United States Environmental Protection Agency Environmental Monitoring Systems Laboratory Las Vegas NV 89114 Research and Development EPA/600/S4-87/012 June 1987 SEPA Project Summary Single Laboratory Evaluation of Phytotoxicity Test W. R. Lower, A. F. Yanders, and W. W. Sutton The phytotoxicity method is a screen- ing test used to predict the potential impact of chemicals on seed germina- tion and early seedling growth. Lethal chemical concentrations are indicated at the seed germination stage while the effects of sublethal concentrations can be assessed from data on early plant development. However, the early seed- ling growth phase is not typically conducted if a particular sample causes a dramatic inhibition in seed germina- tion. For testing purposes, such a result would be sufficient to indicate that the sample was toxic to plants. An evaluation of the phytotoxicity procedure was conducted in order to establish the data quality that could be achieved within a single laboratory and to provide a basis for deciding whether or not the procedure merits collabor- ative testing. The tomato plant Lyco- persicon esculentum was used during this evaluation, and an insecticide, sodium pentachlorophenate, was pre- pared for use as a reference material. For several steps in the procedure, minor alterations were shown to def- initely effect the test result, and the phytotoxicity protocol was conse- quently revised to emphasize those steps where the instructions must be strictly followed. The single laboratory precision capability, expressed as a coefficient of variation, was found to be 27% for the seed germination phase and 23% for early seedling growth. A known test response was established using 50 jug/g and 5 /ng/g of sodium pentachlorophenate for seed germina- tion (EC5o = 65±15) and for early seedling growth (EC5o = 35 + 10), respectively. Limits of reliable measurement for seed germination were determined to be 66.5 /jg/g to above 200 /jg/g (for samples of sodium pentachlorophen- ate). The only statement that could be made for early seedling growth was that the lower limit was determined to be at or above 10.3 /jg/g and that the upper limit was not determined. The seed germination phase was capable of distinguishing between sample con- centrations that differed by at least 50 fjg/g. However, the early seedling growth evaluation did not include enough concentrations to make an effective sensitivity determination. This Project Summary was devel- oped by EPA's Environmental Monitor- ing Systems Laboratory, Las Vegas, NV, to announce key findings of the research project that is fully docu- mented in a six separate volumes of the same title (see Project Report ordering information at bach). Introduction The phytotoxicity test was single laboratory tested in order to establish the data quality that could be achieved within a single laboratory The single laboratory evaluation provides a basis for deciding whether or not the method merits collaborative testing, and it more clearly defines the procedure's potential for inclusion as part of an operational monitoring network. Phases of the single laboratory test typically include the identification of procedural variables that must be carefully controlled (ruggedness testing), evaluation of method sensitivity, identification of the limits of reliable measurement, evaluation of systematic error (bias), and identification of method precision and accuracy. The phytotoxicity method is a screen- ing test used to predict the potential ------- impact of chemicals on seed germination and early seedling growth. Lethal chem- ical concentrations are indicated at the seed germination stage while the effects of sublethal concentrations can be assessed from data on root and shoot development. The test procedure is well documented and, with various modifica- tions, has been used as a bioassay procedure for the last thirty years, e.g., to detect effects of herbicides and metals and to determine salinity tolerance. The final test result for the seed germination phase is presented as the calculated concentration (dilution) of sample mate- rial that will cause a 50 percent reduction in seed germination. A lethal response at the seed germination stage typically means that the early seedling growth phase need not be conducted. The final test result for early seedling growth is also presented as a calculated sample concentration (dilution) that will cause a reduction of approximately 50 percent in seedling growth (i.e., one that results in an approximate ECso). Procedural differences have been noted in some of the previously prepared phytotoxicity protocols, e.g., the number of seeds or seedlings required for a valid