Applications of toxicity curves in assessing the toxicity of diazinon and pentachlorophenol to Lumbricus terrestris in natural soils

Soit Bol. BiochemVol. 29, No. 314, pp. 689-692, 1997

PII: soo3&0717(%)001%-4

0 1997 Elsevier Science Ltd. Al1 rights reserved Printed in Great Britain 0038-0717/97 $17.00 + 0.00

APPLICATIONS OF TOXICITY CURVES IN ASSESSING THE TOXICITY OF DIAZINON AND PENTACHLOROPHENOL TO LUMBRICUS TERRESZ’RIS IN NATURAL SOILS R. P. LANNO,‘* G. L. STEPHENSON2

and C. D. WREN’

‘Department of Zoology, Oklahoma State University, Stillwater, Oklahoma 74078-3052, U.S.A. and ‘Bcological Services for Planning Ltd, 361 Southgate Drive, Guelph, Ontario NlG 3M5, Canada (Accepted 23 June 1996) Summary-Toxicity tests were conducted with Lumbricus terrestris exposed to diazinon in three different natura1 soil types (Brookston Clay, Fox Sand and Guelph Loam) and to pentachlorophenol (PCP) in Fox Sand. Mortalities were monitored at 2, 4, 8, 16, 24, 48, 96 h, and 5, 7, 14, and 21 d. Toxicity curves (t+,/time) were fitted to a one-compartment first-order kinetics (ICFOK) model using non-linear regression analysis to determine incipient lethal levels (ILLs). The ICFOK model provided a reasonable fit to the observed data for diazinon in al1 soil types. Toxicity was markedly influenced by soil type, with lowest toxicity in Brookston Clay. The ICFOK model provided a reasonable fit to the toxicity curve for PCP in Fox Sand up to 14 d, but not for the entire 21-d test period due to increased mortality between 14 and 21 d. The results of this study suggest that toxicity curves and ILLs can be generated from the standard soil toxicity testing protocol by increasing the number of observations taken during the course of a 14-d lethality test. 0 1997 Elsevier Science Ltd

INTRODUCIION

Standardized procedures for assessing the toxicity of chemicals in soil to earthworms have been developed over the last decade (Edwards, 1983; OECD, 1984; ISO, 1991a,b; Greene et al., 1989). The discipline of aquatic toxicity testing has evolved rapidly over the last 25 years and has experienced many problems and pitfalls in its development.’ Since some soil toxicity tests are stil1 in the process of being standardized, there exists a unique opportunity to benefit from the pitfalls encountered in aquatic toxicity testing so that similar problems are not repeated. As an example, the estimation of LC~~S at a given time is only the first step in assessing the toxicity of chemicals to organisms in a test system (Sprague, 1969) and is not an end in itself. The most important comparison comes when the LC~~S are compared in series to produce a toxicity curve, which in turn is used to estimate a lethality threshold. The lethal threshold concentration or incipient lethal leve1 (ILL) is a fundamental concept in toxicity testing (Sprague, 1969) that is often ignored, even in aquatic toxicity testing. It is defined as that concentration of a contaminant at which half of the exposed population wil1 survive indefinitely. The ILL acts to normalize the toxic response so that al1 comparisons of toxicity within *Author for correspondence. Tel.: 405-744-5551. Fax: 405744-7824.

and between chemicals and species of organisms are made at a steady-state with respect to the dose-response curve. Time and soil concentration of a contaminant are intimately linked in soil toxicity tests and, as such, any LCSO value should be qualified by indicating the exposure duration, e.g. 7-d LC~~. However, if 7-d LC~S are used to compare the toxicity of a chemical between species or soils, erroneous conclusions regarding toxicity may result if lethal threshold concentrations have not been established in al1 treatments by 7 d. The objective of this study was to investigate the application of toxicity curves and ILLs (Sprague, 1969) in assessing the toxicity of soil contaminants to Lumbricus terrestris. Toxicity data from diazinon exposure in three natura1 soils (clay, loam, sandyloam) and pentachlorophenol (PCP) exposure in a sandy-loam soil are used as illustrative examples.

