Arch. Environ. Contam. Toxicol. 27, 103-106 (1994) A R C H I V E S O F

E n v i r o n m e n t a l C o n t a m i n a t i o n a n d T o x i c o l o g y © 1994 Springer-Verlag New York Inc.

Acute and Chronic Effects of Four Commercial Herbicide Formulations on Ceriodaphnia dubia

M. P. Oft, J. F. Fairchild, S. E. Finger

National Fisheries Contaminant Research Center, National Biological Survey, Depamnent of Interior, 4200 New Haven Road, Columbia, Missouri 65201-9634, USA

Received: 21 August 1993/Revised: 13 January 1994

Abstract. Toxicity tests with Ceriodaphnia dubia were con- ducted to determine acute (48 h) and chronic (7-day survival and reproduction) effects of four commonly used herbicide formulations. The 48-h LC50s in decreasing order of toxicity were 14.36 mg/L (Micro-TechS), 15.93 mg/L (BicepS), 32.99 mg/L (ExtrazineS), and 35.36 mg/L (LexoneS). Reduced re- production was detected at concentrations below 48-h LC50s for three of the formulations. The 7-day chronic values (ChV) based on reproduction were 17.68 mg/L (Micro-TechS), 8.84 mg/L (BicepS), 17.68 mg/L (ExtrazineS), and 8.84 mg/L (Lex- oneS). The acute-to-chronic ratios (ACRs) for Micro-Tech s (0.81), Bicep s (1.80), Extrazine s (1.86), and Lexone s (4.00) indicate a relatively narrow range between acute and chronic sensitivity in daphnids. A comparison of these response data to environmental concentrations suggests these herbicides are not likely to directly impact invertebrates. Potential impacts on plants and human health should be of primary ecological and regulatory concern.

Herbicide use has increased over the past 20 years due to increased agricultural production pressures and use of soil con- servation practices such as reduced tillage. Approximately 2.09 x 108 kg (460 million lb) of herbicides were applied to U.S. croplands alone in 1991 (Gianessi and Puffer 1991).

Environmental monitoring indicates that herbicides are com- monly found in surface waters (Richards and Baker 1993), groundwaters (Hallberg et al. 1989), and rainfall (Nations and Hallberg 1992). The ecological significance of these herbicides is largely unknown. Acute toxicity values have been summa- rized for numerous herbicides (Mayer and Ellersieck 1986). Chronic effects of atrazine are well documented (deNoyelles et al. 1982; Oris et al. 1991; Pratt et al. 1988); however, little information is available on chronic toxicity of other commonly used herbicides.

Correspondence to: M. P. Oft

Herein results of a study which examined acute and chronic toxicity of four commercial herbicide formulations to Cerio- daphnia dubia are presented. C. dubia was used as a test organism because cladocerans are often one of the more sensi- tive aquatic animals to chemicals (Mount and Norberg 1984; Mayer and Ellersieck 1986) and standard methods for conduct- ing acute and chronic tests have been established (EPA 1989, 1991). Data were compared to published data on measured environmental concentrations to perform an aquatic hazard as- sessment for non-target invertebrates.

Materials and Methods

Test System

Ceriodaphnia dubia neonates (<24 h old) were obtained from cultures at the National Fisheries Contaminant Research Center (NFCRC). Organisms were cultured and tested in contaminant-free well water (hardness 283 mg/L as CaCO 3, alkalinity 255 mg/L as CaCO 3, and pH 7.8) at 25°C.

All tests were conducted in 30-ml glass beakers filled with 15 ml of test solution. Herbicide concentrations in full-strength treatments were 100 mg/L in the 48-h tests and 50 mg/L in the 7-day tests with Micro- Tech ®, Extrazine ®, and Lexone ®. Tests with Bicep ® used 50 mg/L in the 48-h test and 25 mg/L in the 7-d test. Organisms were exposed to 100, 50, 25, 12.5, 6.25, and 0% dilutions of full-strength treatments. Water quality was monitored in high, medium, low, and control treat- ments. Temperature and pH (Orion Research Model 940 meter), and dissolved oxygen (YSI Model 54 meter) were measured daily. Alkalin- ity and hardness (APHA, 1985), conductivity (Cole-Parmer Model 1841-60 meter), and total ammonia (Orion Research Model 940 meter) were measured at the beginning of the tests.

Acute toxicity (48-h) tests were conducted under static conditions using four replicates with five organisms each (20 organisms per con- centration) (EPA 1991). Mortality, defined as lack of movement upon gentle prodding, was measured at the end of all tests. The 7-day survival and reproduction tests were conducted under static-renewal conditions using 10 replicates with one organism each (10 organisms per concentration) (EPA 1989). Reproduction, the number of neonates produced per adult, was recorded at daily test solution renewal and at the end of the test.

