Toxicity characterization of individual chemicals
All experiments were initiated with individual daphnids (>24-h-old) placed in 40 ml of medium. Algae (3.5 × 106 cells) and fish food (0.1 mg dry wt) were provided to each beaker twice daily for the 1st week, after which these amounts were doubled. Medium was changed 3 times weekly and biological endpoints were measured once every 24 h. Mortality was defined as occurring when no discernible movement was visible with the naked eye during 30 s of observation. Growth rates were determined in a manner described previously (Olmstead and LeBlanc 2001). Briefly, the first 4 molted exoskeletons of each daphnid were measured in length from the base of the shell spine to the top of the carapace using an ocular micrometer under the microscope (×4 or ×10 magnification). Molt lengths were plotted against molt numbers and the results fitted with linear regression to yield a slope that was taken as the growth rate. Offspring were removed and counted daily. Experiments were ended once all daphnids had released 3 broods of offspring (17–19 d of exposure).
The 9 chemicals used in this study were chosen from a survey of freshwater streams in the United States (Kolpin et al. 2002). Criteria for selection included frequency of detection, median detected levels, toxicity of the chemicals, and the mechanisms of action for toxicity. Chemicals selected were present at >10% of the sites sampled and, when detected, were found at median levels between 0.04–0.14 μg/L (Table 1). These chemicals all were presumed to have differing mechanisms of toxicity except for the pesticides carbaryl, diazinon, and chlorpyrifos, which all are inhibitors of cholinesterase. Carbaryl, chlorpyrifos, diazinon, and N,N-diethyl-m-toluamide (DEET) were obtained from Chem Service (West Chester, PA, USA). Bisphenol A, fluoranthene, 1,4-dichlorobenzene, and caffeine were obtained from Sigma-Aldrich (St. Louis, MO, USA). The 4-nonylphenol was acquired from Fluka Chemika (Milano, Italy).
The experimental design used for the toxicity assessments of individual chemicals was described previously (Olmstead and LeBlanc 2001). Each experiment consisted of 50 different exposure concentrations. The concentrations of chemicals in the exposure treatments were determined by starting with the lowest discerned acutely lethal level based upon preliminary experiments, or 1,000 μg/L, whichever was lower, as the highest treatment. Subsequent treatments were prepared at concentrations 90% of the next highest treatment level. A single female daphnid was exposed continuously to each treatment. Experimental conditions ensured that these females would reproduce asexually (parthenogenesis). All treatment solutions within an experiment contained the same concentration of ethanol (≤0.01%) that was used to deliver the chemicals. Ten control daphnids exposed to the appropriate amount of ethanol were monitored with each experiment.
Data from each toxicity assessment was transformed to a 0 to 100% scale to normalize results among experiments and to allow for the same concentration-response fit to be performed on all endpoints. Survival raw data were transformed using the following equation:
where S is the percent lifespan reduction and M is the day on which the daphnid died. Percent lifespan reduction represents the degree to which a chemical or mixture reduced the lifespan of a daphnid from a maximum of 18 d. Percent lifespan reduction among control daphnids typically was 0. The growth and reproductive raw data was transformed by dividing each treatment response by the average control response and multiplying by 100%. Data then were graphed and fit with a sigmoidal line using Origin™ software (Microcal™ Software, Northampton, MA, USA) with the following equation:
where R is the endpoint response and C is the concentration of the chemical. The power or slope of the curve (ρ) and the center of the sigmoid curve (Cm) were determined from fits of the experimental data and used to generate model predictions of mixture toxicity as described below. Concentrations of a given chemical expected to yield a 5% response (EC05) and a 50% response (EC50) were interpolated from these fits, with 95% confidence intervals calculated using a bootstrap approach (Efron and Tibshirani 1993) with SAS® 9.1 software (SAS institute, Cary, NC, USA). The center of the curve (Cm) corresponds to the EC50 of a given chemical.
