.S.: (Wet Wt. Basis) Test Temp.:. I;mt . T.S./Rep. g (Dry Wt.) Start Date:. Time: No. Worms/Rep. No.Worms/Cone.:. Batch # A.S. I Rep.IInit. IInit. 7 Days 14 Days Final Conc.^ No. lEiL [Weight # Dead % Effect # Dead % Effect PH

1) % of S.S. (dry wt.) 2) grams

62 iittachm^2n t # 5 Calculation Worksheet For Eisenia Definitive Site Soil Bioassav

1 2 3 4 5 6 7 8 9 10 % wt. wt. water wt. water total water total wt. SS dry SS wet SS needed dry AS needed water in water wet TS needed needed for needed for needed wet SS to add per dry SS drv AS for TS to TS iar

1

Ml weicjht s in cjrams anci all vo].ume s in millilil:ers .

<53 F.eu±hwDnn exposure chairiaer location -—fv}-— Zo'-^|.-rw Vl ex IT POOTE. T

4 4 4 4­ i­ 4­ ] • * » »- •- * 1- .? ^ • ^ 4 4- ' 4- f ]

•».. <

<:;tt&eMHou&K

Moa.f«v APPstweo ASSICMMUTV ^ ^L

waste nbjraqe location tiMfio mtttm I

3 tor a le eluter location

s 0 10 IS }0 •"-/cfn/d tcola '^r 'Oi, •3S3HEEE: ^^r 27,7^ ^^ Attachment # 7 Observation Sheet For Eisenia Definitive Site Soil Bioassay Jample No, Project:. Species:. uration Of Exposure:, Type Of Test:. D ate Test Started: Investigator.

I Cone. ^ I Rep. Observation I No.

1) % S.S. (dry wt.)

65 I Attachnent # 8 ULD YOD LIKE TO CREATE A PLOT AND SUMMARY FILE(Y/N)? fME OF PLOT AND SUMMARY FILE TO BE CREATED: PH110787 ENTER DATE OF TEST: 110787 TER TEST NUMBER: PHN45002 AT IS TO ESTIMATED: « (L) LC50 OR (E) EC50 ? L •TER SPECIES NAME: Eisenia foetida IITER CHEMICAL NAME: Poison Hollow (40-22A) ENTER UNITS FOR CONCENTRATION OF CHEMICAL: Percent TER THE NUMBER OF CONCENTRATIONS: 5 TER THE 5 CONCENTRATIONS (IN INCREASING ORDER) f 5

1»( JO E THE NUMBER OF INDIVIDUALS AT EACH CONCENTRATION EQUAL(Y/N)? « TER THE NUMBER OF INDIVIDUALS AT EACH CONCENTRATION: 30 ENTER UNITS FOR DURATION OF EXPERIMENT(HOURS.DAYS.ETC.): Days MTER DURATION OF TEST: 14 ^TER THE NUMBER OF MORTALITIES AT EACH CONCENTRATION: I JO

% iuLD YOU LIKE THE AUTOMATIC TRIM CALCULATION(Y/N)? Y I I

4uLD YOU LIKE TO HAVE A COPY SENT TO THE PRINTER(Y/N)? Y WOULD YOU LIKE TO SAVE THESE RESULTS IN THE PLOT/SUMMARY FILE(Y/N)? Y mi)UL D YOU LIKE TO CONTINUE (Y/N)? N I I I I I 66 I Attadment # 9 IMMED SPEARMAN-KARBER METHOD. MONTANA STATE UNIV

OR REFERENCE, CITE: AMILTON, M.A., R.C. RUSSO, AND R.V. THURSTON. 1977. (RIMMED SPEARMAN-KARBER METHOD FOR ESTIMATING MEDIAN LETHAL CONCENTRATIONS IN TOXICITY BIOASSAYS. NVIRON. SCI. TECHNOL. 11(7): 714-719; ORRECTION 12(4):417 (1978).

LATE: PHI10787 •EST NUMBER: PHN45002 iURATION: 14 CHEMICAL: Poison Hollow (40-22A) PECIES: Eisenia foetida

AW DATA: CONCENTRATION(percent ) 6.00 10.00 20.00 40.00 80.00 .00 .00 .00 NUMBER EXPOSED: 30 30 30 30 30 0 0 0 0 MORTALITIES: 0 3 27 30 30 0 0 0 0 SPEARMAN-KARBER TRIM: .00% SPEARMAN-KARBER ESTIMATES: LC50: 14.14 VAR OF LN OF EST. : .28827D-02 95% LOWER CONFIDENCE: 12.70 95% UPPER CONFIDENCE: 15.75

67 Attachment #10

HAZARDOUS HATERIALE DISPOSAL L06

HIGH TOTAL NUriBER •DATE MATERIAL CONCENTRATION VOLUHE OF FACTOR NAME (M6/L ) (L) CONCENTRATIONS

H

68 Att achment § 11 TRAY #

Ftood Soil Feed Pood Date Remaining Moisture Turned Added Batch ODraments

^H

1 1 1 1 1 1 .JL. • '• 1 WASTE FLOW CHART Nontoxic to Dumpster Waste to 8-23 (Pending) WHC^ •^Toxic to 8-23, Packaged, Waste to 8-23 (Pending) Determination Logged, Etc. (Next to TSCO)

Waste to Original Container North Hood (Packaged, Tagged \ Etc.)^ Excess Soil Lab 250 Spent Soil Rec'd Eluted Processing Eluate Eluate Eluate (300F) Test" to North o 300F > Hood (Pending) Excess Nontoxic ^/ ^ Soil Test Eluate""" to sewer (S.G, E.A.) I Spent Materials ­ Toxic to 8-23 -Toxic to to 8-23 (Pending) Logged, (Next Communal 4»: Packaged, Tagged, Receptacle 4-" to TSCO) c Etc. (Logged) g u

Nontoxic to Dumpster