test result, the number of different plant species required for testing, and the required plant culture conditions (temperature, light, etc.). Prior to the current evaluation, the phytotoxicity protocol was revised to improve the completeness and clarity of all proced- ural instructions and was peer reviewed as a step toward achieving a consensus procedure. The single laboratory test was then conducted using the tomato plant Lycopersicon esculentum (Solanaceae) and an insecticide, sodium pentachlo- rophenate, was prepared for use as a reference material. Procedure Phytotoxicity Test The phytotoxicity protocol tested dur- ing this evaluation was based on the 1982 procedure prepared by the EPA Office of Toxic Substances and on the 1978 procedure presented in the Federal Register for the identification and listing of hazardous wastes. However, during the current effort several technical improvements and modifications were made to the existing method instructions, a peer review was also conducted as a step toward achieving a "consensus" procedure, and, finally, some revisions were required based on results of the ruggedness test. The final protocol also contains some comparatively non- specific instructions for (a) assessing phytotoxic effects of volatile compounds and for (b) acquiring extracts of solid material, but these procedures were not evaluated during the current single laboratory effort. The seed germination and early seed- ling growth phases each progress through a preliminary and definitive test sequence, but it is not always necessary to conduct both tests under routine operating conditions. A very low or a very high toxicity result for a respective preliminary test would constitute a final test result for the respective phase. In addition, the early seedling growth phase would not be conducted at all if a particular sample caused a 50 percent or more inhibition to seed germination. For testing purposes, such a result would sufficiently indicate that the sample material is toxic to plants. However, a very low toxicity, or a no effect test result, for seed germination does not mean that the sample would not cause sublethal toxic effects and, under these conditions, testing would continue with the early seedling growth phase. For both seed germination and early seedling growth, three different concen- trations (dilutions) of the sample material are tested during the preliminary effort, i.e., 0.01, 1.0, and 100 percent of the original sample concentration. (Note: The essentially undiluted sample material, designated as the 100 percent sample, is usually somewhat less than 100 percent due to the Hoagland's solution, solvents, etc.) Based on the results from the respective preliminary test, four concentrations are selected for the definitive effort. The selected concentra- tions must be chosen from the following list of seven dilution options, and they must be four adjacent or sequential concentrations. The seven dilution options are 0.01, 0.33, 0.66, 1.0, 33.0, 66.0, and 100 percent of the original material. No attempt is made to continue testing at dilutions less concentrated than 0.01 percent of the original sample. No additional testing is done to confirm the validity of the respective ECso esti- mate, and no additional tests are used to more thoroughly characterize a dose response curve. As mentioned above, positive controls, negative controls, and, if applicable, a solvent control, are routinely used during the test. Acceptance criteria, associated with each type of control, must be achieved before the overall test response is recorded as a valid result. Various species of plants can be used for the phytotoxicity test, including lettuce, cucumber, tomato, soybean, cabbage, carrot, oat, corn, perennial rye grass, onion, and winter rye. The current evaluation was conducted using the tomato and it is likely that some addi- tional testing (precision, sensitivity, etc.) would be necessary if another plant species were selected. During the seed germination phase, the pH adjusted test sample (or control sample) is first added to a stainless steel seed germinator. Filter paper (4 sheets of qualitative course paper placed together and cut to a predetermined size) is soaked in the test material, removed, allowed to drain, and then reversely oriented (top becomes the bottom) to minimize any chromato- graphic effect. The seeds are thinly spread on a tray so that they can be either picked up using a vacuum planter or manually transferred to the wetted filter paper. Fifty seeds are placed on the filter paper in three horizontal rows at approx- imately 18, 36, and 54 mm from the top. Seeds must be alternately spaced from one row to the next