Mixture toxicity modeling
The toxicity of a mixture of the 9 chemicals was modeled with each chemical present in the mixture at the median concentration measured in those waters where the chemical was detected (Kolpin et al. 2002; Table 1). The toxicity of this mixture was modeled at 50 different levels representing dilutions or fortifications of the base level (median detected concentration). Mixture levels were designated by their percentage of the base level concentrations. For example, at 200%, all chemicals were present at twice the base level and, at 50%, all chemicals were present at half of the base level. The toxicity of all mixture levels was modeled with respect to reduced lifespan, growth rate, and fecundity.
Toxicity of the mixture was modeled by combining the concepts of concentration addition and independent joint action. Chemicals were assigned to cassettes based upon their presumed mechanisms of action. Chemicals having the same mechanism of action were assigned to the same cassette. The acetylcholinesterase inhibitors diazinon, chlorpyrifos, and carbaryl all were placed in the same cassette. The other 6 chemicals all were presumed to have different mechanisms of action and were assigned to separate cassettes. The joint toxicity of chemicals within a cassette was calculated using the concentration addition approach, while the joint toxicity of different cassettes was calculated using the independent joint action paradigm. All parameters used in the models were derived from the toxicity evaluations of the individual chemicals and are presented in Table 2.
The toxicity or response (R) to chemicals within the same cassette was calculated by concentration addition. Equation 4 was rearranged to
where R is the response of a mixture and C is equivalent to ECx in Equation 1. The right side of Equation 5 can be substituted into Equation 1 to yield
The average power (ρ′) for the individual chemicals within a cassette was used in place of ρi because the powers of the concentration-response curves for chemicals having the same mechanism of toxicity are comparable (USEPA 1986). Solving this equation for R yields
where Cm,i and Ci are the center and concentration of the ith chemical, respectively.
Table Table 2.. Parameters derived from toxicity evaluations of the individual chemicals that were used in the mixture model. These parameters were used in Equation 7 in order to calculate expected responses from mixtures of chemicals. Values are represented as the parameter estimate plus or minus the error of that estimate. The average power (ρ′) is reported for the 3 cholinesterase inhibitors. An NA indicates that the respective chemical did not exhibit a response in the given endpoint at the concentrations examined and, therefore, would not contribute to the toxicity of the mixture. Caffeine, N,N-diethyl-m-toluamide, and dichlorobenzene did not exhibit any toxicity at the concentrations tested and were assumed to not contribute to any toxic effects of the mixtures
|Chemical||Cm (μg/L)||ρ||Cm (μg/L)||ρ||Cm (μg/L)||ρ|
|Bisphenol A||NA||NA||365,000 ± 648,000||0.663 ± 0.255||7,310 ± 330||7.96 ± 2.96|
|Carbaryl||10.4 ± 0.5||3.37 ± 0.86||13.6 ± 1.4||4.21 ± 1.14||9.18 ± 0.76||4.10 ± 1.30|
|Chlorpyrifos||0.190 ± 0.018||3.37 ± 0.86||NA||NA||NA||NA|
|Diazinon||0.522 ± 0.036||3.37 ± 0.86||NA||NA||NA||NA|
|Fluoranthene||NA||NA||194 ± 11||1.85 ± 0.17||85.9 ± 6.0||3.64 ± 0.88|
|4-Nonylphenol||195 ± 0||361 ± 0||205 ± 14||5.66 ± 1.72||NA||NA|
The combined toxicity (Rmix) of cassettes that comprised the mixture was modeled using independent joint action (Eqn. 2) for each endpoint (reduced lifespan, growth, and fecundity) at each mixture level. Effect levels (EL05 or EL50) were calculated from the model. The EL05 is the mixture level calculated to elicit a 5% response and was used as an estimate of the lowest level of the mixture at which toxicity would be evident (i.e., lowest observed effect level). The EL50 is the mixture level calculated to elicit a 50% response and provides a characterization of the toxicity of the mixture with the greatest statistical confidence.