Acute toxicity of a synthetic oil, aniline and phenol to laboratory and natural populations of chironomid (diptera) larvae

Environmental Pollution (Series A) 34 (1984) 321-331

Acute Toxicity of a Synthetic Oil, Aniline and Phenol to Laboratory and Natural Populations of Chironomid (Diptera) Larvae P. J. F r a n c o , K. L. Daniels,* R. M. C u s h m a n & G. A. K a z l o w t Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830, USA

ABSTRACT The acute toxicity o['a water-solublej?action of a coal-derived oil and two oj its components, aniline andphenol, to larvae of[bur chironomidspecies was determined in 48-h bioassays. Three species, Clinotanypus pinguis, Einfeldia natchitocheae and Tanypus neopunctipennis, were collected from natural populations and the fourth, Chironomus tentans, was reared in laboratory culture. Bioassay data were examined using weighted least squares and probit analyses. The LCso values calculated .[rom regression equations jbr the water-soluble fraction ranged from 1"1 °/ojbr E. natchitocheae to 2.0 % Jbr C. pinguis," Jbr aniline, 287 mg litre- I jor T. neopunctipennis to 442 mg litre- ~for E. natchitocheae; andJor phenol, 73 mg litre-l jbr T. neopunctipennis to 187 mg litre-1 jbr C. tentans. Comparisons from sensitivity analyses revealed that the closer the taxonomic relationship between species, the more similar the response to a given toxicant. The data suggest that the laboratory population may be representative oJ" natural midge populations in bioassays.

INTRODUCTION Acute bioassays using laboratory reared organisms are useful as screening tools in toxicological studies because they are rapid and * Present address: Industrial Safety and Applied Health Physics Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830, USA.

t Present address: Emory University, Atlanta, Georgia 30322, USA. 321 Environ. Pollut. Ser. A. 0143-1471/84/$03.00 © Elsevier Applied Science Publishers Ltd, England, 1984. Printed in Great Britain

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P. J. Franco, K. L. Daniels, R. M. Cushman, G. A. Kazlow

inexpensive and because test populations are relatively uniform. However, because species may vary in their tolerance of pollutants (Resh & Unzicker, 1975), bioassay results for one species may not be applicable to another. Predicting the response of natural populations is ditficult because laboratory culture conditions often differ greatly from nature. Concerns about the selection of tolerant or adaptable species as test organisms (USEPA, 1980) and possible genetic changes induced by culturing (Salt, 1969) also limit the extrapolation of most bioassay data to natural populations. In many studies only the concentration of a toxicant that produces a response in a given proportion of the population, e.g. the',concentration lethal to 50 ~ of the test organisms (LCso), is reported. Examination of such values may be inadequate for comparing the responses of two species since they may have similar LCso values yet differ in their overall response (Loomis, 1978). Recently, however, Daniels et al. (1982) described a weighted least squares method useful in comparing the total responses of several species to toxicants. In this study we compare the responses of larvae of three species of chironomid midges, Clinotanypus pinguis, Einfeldia natchitocheae and Tanypus neopunctipennis, from outdoor ponds, and one species, Chironomus tentans, from laboratory culture, to a water-soluble fraction (WSF) of a coal-derived oil and two of its major chemical components, aniline and phenol. Coal liquids are of interest because (1) they are chemically different from the petroleum products they are intended to replace (Guerin et al., 1981); (2) commercial production of synthetic fuels could result in accidental releases into aquatic waterways (Leggett et al., in press) and (3) water-soluble components of coal oils are more toxic to aquatic organisms than those of petroleum (Giddings et al., 1980; States et al., 1981; Giddings & Washington, 1981; Gray et al., 1982; Ullrich & Millemann, 1983). Our objectives were to compare critically the overall responses of the four species using regression analysis, to determine how representative a laboratory culture is of natural midge populations and to expand the existing toxicity data base for synthetic fuels.