so that the roots can grow past seeds in the adjacent row. The filter paper and seeds are then placed between two glass plates. Spac- ers and clamps are added to prevent damage to the seeds and to hold the glass in a fixed position. The assembly is then returned to the seed germinator where the bottom of the plates and filter paper are immersed in the test solution to a depth of 1 cm (Figure 1). The germinators (positive control, negative control, sol- vent control, and various test sample dilutions) are then placed in an incubator and kept at 25°C, in total darkness, for 96 hours (germination time for tomato). After 96 hours, the number of germi- nated seeds is counted. For purposes of this test, the primary root (radicle) must be at least 3 mm long in order for the seed to be counted as having germinated. The germination results are then com- piled for groups of seeds that were exposed to the different sample dilutions and to the various control samples. The same plant species must be used during the early seedling growth phase as was used for the seed germination phase. Seedling growth is presented as an increase in plant biomass (dry weight] and is based on the difference between seedling dry weight at time of initial exposure (initial negative control) and ------- seedling dry weight at the completion of exposure 168 hours later. Plants must reach a predetermined stage of development before the actual test begins. Approximately 70 tomato seeds are added per germinator and are allowed to germinate in full strength Hoagland's solution. With the appearance of the first true leaf (7 to 10 days for tomato plants), the number of seedlings is reduced to 50 plants per germinator. There will usually be seven germinators (Figure 2). The various groups of seedlings then receive respective dilutions of the sample material, the positive control sample, or, if required, the solvent control sample. One group of 50 seedlings (initial neg- ative control) is harvested at the start of the test, dried, and weighed. The remain- ing groups are kept in temperature, light, and humidity regulated growth chambers for 168 hours. A simulated day/night cycle of 16 hours light, 24°C, 50 to 70 percent humidity x 8 hours dark, 21 °C, 70 to 80 percent humidity is maintained throughout the test. Fluorescent and incandescent lights are used to maintain proper lighting. After 168 hours, all plant groups are harvested. The initial negative control dry weight is used as an approximation of plant biomass at the beginning of exposure (for all seedling groups). The weight differ- ence between initial and final negative controls represents the increase in plant biomass that would occur in the absence of either the sample material, the positive control, or, if applicable, the solvent control. Sample Material The phytotoxicity test was conducted several times using different chemicals before a compound was selected for the single laboratory evaluation. These preliminary determinations not only allowed for the selection of a suitable test compound, but they also allowed the laboratory personnel to become more familiar with the "consensus" protocol instructions. Many compounds were available that would inhibit both the germination and the early growth of tomato plants, but not all of these chemicals were available as reference materials in suitable quantities for single laboratory testing. A reference material is not required for the ruggedness evaluation but, if available, should be used for determinations of precision, method sensitivity, and limits of reliable 1 7/8" 2 7/2" 183 /a" fl »„ ill .« •J- f L_ pa Filter Glass 4" cer , oajn»;^'''A4 - - - 1 1 i 1 J :^ (teflon) f\' 7 313/16"l Basic Test Container 181/4" 13/4" Figure 1. Seed germination unit for phytotoxicity test. Figure 2. Basic Test Container Early seed/ing growth unit for phytotoxicity test. measurement. Reference samples should definitely be used for determina- tions of accuracy and systematic error. An insecticide, sodium pentachloro- phenate, was eventually selected. Ampules of the compound, prepared as a reference material, were obtained from the Environmental Monitoring and Sup- port Laboratory, Cincinnati, Ohio(EMSL- Cincmnati). The reference samples were subsequently diluted to the required concentration. Even though the samples were diluted, the material was eventually exhausted and, unfortunately, this par- ticular reference compound was not being prepared on a continuing basis. Concluding portions of the phytotoxicity evaluation were conducted using analyt- ical grade sodium pentachlorophenate dissolved in deionized water. Single Laboratory Evaluation Prior to beginning the single laboratory test, the