104 M. P. Ort et al.

Test Mater ial

Four commercial herbicide formulations were used in these tests. Mi- cro-Tech s (Monsanto Agricultural Company) is an emulsifiable con- centrate with 41.5% alachlor [2-chloro-N-(2,6-diethylphenyl)-N-(meth- oxymethyl)acetamide]. Bicep 6L s (Ciba-Geigy Corporation Agricultural Division) is an emulsifiable concentrate with 27.4% atrazine[6-chloro-N- ethyl-N'-(1-methylethyl)-l,3,5-triazine-2,4-diamine], 1.5% atrazine related compounds, and 36.1% Metolachlor [2-chloro-N-(2-ethyl-6- methylphenyl)-N-(2-methoxy-l-methylethyl) acetamide]. Extrazine II DF ® (Dupont Agricultural Products) is dispersible granules with 21.4% atrazine, 67.5 % cyanazine [2- [[4-chloro-6- (ethylamino)- 1,3,5- triazine-2-yl]amino]-2-methylpropanetril], and 1.1% cyanazine re- lated compounds. Lexone DF ® (Dupont Agricultural Products) is dis- persible granules with 75% metribuzin [4-amino-6-(1,1-dimethylethyl)-3- (methylthio- 1,2,4- triazin-5(4H)-one].

Concentrations of stock solutions were nominally defined. Stock solutions were prepared by dissolving 100 mg of an herbicide formula- tion in 1 L of well water. After mixing for 1 h, stock solutions were stored overnight at 4°C in the dark, and mixed and aerated for 1 h before tests were started.

Statistical Methods

The 48-h LC50s were estimated using the trimmed Spearman-Karber method with ot set at 5 (Hamilton et al. 1977). When a 100% kill or a partial kill was not observed, nonlinear interpolation was used, and binomial confidence limits (>95%) were reported. Survival data from all tests were analyzed using Fisher's Exact Test (P < 0.05) to deter- mine lowest observed effect concentrations (LOEC) and no observed effect concentrations (NOEC) for survival.

Reproduction data from the 7-day tests were analyzed using hypoth- esis testing to obtain a chronic value (ChV). Treatments inducing significant mortality (i.e., ~>LOEC) were excluded from the analysis. Prior to statistical analyses, the data were tested for normality and homogeneity of variance. Analysis of variance was performed with a General Linear Models (GLM) procedure. Dunnett's test was used to compare the treatment mean with the control mean (p < 0.05) to determine NOECs and LOECs for reproduction. The ChV was defined as the geometric mean of the LOEC and NOEC. We divided the 48-h LC50 by the ChV to obtain the acute-to-chronic ratio (ACR).

Results

Control survival was />90% after the 48-h exposure (Table 1). After the 7-day exposure, control survival was 100% and mean reproduction was 25.8 young per female (Table 1). The 48-h LC50s ranged from 14.36 to 35.36 mg/L (Table 2). The chronic values (ChV) ranged from 8.84 to 17.68 mg/L (Table 2). Acute-to-chronic ratios (ACR), calculated from 48-h LC50s and 7- day ChVs, indicate that reproduction was a more sensi- tive endpoint for three of the herbicides tested (Table 2). The ACR values ranged from 0.81 to 4.00 (Table 2).

Discussion

Comparison of results with those from other investigations indicates that C. dubia is of similar sensitivity to herbicides as other commonly tested fish and invertebrates (Table 3). Rela- tive sensitivities generally range from 3 to 100 mg/L for these herbicides. Results of 48-h and 7-day tests exposing C. dubia to

Table 1. Survival and reproduction data for Ceriodaphnia dubia ex- posed to several herbicides formulations for 48 h and 7 days

% Survival Mean Nominal young/female

Herbicide concentration 48 h 7 days at 7 days formulation (mg/L) (n = 20) (n = 10) (n = 10)

Micro-Tech s 100 0 NT a NT 50 10 10 0.1 25 10 20 b 2.3 b 12.5 55 b 90 19.5 6.25 95 90 23.1 3.12 NT 1130 26.5 0 90 100 25.8 c

Bicep s 50 0 NT NT 25 0 40 b 0.2 12.5 90 b 100 14.6 b 6.25 95 100 22.4 3.12 NT 100 25.0 1.56 NT 100 23.5 0 100 100 25.8

Extrazine s 100 0 NT NT 50 0 b 0 0.0

25 95 40 b 0 .8 b

12.5 95 100 21.8 6.25 100 90 27.4 3.12 NT 90 32.6 0 100 100 25.8

Lexone s 100 0 NT NT 50 o b o b o.o

25 100 i00 0.0 12.5 90 90 1.4 b 6.25 100 100 29.7 3.12 NT 100 28.1 0 100 100 25.8

a Not tested because concentrations were adjusted after 48-h test bLowest observed effect concentration (P < 0.05) Cn = 20 in control for 7-day test

Table 2. The 48-h LC50s, 7-day chronic values (ChV) for reproduc- tion, and acute-to-chronic ratios (ACR) of several herbicide formula- tions for Ceriodaphnia dubia reported as nominal concentrations (mg/L)