MATERIALSAND METHODR

Adult L. terrestris purchased from commercial suppliers were refrigerated (3 f 2°C) in black loam soil for 2-3 wk prior to testing. Worms were transferred into natura1 test soil 2-3 d prior to the beginning of the test and stored at 8-9°C for 1 d and at 15°C for 1-2 d for acclimation. Natura1 soils were selected to differ in their particle-size distribution and cation-exchange capacity, representing three soil types (Brookston Clay, Agriculture Canada 689

690

R. P. Lanno el al Table I. Chemical characterization

of the natural soils used in Lumbricus rerrrsrris toxicity tests Soil type

Parameter

Guelph Loam

Brookston Clay

Fox Sand

Phosphorus (mg I-‘) Potassium (mg I-‘) Magnesium (mg I-‘) PH Cation exchange Capacity (cmol ’ kg-‘) Soil texture (% by weight) Gravel Very coarse sand Coarse sand Medium sand Fine sand Very fine sand Sand Silt Clay Organic matter (%) Moisture content after sieving (%)

4 136.5 238 7.5 I I .92

29 252.4 323 6.4 34.17

53 16.1 68 7.8 6.32

0 0.5 2.4 1.7 18.4 25.3 54.3 38.2 1.6 2.7

0 0.3 2.4 8.5 15.3 6.9 33.4 36.3 30.3 IO.1

0 0.1 4.3 43.8 33.9 5.6 87.7 8.2 4.1 1.5

12.8

25.2

7. I

Moisture content during tests (%)

21.1

33.5

16.5

Harrow Research Station; Guelph Loam, University of Guelph Arkell Research Station; Fox Sand, Agriculture Canada Delhi Research Station) found in southwestern Ontario. The collection sites had known land-use histories and were considered to be free of pesticide contamination. Soils were sieved through a 2-mm stainless steel mesh and appeared uniform in colour and texture. Organic matter content, fertility, pH, cationexchange capacity, texture and moisture content were determined according to standard methods at the Soils Testing Laboratory, University of Guelph (Table 1). The water-holding capacity of each soil was estimated by adding water in 10 ml increments to sieved soil until pooled water was visible, mixing with a stainless steel spoon after the addition of each aliquot of water. Water was added to approximate 80% of the water-holding capacity of each soil. Test soils (487.5 g dry wt) were weighed into 2-l clear glass jars (OECD, 1984). The toxicant solutions were prepared by serial dilution of a stock PCP preparation (Aldrich Chemical Co., St. Louis, MO) or a commercial diazinon formulation (Basudin SOOEC, 500 g 1-l ai.; Ciba-Geigy Canada Ltd, Cambridge, Ont.) with distilled-deionized water. The PCP stock solution was prepared by dissolving in a NaOH solution. Appropriate volumes of stock solutions were diluted and mixed manually until the soil appeared uniform in colour, texture and moisture content. Chemical concentrations in soil are reported as nominal levels (mg a.i. kg-’ dry wt). Tests comprised seven or eight chemical concentrations on a logarithmic scale and a control, with three replicates per treatment and 10 worms per jar. PCP tests included a solvent control containing the maximum amount of NaOH solution used in dispensing PCP to the soil. Test duration was 21 d and mortalities were monitored, when

possible, at 2, 4, 8, 16, 24, 48 and 96 h, 5, 7, 14 and 21 d. Worms were presumed dead when no contractile response was observed when prodded lightly with a blunt probe. Nominal initial soil concentrations were used to determine LC~~S from mortality data by trimmed Spearman-Karber analysis (Hamilton et al.. 1977) for each time where > 50% mortality occurred in at least one concentration. ILLS were estimated by fitting a one-compartment, first-order kinetics (ICFOK) model to the Lcso/time data using nonlinear regression analysis. ILLS for diazinon were analysed by ANOVA, followed by Tukey’s Honestly Significant Difference test for a posteriori comparison of means (u = 0.05).