method protocol was peer reviewed as a step toward achieving a "consensus" procedure. Throughout the single laboratory effort, the method procedure and method requirements ------- (experimental conditions, reagents, laboratory equipment, testing sequence, controls, etc } were strictly followed as they were written in the protocol. It was therefore of considerable importance that these written instructions be tech- nically correct, complete, and as unam- biguous as possible The initial phase of the single labor- atory test was to identify procedural variables that must be carefully con- trolled If a procedure is "rugged" it will not be susceptible to the inevitable, modest departures in routine and the final test result will not be altered by these minor variations When a final test result is altered by small procedural variations, the method protocol must be revised to emphasize that a specific step must be followed or, in some instances, to provide an associated step for quality control A single concentration of sample material was used, and seven variables were selected for testing. The protocol variations were studied simultaneously thus requiring only eight separate anal- yses Basically, the difference between the "protocol directed" result and the "protocol altered" result were compared. When the ruggedness test was com- pleted, the remaining phases of the single laboratory evaluation (precision, method sensitivity, etc.) were conducted using the revised method procedure Ten separate determinations were conducted for each test phase to deter- mine the method's capability for preci- sion (using 50/yg/g and 5/vg/g of sodium pentachlorophenate respectively for seed germination and early seedling growth) Each determination represented a valid test response as required by the method protocol. The determinations were conducted sequentially using ahquots of the respective sample, i.e., a valid test result was obtained from one determination before proceeding to the next until all 10 of the respective assays had been completed. The precision estimate was then expressed as a coefficient of variation. To determine the method's single laboratory capability for accuracy (and for systematic error), the testing laboratory would need both a standard reference material and a known method response (true response)to this reference material. The Student t-test would then be used to determine the significance of the difference between the observed single laboratory test result and the known true value. Similarly, to detect a consistently 4 present systematic error, a series of reference material dilutions would be used. However, there was no known test response for the phytotoxicity procedure and, consequently, the current evalua- tion could not determine the method's capability for accuracy. To provide a known test response for future efforts, an average response was established based on 20 separate determinations, i.e., 20 runs for seed germination using 50pg/g sodium pentachlorophenate and 20 runs for early seedling growth using 5 /jg/g sodium pentachlorophenate. This effort required only 10 additional deter- minations for each phytotoxicity test phase since the respective 10 responses from the precision effort could be used as part of the data needed to obtain this average response to a reference sample. For purposes of the single laboratory test, the method's sensitivity was defined as the method's capability to detect (or distinguish between) small changes in sample concentration, i e., concentration of analyte. Sodium pentachlorophenate concentrations were selected that were greater than, and less than, the concen- tration used during the method precision determination. If the procedure distin- guished between samples at the origi- nally selected concentration interval, the concentration interval was reduced by approximately one-half and the addi- tional samples were tested. Similarly, if the procedure could not distinguish between samples at the initial interval, the concentration interval was increased. Ten sequential assays were conducted using each of the selected concentrations Under routine operating conditions, the assay would only be conducted one time per sample and, consequently, when tabulating data for sensitivity, non-overlapping standard deviations (rather than non-overlapping standard errors) were used to indicate whether or not the method could distin- guish between the different samples. To determine the method's limits of reliable measurement, an attempt was made to verify that the method capabil- ities for sensitivity and precision did not deteriorate at the upper and lower extremes of the detection range No attempt was made