MATERIALS AND METHODS A culture of C. tentans has been maintained for 3 years in aerated 35-1itre aquaria containing 10cm of water and shredded paper towels for a

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burrowing substrate (Cushman & McKamey, 1981). Each aquarium receives 20 ml of a ground dry dog food and water slurry weekly with a monthly supplement of 4g of ground Dog Kisses (Hartz Mountain Corp.). The culture is kept at 20 to 25 °C with 12 h light per day. For collection, the aquaria contents were stirred and 4th-instar larvae were lifted from the water surface with a blunt curved dissection needle. Larvae of C. pinguis, E. natchitocheae and T. neopunctipennis were collected from ponds on the US Department of Energy Reservation at Oak Ridge National Laboratory. Two-litre bottom samples were taken with an Ekman sampler, and larvae were separated by gently sieving the sediment through a 500-/~m wire mesh. Larger individuals (3rd- and 4thinstars) were used in the bioassays. A coal-derived fuel oil blend was obtained from Hydrocarbon Research Inc., Lawrenceville, New Jersey. This oil, designated PDU-9, was produced at a process development unit and is not necessarily representative of future commercial synthetic fuels. A water-soluble fraction (WSF) of the oil was prepared by layering 100 ml oil over 800 ml filtered well water in a darkened, sealed 4-1itre bottle. The well water (pH = 7.8, alkalinity = 150mg litre-1 as CaCO3) was filtered through 0.45-pm Millipore HA membranes. The solution was slowly stirred for 16 h at 20 °C. The aqueous phase was separated from the residual oil and passed through glass-fibre filters to remove suspended oil droplets. Test concentrations were made by dilution of the WSF with filtered well water. Solutions of aniline and phenol were prepared by the addition of known amounts of reagent-grade chemicals to filtered well water. The bioassay used was similar to that of Cushman & McKamey (1981). Four to seven larvae were put into 125-ml bottles containing 0.25 g glass wool and 80 ml filtered well water. They were acclimated to test conditions of 17 ___0.5 °C and 12 h light (gold fluorescent lamps to prevent chemical photolysis) per day for 24 h before exposure to test solutions. At the end of acclimation, any dead or pupated individuals were replaced with other acclimated larvae. The well water was decanted from the bottles and test solutions were added. For each toxicant, at least four concentrations were tested plus a well water control, with four replicate bottles per concentration. The bottles were capped and, 48 h later, larval mortality, defined as failure to respond to mechanical probing, was determined. LCso values and 9 5 ~ fiducial limits were calculated using the Statistical Analysis System (SAS) (1982a) probit analysis. Data were also examined using the SAS (1982b) general linear models (GLM) procedure

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P. J. Franco, K. L. Daniels, R. M. Cushman, G. A. Kazlow

for conducting a least squares analysis with a covariable (Daniels et al., 1982). Mortalities at each concentration were corrected for pupation and control deaths ( < 10 ~ in all tests) according to Abbott (1925). Per cent response was transformed to the probit function (Finney, 1978) and concentrations were logarithmically transformed. For concentrations that produced either 0 ~o or 100 ~o response (where the probit function is undefined), the responses were estimated using Berkson's (1953) formula. Because the error variance of the transformed response data was not constant, the model was fitted by assigning weights to parameter estimates that were inversely proportional to their variances (Neter & Wasserman, 1974). Thus, responses near 0 ~ or 100 ~o contributed less to the model than those about the response mean. LCso values were also calculated from the resulting regression equations. The comparative method requires that each concentration-response relationship is linear and the lines are parallel. The G L M procedure was used to test for linear relationships between toxicant concentration and response for each species and for equality of slopes and intercepts among species for each toxicant at the 0.95 confidence level (Daniels et al., 1982). Where significant differences were found among either slopes or intercepts, multiple comparisons were made between species or species pairs to identify the sources of difference. The comparisons were based on Student's t-distribution with Bonferroni's adjustment (Chew, 1977) to guarantee at least a 0.95 level of confidence. The t-statistic numerator was the difference between estimates of slope or intercept, and the denominator was the pooled standard deviation about the estimates. If slopes for two or more species were not significantly different, a comparison of sensitivity was made by estimating a c o m m o n slope and calculating intercepts for each species based on the c o m m o n slope. The relative sensitivity among species was calculated from the horizontal distance between regression lines (Hewlett & Plackett, 1979).