RESULTS

The equations estimated by non-linear curve fitting of the 1CFOK model to the toxicity curve provided a reasonable fit to the observed data for diazinon in all soil types tested (Fig. 1; R2 range = 0.853-0.931). ILLS for diazinon decreased significantly in order: Brookston Clay, Guelph Loam, Fox Sand (Table 2). Half-lives for toxicity estimated from the model for diazinon in Brookston Clay, Guelph Loam, and Fox Sand were 143, 33 and 7.3 h, respectively. The 1CFOK model provided a reasonable fit to the toxicity curve for PCP in Fox Sand up to 14d (Fig. 2; R* = 0.932). Between 14 and 21 d, a marked inflection point was observed in the toxicity curve. If the model was applied to the data up to 21 d, no convergence of the iterations was observed, and no ILL could be estimated. Upon closer observation of the data, this inflection point in the toxicity curve was due to almost complete mortality of worms exposed to the lowest concentration of PCP

Toxicity curves in earthworm toxicity tests

691

Tlme (hours) Tlme (hours)

Fig. 1. Toxicity curves generated using Lc,/time data for L. terrestrisexposed to diazinon in Brookston Clay (+), Guelph Loam (+), and Fox Sand (U). Symbols are observed values representing means of triplicate tests. Solid lines indicate the theoretical curve generated by the 1CFOK model fitted to the data. Numerical values associated with each curve are the ILL values estimated from the model (expres& as mg diazinon kg-‘).

used in the tests between days 14 and 21. NO mortality was evident in either of the control groups.

DISCUSSION

Diazinon was about lO-fold more toxic to L. terFox Sand (ILL, 27 mg kg-‘) than in Brookston Clay (ILL, 262 mg kg-‘), while toxicity in Guelph Loam was intermediate (ILL, 63 mg kg-‘) (Table 2). The three different soil types used in this study significantly influenced the toxicity of diazinon to L. terrestris. The toxicity of a chemical in soil is modified by the interaction between the physical and chemical properties of the compound (e.g. octanol/water partition coefficient, solubility) with those of the different soil types (e.g. pH, particle size and organic matter content) and test organisms (e.g. size and metabolism). Reduced toxicity of diazinon in Brookston Clay was likely a function of the high organic matter and clay content relative to the other soils. The toxicity of diazion was lower in Guelph Loam relative to Fox Sand, possibly due to the greater fraction of silt and clay, since the organic matter content of the two soils was very similar. van Gestel and Ma (1988) also found the toxicity of PCP to Eiseniu andrei and L. rubellus was lowest in a soil with a restris in

higher

organic

matter

and clay content

when com-

Fig. 2. Toxicity curve generated using Lcse/time data for 15. terrestrisexposed to PCP in Fox Sand. Symbols are observed values representing means from triplicate tests; the dotted line indicates the theoretical 1CFOK model fitted to the data up to 336 h. The ILL estimated from the 1CFOK is expres& in mg PCP kg-‘. Numerical value associated with the dotted curve is the ILL value estimated from the model (expressed as mg PCP kg-‘). Note inflection point in the curve at 336 h.

pared with two sandy soils with less clay and organic matter. In both these experiments, organic matter content and the fraction of clay and silt particles were highest in the least toxic soils, making it difficult to distinguish which ameliorative factor(s) reduced toxicity. When comparisons of acute lethality data are made, the values that are often compared are LQ~S at a given time, with 14 d as the standard for earthworm tests (Greene et al., 1989). The basis of this comparison is that the LC~~ values are a good approximation of the time-independent lethality threshold or incipient lethal level. The OECD (1984) protocol for earthworm toxicity tests suggests that ~~50s be calculated for 7 and 14 d, but no reason is given for these times. This sampling regime is often sufficient to allow an ILL to be established, but due to differences in toxicant kinetics, the time to reach an ILL wil1 vary with the chemical, the test species, and the substrate being tested. More hydrophobic compounds, such as PCBs or dioxins, may take longer than 14 d to reach an ILL. In al1 three soils, the 7- and 14-d LQ~S for diazinon compared favourably with their respective ILL estimates, suggesting that an ILL was reached within 14 d. An ILL was estimated for PCP in Fox Sand at 14 d, but then ~~50 values decreased dramatically by 21 d (Table 2). This