to establish an upper and lower detection limit. Sodium pen- tachlorophenate concentrations were selected at what was thought to be an approximate upper and lower limit of detection. Ten analyses were conducted using each concentration to provide precision data (expressed as a coefficient of variation). Two additional concentra- tions were then selected, one at each extreme of the estimated response range in order to assess method sensitivity, i.e.,, a total of two sample concentrations were now available at each extreme of the response range. As before, ten determinations were made using the newly selected concentrations, and the method's ability to distinguish between the respective concentrations was pre- sented as non-overlapping standard deviations. Results and Discussion Ruggedness Table 1 provides a summary of the seven variable ruggedness approach which was used during this evaluation. An individual varied condition was either slightly above or slightly below a "pro- tocol directed" condition. As noted in the table, the "protocol directed" conditions were designated as A through G, and the varied conditions were designated as a through g. The evaluation was concerned with identifying variations in the test result due to the specific procedural differences, i e., A-a, B-b, C-c, D-d, E-e, F-f, and G-g. Each of the eight determi- nations consisted of a single assay conducted using eight respective ali- quots of a single test material. The assay results are indicated as s, t, u, v, w, x, y, and z. The average ofA = (s + t+u+ v)/4 compared with the average of a = (w + x + y + z)/4 served as a means of Table 1. Experimental Design fora Seven Variable Ruggedness Test* Determination Assay Number Variables Result 1 2 3 4 5 6 7 8 A B C D E F G A B c D e f g A b C d E f g A b c d e F G a B C d e F g a B c d E f G a b C D e f G a b c D E F g s t u V w X Y z *Based on W. J. Youden, 1969. The Collaborative Test, p. 151-158. In Precision Measurement and Calibration. H. H Ku, Editor, U.S Department of Commerce, National Bureau of Standards, 436 pp ------- assessing the effect of changing variable A to a. Since each of the two groups of four determinations contained the other six variables, twice at the upper case level and twice at the lower case level, the effect of these variables (if present) tended to cancel out leaving only the effect of changing variable A to a. The relative effect of the other variables was also estimated by examining the follow- ing averages: B _ (s + t + w + x) b_ (u + v + y + z) 4 4 „ _ (s + u + w + y) _ (t + v + x + z) \j ~~ ~ C — 4 4 n = (s + t + y + z) . _ (u + v + w + x) 4 4 E = (s + u + x + z) _ (t + v + w + y) F_ (s + v + w+z) f _ (t + u + x + y) 4 4 „ _ (s + v + x + y) _ (t + u + w + z) 44 After tabulating the above averages, the differences between each respective variable was computed, e.g., . =_ (s + t+u + v) . (w + x + y + z) r\ — 3 — * ~ 4 4 If one or two variables were having an effect on the test result, their individual differences (directed vs. altered) would be substantially larger than the group of differences associated with the other variables. Most of the modest procedural alterations should have little effect on the test result since, with few exceptions, the variations were only of a magnitude that might have been made by a qualified laboratory following the written method protocol Since the phytotoxicity test was com- posed of a seed germination phase and an early seedling growth phase and si nee both phases contained a preliminary and definitive segment, the ruggedness evaluation was conducted four separate times in order to provide a thorough evaluation for each test component. Seven protocol variables were selected for each respective component, i e , seed germination preliminary test, seed germination definitive test, early see- dling growth preliminary test, and early seedling growth definitive test. However, since the preliminary and definitive tests were conducted separately for each phase, the overall effect of a single variable was more difficult to assess (i e , effect on final test result). The ruggedness test indicated several critical steps in the protocol that must be conducted exactly as directed. In the case of seed germination, a consistent seed size and a consistent number of seeds were critical for all treatment and control groups. The specified amount of solvent (0 01 percent of volume) was also critical as was maintaining a consistent exposure time for the sample dilution groups (96 hours). For the early seedling growth phase, it was critical that all harvested plant material be dried for a consistent amount of time (at the stated temperature) and that four sample dilutions