RESULTS The concentration response relationships for the midge species and three toxicants are shown in Figs 1 to 3, and the LCso values estimated by both the probit and least squares analyses are given in Table 1. Generally, there was close agreement between the mean response concentrations predicted by the two methods. For C. pinguis and aniline and E. natchitocheae and

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Fig. 1. Response (probit of per cent mortality) of larvae of four chironomid species to a water-soluble fraction of a coal-derived oil. Regression lines are from weighted least squares analysis.

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Fig. 2.

Response of larvae of three chironomid species to aniline, as in Fig. I.

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Fig. 3. Response of larvae of three chironomid species to phenol, as in Fig. I.

phenol, the least squares analysis did not yield LCso estimates because the regression relationships were non-linear. These bioassay data also poorly fit the probit model, as seen from the 95 ~o fiducial limits. All slopes of species response for the W S F (Table 2) were significantly different from zero but were not different from one another (F3,14=2"28). The test for equality of regression lines showed a significant difference a m o n g the intercepts of the four species (Fa, 7 = 4.37). The multiple comparisons failed to indicate a difference between the intercepts of C. tentans and E. natchitocheae (subfamily Chironominae) or between C. pinguis and T. neopunctipennis (subfamily Tanypodinae); however, a difference was found between the mean intercepts of the species pairs. Based on the sensitivity analysis, the WSF was approximately I-8 times more toxic to the Chironominae than to the Tanypodinae. Because there was not a linear c o n c e n t r a t i o n - r e s p o n s e relationship for C. pinguis and aniline, these data could not be included in additional comparisons. The slopes for aniline and the remaining species were equal (F2,12 =2.70). The intercepts were different (F2, 7 = 13.8), but paired

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TABLE 1 The M e a n Response C o n c e n t r a t i o n s (LC~0) Estimated by the General Linear Models ( G L M ) and Probit Procedures

Toxicant

Species

GL M LCso

Probit 95 °/o Fiducial limits LCso

C. E. C. T. C. E. C. T. C. E. C. T.

WSF ('!Jo)

Aniline (mg litre- 1)

Phenol ( m g l i t r e

1)

tentans natchitocheae pinguis neopunctipennis tentans natchitocheae pinguis neopunctipennis tentans natehitocheae pinguis neopunctipennis

1.30 I. 12 1-98 1.87 412.2 442.5

a 287'2 187.2

a 80"5 72.7

1.24 1-09 1.98 1.84 399"9 427.9 477.9 272.1 187.1 69.8 80.5 70.0

Lower 1.19 0.91 1.76 1-69 363.3 377.3 397.5 230"9 172.2 b 62.0 44.8

Upper 1.44 1.34 2.47 1-98 441-5 517.7 1 100"0 309"9 216"9 b 96"6 93.2

Regression non-linear; LCso not determined by the G L M procedure. b P o o r fit o f data to p r o b i t model precludes estimation o f fiducial limits.

TABLE 2 Regression Coefficients from a Weighted Least Squares Analysis of 48-h Bioassay D a t a for Larvae of F o u r Midge Species

Toxicant WSF

Aniline

Phenol

Species

Slope

Intercept

r2

C. tentans E. natchitocheae C. pinguis T. neopunctipennis C. tentans E. natchitocheae C. pinguis T. neopunctipennis C. tentans E. natchitocheae C. pinguis T. neopunctipennis

5.71 4-15 10-68 10.89 6.86 5' 14 5"23 8.38 7"31 6"41 8"06 3.54

-0"65 - 0"21 - 3.17 - 2.97 - 17.94 - 13.60 - 14"04 -20.60 - 16.61 - 11-80 - 15.36 - 6.59

0.88 0' 88 0.98 0.95 0-95 0.98 0.79 a 0.99 0.93 0.79 a 0.98 0-94

a Regressions non-linear.