Mortality Reproduction 48-h LC50 ChV Acute-to-chronic

Herbicide (95% C.I.) (NOEC-LOEC) ratio

Microtech s 14.36 17.68 0.81 (11.60-17.78) (12.5-25)

Bicep s 15.93 8.84 1.80 (14.20-17.87) (6.25-12.5)

Extrazine s 32.99 17.68 1.86 (29.98-36.29) (12.5-25)

Lexone s 35.36 8.84 4.00 (25-50) a (6.25-12.5)

a >95% binomial confidence interval

nine pesticides (Otis et al. 1991) were used to calculate ACRs which ranged from 1.3 to 10.2. The ACR for alachlor calcu- lated from other studies (2.0) is greater than for data from this research (0.8), but overall the range of ACRs were similar

Toxicity of Four Herbicides to Ceriodaphnia dubia 105

Table 3. Comparative toxicity of herbicide active ingredients to aquatic organisms

Toxic Chemical concentration organism (mg/L) Endpoint Reference

Alachlor Ceriodaphnia dubia

Chironomus riparius Daphnia magna Atrazine Ceriodaphnia dubia

Salmo gairdneri Metolachlor Daphnia magna 23.5 Pimephales promelus 8.0 Cyanazine Salmo gairdneri 9.0 Pimephales promelas 16.3 Metribuzin Ceriodaphnia dubia

Daphnia magna Salmo gairdneri Chironomus riparius

6.0 48-h LC50 a 7.3 7-day ChV a 7.9 48-h LC50 b 4.0 7-day ChV b

10.0 48-h EC50 c 21.0 48-h LC50 d

>30 48-h LC50 b 3.5 7-day ChV b

24 96-h LC50 d

48-h LC50 d 96-h LC50 d

96-h LC50 d 96-h LC50 d

26.5 48-h LC50 a 7.1 7-day IC50 a

>100 48-h LC50 d 42 96-h LC50 d 43.5 48-h EC50 c

(immobilization)

aThis study bOris et al. (1991) CBuhl and Faerber (1989) dMayer and Ellersieck (1986)

between studies. The ACRs for formulations tested in this study indicated a relatively narrow range between acute and chronic sensitivity when compared to a 21-day ACR (>30) reported for Daphnia magna exposed to the insecticide fonofos (Fairchild et al. 1992).

Algae and vascular plants are more sensitive to herbicides than fish or invertebrates. Previous studies at the NFCRC deter- mined that the unicellular green algae Selenastrum capricornu- tum were about 50-630 times more sensitive than C. dubia. The 96-h LC50s for Extrazine ®, Lexone ®, Micro-Tech ®, and Bicep ® were 28, 48, 87, and 181 ixg/L, respectively (Fairchild et al. 1993). St. Laurent et al. (1992) determined that S. capri- cornutum was sensitive to cyanazine and metolachlor at con- centrations of 18 and 55 p~g/L (96-h LC50s), respectively. Atrazine reduced photosynthetic carbon uptake and biomass in algae at concentrations ranging from 25 to 131 Ixg/L (deNoy- elles et al. 1983; Larson et al. 1986). Correll and Wu (1982) observed significant decline of Vallisneria americana after a 47-day exposure to 12 I~g/L atrazine in simulated estuarine microcosms. Similarly, Kemp et al. (1985) found that atrazine reduced biomass of Potamogeton pectinatus and Myriophyllum spicatum in microcosms at 30 and 91 Ixg/L, respectively.

Comparison of environmental concentrations of these herbi- cides to the sensitivity of fish, invertebrates, and plants sug- gests that aquatic plants should be of primary ecological con- cern. Richards and Baker (1993) recently published an 8-year data base of average herbicide concentrations in seven major Lake Erie tributaries. The maximum peak concentrations they

reported were 64.94 (alachlor), 68.40 (atrazine), 96.92 (me- tolachlor), 24.77 (cyanazine), and 25.15 vg/L (metribuzin), but the annual average concentrations were generally < 2 Ixg/L. Thus, peak concentrations of these herbicides can exceed con- centrations known to impact aquatic plants, but they are below acutely lethal concentrations for fish and invertebrates such as C. dubia.

Current measured ambient herbicide levels frequently exceed levels of human health concern. For example, the maximum contaminant level (MCL) in drinking water for alachlor and atrazine established by the USEPA are 2 and 3 Ixg/L, respec- tively (Richards and Baker 1993). Thus, herbicide formulation, use, and application rates will undoubtedly undergo further environmental scrutiny in spite of the relative insensitivity of fish and invertebrates.

Acknowledgements. We thank Doug Hardesty, Paul Heine, Robin Hur- tubise, Phil Lovely, Ann Rueff, Shane Ruessler, Mike Tomasovic, Kim Turner, and Dave Whites for technical assistance with this project.

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