be used for the definitive test As was noted for seed germination, it was essential that the protocol directed exposure time for treatment and control groups be carefully maintained and that no more than the protocol directed amount of solvent be added to the test sample. Based on the current rugged- ness evaluation, the phytotoxicfty test was not considered to be a particularly rugged technique and, for several of the instructions, a minor procedural altera- tion will definitely affect the test result The phytotoxicity protocol was conse- quently revised to emphasize that, for the above mentioned steps, no procedural latitude is allowed and the instructions must be strictly followed. Precision Table 2 provides the results for pre- cision, sensitivity, and limits of reliable measurement. Based on 10 separate determinations using 50 /ug/g of sodium pentachlorophenate, the seed germina- tion phase's single laboratory capability for precision (expressed as a coefficient of variation) was 27 percent Based on 10 separate determinations using 5 /ug/ g of sodium pentachlorophenate, the early seedling growth capability for precision was 23 percent For both test phases, there was some indication that the precision was actually better than these values suggest In the case of seed germination, the 50 fjg/g concentration was subsequently shown to be approach- ing the procedure's lower limit of reliable measurement Determinations made using 100 /ug/g samples resulted in an improved method precision of 16 per- rent The 5 /ug/g concentration used to determine precision for early seedling growth was definitely at or below the method's limit of reliable measurement Subsequent determinations using 1 /jg/ g concentrations revealed a dramatic deterioration in precision to 1 77 percent In retrospect, the 5 /jg/g concentration was obviously a poor selection, and the precision assessment would probably have been more valid using a greater concentration of sodium pentachloro- phenate. Accuracy Since a known test response was not available to assess the method's single laboratory capability for accuracy and for systematic error, the current evaluation established a known response based on 20 respective determinations for seed germination and for early seedling growth When using 50 /ug/g sodium pentachlorophenate samples, the seed germination EC5o response was 65 ± 15 and, when using 5 /ug/g samples, the early seedling growth EC50 response was 35 ± 103. Since the sodium pentach- lorophenate was not an actual reference material, these results are not as bene- ficial as had been originally intended. However, the documented response to analytical grade sample material will provide some basis for comparison when other laboratories are conducting this procedure. Method Sensitivity Method sensitivity was assessed by non-overlapping standard deviations as opposed to non-overlapping standard errors (Table 2). Concentration intervals used to evaluate the seed germination phase were approximate differences of 10, 20, 40, 50, and 1 50 /ug/g Since the 12 5 /ug/g concentration was obviously below the limit of reliable measurement and since the 42 fjg/g concentration was also close to the lower limit, there were not as many specific concentrations for comparison as had been originally intended The seed germination phase effectively distinguished between 200 fjg/g and 100 /ug/g (interval of 100 /jg/ g), between 200 /ug/g and 83 7 /ug/g (interval of 11 6 /ug/g), and between 83 7 /ug/g and 42 /ug/g (interval of 42 /ug/g) However, there was slight overlap between 100 /ug/g and 50 /ug/g which suggests that a 42 /ug/g interval could not be consistently achieved The seed germination procedure's capability for sensitivity is therefore presented as 50 /ug/g (using sodium pentachlorophenate as sample material) The early seedling growth evaluation did not include enough concentrations to 5 ------- Table 2. Phytotoxicity Test Results Using Concentrations of Sodium Pentachlorophenate in Distilled Water (Values Given are Final Test Results for Either Seed Germination or for Early Seedling Growth) Seed Germination Phase Sequential Assay Number 1 2 3 4 5 6 7 8 9 10 n mean (ECsd S.D. C. V. (%) S.E. mean ± S.E. mean ± S.D. 12.5 V9/9* nontoxic nontoxic nontoxic nontoxic nontoxic nontoxic nontoxic nontoxic nontoxic nontoxic 10 — — — — — — 42.0 tJ9/9 79 170 97 88 95 90 75 97 84 81 10 96 27 28 8.5 105-88 123-69 50.0 V9/g 60 58 91 105 60 53 78 57 b b 8 70 19 27 6.7 77-63 89-51 66.5 P9/9 58 60 71 72 65 64 55 66 71 75 10 66 7 11 2.2 68-64 73-59 83.7 V9/9 34 34 47 49 41 58 46 43 59 56 10 47 9 19 2.8 50-44 56-38 100.0 V9/9 46 40 50 37 33 54 52 47 50 38 10 45 7 16 2.2 47-43 52-38 200.0 ug/9 32 30 22 