Table 2. Comparison of 7-, 14- and 21-d I_C~Swith incipient lethal levels (ILL) (mg kg-’ dry wt) estimated from non-linear curve tìts for diazinon in three types of soil and PCP in Fox Sand. Values represent means from triplicate tests. Standard error is given in parentheses. ILL values in rows with different superscripts are significantly different (Tukeys’ Honestly Significant Differente, a = 0.05) Chemical 7-d Diazinon Pentachlorophenol

Brookston Clay 14-d 21-d 233 (3.6)

233 (3.6)

ILL

7-d

Fox Sand 21-d 14-d

262a (6.3)

ILL

7-d

b

(Yl < 10 )

113 (4.3)

< 10

(lY1)

Guelph Loam 21-d 14-d 59 (7.7)

ILL

692

R. P. Lanno e/ UI.

would suggest that 7- and 14-d ~csns are not good estimates of the toxicity of PCP to L. terrestris in Fox Sand and that the test should be continued for a greater duration than 21 d, conducted with a range of chemical concentrations which include lower levels, and/or repeated to duplicate the results. Karnak and Hamelink (1982) exposed L. terrestris to Benomyl and estimated 7- and 14-d LC~~Sof 1.70 to 0.42 mg kg-‘, respectively. The significant decrease in ~~50 values between 7 and 14 d suggests that a lethality threshold may not have been established by 14 d and the test duration should be extended until an ILL was established. An inflection point in an established toxicity curve is indicative of a secondary mode of toxic action (Sprague, 1969). Since PCP interferes with oxidative phosphorylation and no secondary mode of toxic action is known, this may be indicative of some other factor contributing to mortality in the test protocol. One potential problem is that Fox Sand is an unsuitable test medium for L. terrestris beyond 14d due to physical and chemical parameters such as particle size or pH. Additionally, the OECD (1984) test method was designed for use with E. ferida and not L. terrestris. These worms differ in size by an order of magnitude, and the Fox Sand may have been unable to meet the energetic requirements of L. terrestris beyond 14 d at the test temperature of 15°C. In this context, for the adaptation of existing testing protocols to other species, toxicity curves could prove an invaluable tool. The toxic response of L. terrestris to diazinon in Fox Sand was different than in the other two soils as judged by the shape of the toxicity curve. Although an ILL could be estimated with the ICFOK model, no distinct ILL was reached by 14 d, as in tests conducted in Brookston Clay and Guelph Loam (Fig. 1). In order to estimate the ILL by applying various models, a minimum number of observations are necessary, with two often not being enough to define the curve. One way of improving the accuracy of the toxicity estimates in soil toxicity tests is to increase the number of observations. Observation times should be distributed so that more frequent observations are made at the beginning of the test, as typified by the logarithmic sequences suggested by Sprague (1973). An example of an improved observation regime for acute lethality tests with earthworms would be as follows: 0.5, 1, 2, 4, 8, 14 d. The extra observation times during the test would contribute greatly to understanding the kinetics of soil contaminant uptake by earthworms. The results of this study suggested that toxicity curves and ILLs can be generated from the standard soil toxicity testing protocol by increasing the

number of observations taken during the course of a 14-d lethality test. The 14-d exposure regime suggested for earthworm lethality tests appears to be of sufficient duration to achieve a lethality threshold with most of the chemicals of concern that have been tested. However, caution should be taken when tests are conducted with extremely hydrophobic chemicals (PCBs, dioxins and some pesticides), as a 14-d exposure may not be sufficient to achieve a lethality threshold.

Acknowledgements--Partia financial support for this project was provided by a Natura1 Scienc& and Engineering Research Council Industrial Research Fellowshin to R. P. Lanno. The authors gratefully acknowledge the support of Dr Erven MacIntosh, Ecological Services for Planning Ltd. The technical support provided by Mr Ian Feir was invaluable.

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