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P. J. Franco, K. L. Daniels, R. M. Cushman, G. A. Kazlow

comparisons could not isolate the source of this difference. The sensitivity analysis, however, showed that aniline was equally toxic to C. tentans and E. natchitocheae but T. neopunctipennis was 1.5 times more sensitive. The data for E. natchitocheae and phenol were also eliminated from further analysis by the least squares procedure because of non-linearity. The test for equality of slopes for the other species revealed a difference between the slopes of C. tentans and T. neopunctipennis but not between C. tentans and C. pinguis. Because of the inequality of slopes, a sensitivity analysis could not be performed for C. tentans and T. neopunctipennis, but phenol was 2.5 times more toxic to C. pinguis than to C. tentans.

DISCUSSION The most sensitive invertebrate of the organisms used by our group to study the aquatic effects of synthetic fuels was Daphnia magna, while the freshwater snail Physa gyrina was the most tolerant. The 48-h LCso values for the two species were 0.4 and 3"0~o for the coal-oil WSF (Giddings et al., 1983) and 19.8 mg litre- 1 and 260rag litre- 1 for phenol (Millemann et al., 1984) (a similar range of LCs0 values for aniline is not available). Although such comparisons are at best approximate, when these LCs0 values are compared, D. magna is about 7-5 times more sensitive than P. gyrina to the WSF, and thirteen times as sensitive to phenol. The comparably small ratio of sensitivities from the sensitivity analyses for our midge species (1.8 for the WSF, 1.5 for aniline, and 2.5 for phenol) indicates that any one of the chironomids will give a reasonable response estimate for the others for either toxicant. The Chironominae species, C. tentans and E. natchitocheae, are similar in many aspects of their ecology; for example, both are detritivores and burrow into sediments. Beck (1977) reported that they both show a wide tolerance to pH and nutrient concentration and to organic pollution. C. pinguis and T. neopunctipennis, however, are predacious and freeswimming. Less is known of them, but C. pinguis is generally found in clear, unpolluted water and prefers a narrower pH range than do either of the two Chironominae (Beck, 1977). The species of the two subfamilies also differed in their responses to the toxicants we tested. When a direct comparison of sensitivity was possible, responses of species within a subfamily were in close agreement, but differences between the two groups of species were present. Even though the two Tanypodinae species

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had unequal response slopes and relative toxicity could not be quantified, the overall response of T. neopunctipennis was nearer that of C. pinguis than that of C. tentans (Fig. 3). Suter & Vaughan (in press) also found that, in toxicity studies with fish and invertebrates, the closer the taxonomic relationship between species, the better the agreement in response. With aniline and phenol, the Chironominae were less sensitive than the Tanypodinae, but the relationship was reversed for the coal-liquid WSF; the Tanypodinae were about half as sensitive as the Chironominae. The WSF is a mixture of acidic (phenol and derivatives), basic (aniline and derivatives) and neutral compounds (Herbes et al., 1983). Although phenols have been demonstrated to be additive in their toxicities to several aquatic species (Parkhurst et al., 1979, 1981), Herbes & Beauchamp (1977) reported that the toxicity to D. magna of a mixture of a dihydric phenol and an aromatic amine (present in coal liquefaction effluents) is less than additive, i.e. the chemicals have an antagonistic interaction. The reversal in the order of sensitivity of the two subfamilies to the WSF, as compared with their relative responses to the single chemicals, may reflect either a difference among species in the additivity of toxicities or differences in sensitivities to those WSF components we did not test. Our results indicate that the cultured population, despite its isolation in the laboratory for more than thirty generations, is still representative of natural midge populations in its response to several toxicants. We know of no other investigations in which similar comparisons have been made. Although other tests may be more sensitive to culture-induced changes, the data suggest that laboratory cultures are valid surrogates for natural populations in acute toxicity bioassays.

ACKNOWLEDGEMENTS This study was sponsored by the Office of Health and Environmental Research, US Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation. W.L. Goldman, Hydrocarbon Research Inc., provided the coal liquid used in this investigation. We thank J.J. Beauchamp, J. M. Giddings and R.E. Millemann for their helpful comments during the preparation of this manuscript. Publication No. 2263, Environmental Sciences Division, ORNL.

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