33 27 22 24 38 32 30 10 29 5 18 1.6 31-27 34-24 10.3 V9/9 27 16 53 28 40 12 13 39 10 20 10 26 14 55 4.4 30-22 40-12 Early Seedling Growth Phase 5.0 fjg/9 48 43 33 33 51 38 33 35 21 41 10 38 9 23 2.8 41-35 47-29 1.0 V9/9* nontoxic nontoxic 80 nontoxic nontoxic 94 nontoxic nontoxic c c 8 — — — — — — S.D. = standard deviation. S.E. = standard error. C. V. = coefficient of variation. = Calculated sample dilution that will cause either a 50 percent reduction in seed germination or a 50 percent reduction in seedling growth. dilution estimates greater than 100 percent indicate a nontoxic response. A comparison of specific no effect values would be meaningless. "Test response not considered valid based on agreement criteria for positive control results. ^Determinations 9 and 10 not conducted. make an effective sensitivity determina- tion. The 1 ug/g concentration noted in Table 2 was below the limit of reliable measurement, and the early seedling growth procedure was not capable of distinguishing between concentrations of 10.3 fjg/g and 5.0 ug/g (i.e., essen- tially a 5 fjg/g interval). The only state- ment that can be made about the sen- sitivity for early seedling growth is that it is greater than 5 /ug/g. Limits of Reliable Measurement The limits of reliable measurement are typically determined through an assess- ment of accuracy, precision, and sensi- tivity at the upper and lower limits of the detection range. As stated previously, the method's capability for accuracy could not be determined and, therefore, the limits of reliable measurement were based on an assessment of precision and sensitivity. For both phases of the phytotoxicity test, the concentrations ranged from 200 fjg/g to 1 ug/g {Table 2). These concentration selections proved to be adequate for an assessment of the lower limit, but they clearly did not allowfor a determination of the upper limit. For seed germination, the lower limit was probably 66.5 /ug/g because of the noted deterioration in the coefficient of variation between test results for 66.5 ug/g and for 50.0 ug/g (i.e., 11 percent as opposed to 27 percent). In terms of sensitivity, a concentration of 66.5 ug/ g could not be distinguished from less concentrated samples of 50 ug/g and 42 ug/g (i.e., respective concentration intervals of approximately 17 and 25 ug/ g). However, a sample concentration of 66.5 ug/g could be distinguished from samples of 83.7 ug/g (i.e., interval of approximately 17 ug/g). The 12.5 ug/g concentration was definitely below the method's lower limit. Limits of reliable measurement for the seed germination phase have consequently been pre- sented as 66.5 ug/g to above 200 ug/ g (for samples of sodium pentachloro- phenate). In the case of early seedling growth, there was even less information available. The 1 ug/g concentration was clearly below the limits of reliable measurement, and the procedure did not distinguish between concentrations of ------- 10.3 /jg/g and 5.0 //g/g. Consequently, the only statement that can be made for early seedling growth is that the lower limit is at or above 10.3 jt/g/g and that the upper limit has not been determined. Conclusions As a result of this single laboratory evaluation, the phytotoxicity procedure is much closer to a collaborative testing stage than it was previously, especially if the tomato plant continues to be used as the test species. It is essential that a reference material (preferably sodium pentachlorophenate) be available for use by the collaborating laboratories and that the known test response, determined during the current effort, be verified using the reference samples. It will also be necessary to acquire a better estimate for the procedure's limits of reliable measurement before a collaborative study is conducted. All assay data from the current single laboratory evaluation and a complete copy of the phytotoxicity method protocol are included in the project report. W. R. Lower and A. F. Yanders are with the University of Missouri. Columbia, MO 65203. W. W. Sutton is the EPA Project Officer (see below/. The complete report, entitled "Single Laboratory Evaluation of Phytotoxicity Test." (Order No. PB 87-188 694/AS: Cost: $24.95) will be available only from: National Technical Information Service 5285 Port Royal Road Springfield. VA 22161 Telephone: 703-487-4650 The EPA Project Officer can be contacted at: Environmental Monitoring Systems Laboratory U.S. Environmental Protection Agency ' P.O. Box 15027 Las Vegas. NV89114 ------- United States Center for Environmental Research Environmental Protection Information Agency Cincinnati OH 45268 Official Business Penalty for Private Use $300 EPA/600/S4-87/012 0000329 PS gI| "REET 60604 ------- |