Phthalates are animal carcinogens. Potassium hydrogen phthalate (KHP), which has the least complicated structure among phthalates, is used for the analysis of total organic carbon and formaldehyde. However, its toxicity has not been confirmed. A 24-hour acute toxicity test was performed using Daphnia magna, a water flea used to evaluate aquatic toxicity owing to its high sensitivity. The lowest observed effect concentration of KHP was found to be 240 mg/L. The effects of phosphorus, nitrogen, and Cr(6+), which are able to be discharged along with KHP, were also confirmed using tests. At 240 mg/L KHP, toxicity increased as phosphorus, nitrogen, and Cr(6+) increased. In addition, tests were performed to confirm the half maximal effective concentration of KHP. Through 10 test repetitions, the average ecotoxicity value was found to be 0.3, the average half maximal effective concentration was 327.75 mg/L, and the coefficient of variation (%) was 3.16%; because the latter value is lower than 25%, which is what is generally suggested for the water pollution standard method, the reproducibility of the tests is sufficient to replace the existing standard reference toxicity test that uses potassium dichromate. In addition, the half maximum effective concentration of potassium hydrogen phthalate is approximately 218 times more than that of potassium dichromate; therefore, toxicity is relatively low. In conclusion, KHP is a feasible alternative to the highly toxic potassium dichromate for performing the standard reference toxicity test.