fish showed curved spine from 4 hours onward. At 24.5 mg/L, one fish showed darkened

p igmentation at 4 and 24 hours, one fish showed loss of body color from 4 hours onward, and three fish showed loss of co-ordination and spiral swimming at 24 hours only. N o mortality occurred during the 96 hour test; therefore, the 96-hour LC50 was greater than the mean measured concentration of 24.5 mg/L. This study is acceptable. 96-hour LC50 > 24.5 mg/L

Daphnid Ecotoxicity Test: conducted a 48-hour toxicity test in water fleas (Daphnia magna) with P-14-0198 (99.5% p urity) under static conditions. This study was reported to follow EC Methods for Determination of Ecotoxicity, Annex to Commission Regulation (EC) N o 440/2008 Part C, Method 2 “Daphnia sp. Acute Immobilization Test” and the OECD Guideline for Testing of Chemicals No. 202, “Daphnia Acute Immobilization Test” (2004). Following a range-finding test, four replicates of five D. magna were exposed to a dilution water control (Elendt M4 medium) or the test substance at nominal concentrations of 0.52, 1.15, 2.54, 5.58, 12.3 and 27.0 mg/L. Corresponding arithmetic mean measured concentrations were 0.471, 0.999, 2.25, 4.82, 10.9 and 24.8 mg/L, respectively, as determined by LC-MS/MS (LOQ = 0.1 mg/L; LOD = 0.04 mg/L). The test substance (54.2 mg) was ground into a smooth pourable paste on an agate mortar with the dropwise addition of Elendt M4 medium. The paste was then added to a volumetric flask (2 L) containing Elendt M4 medium (1700 mL), which was then shaken before being made up to volume and stirred for ca. one hour until no particulate matter remained. The

p reparation was then filtered (0.45 μm pore size) to provided an aqueous solution with a nominal concentration of 27.0 mg/L; this was either used directly at the highest test concentration or aliquots were serially diluted in volumetric flasks (1 L) containing Elendt M4 medium to provide test media with the desired nominal concentrations. The glass dishes were loosely covered. At the start and end of the test, the test media were colorless at all concentrations. Over the course of the test, temperature ranged from 19.6

–20.4  ̊ C, p H ranged fro m 7.62

–7.90 and dissolved oxygen ranged from 97

–101% ASV. Dilution water hardness was 245 mg CaCO3/L. A loading of 41.67 daphnids/L was calculated. N o immobilization was observed during the test; therefore, the 48-hour EC50 was greater than the mean measured concentration of 24.8 mg/L. This study is acceptable. 48-hour EC50 > 24.8 mg/L

Algal Ecotoxicity Test: conducted a 72-hour toxicity test in green algae (Pseudokirchneriella subcapitata) with P-14-0198 (99.5% p urity) under static conditions. This study was reported to follow EC Methods for Determination of Ecotoxicity, Annex IV to Commission Regulation (EC)

N o 761/2009 Part C, Method 3 “Freshwater Algae and Cyanobacteria, Growth Inhibition Test” and Procedure 201 of the “Guidelines for Testing of Chemicals” of the Organization for Economic Co-operation and Development: Freshwater Alga and Cyanobacteria, Growth Inhibition Test” (2006, corrected 2011). Following a range-finding test, three replicates of P. subcapitata (1 x 104 cells/mL) were exposed to the test substance at nominal concentrations of 0.008, 0.03, 0.08, 0.26, 0.82, 2.64 or 8.44 mg/L. Corresponding geometric mean measured concentrations were 0.00668, 0.0221, 0.0589, 0.212, 0.719, 2.39, 7.56 and 28.9 mg/L, respectively, as determine by LC-MS/MS (LOQ = 0.001 mg/L; LOD = 0.0004 mg/L). Additionally, six replicates of P. subcapitata were exposed to an OECD medium control. Thealgae were illuminated with a mean light intensity ranging from 5790

–7040 lux, with constant shaking. The test substance (54.3 mg) was ground into a smooth pourable paste on an agate mortar with the dropwise addition of OECD medium. The paste was then added to a volumetric flask (2 L) containing OECD medium (1500 mL), which was then shaken before being made up to volume and stirred for ca. one hour until no particulate matter remained. The formulation was then filtered (0.45 μm pore size) to provide an aqueous solution with a nominal concentration of 27.0 mg/L that was used directly for the highest test concentration and subsequent dilution to nominally 8.44 mg/L. Aliquots were serially diluted in volumetric flasks (1 L) containing OECD medium to provide test media with the desired nominal concentrations. Each test flask was loosely plugged with a foam bung. At the start of the test, the test media at all concentrations were colorless. Over the course of testing, temperature ranged from 22.3

– 23.4  ̊ C and pH ranged from 7.70

–9.54. The cell density in control cultures increased by a factor of 201.6 within 72 hours. Based on mean measured concentrations, the 72-hour EC50 values for growth rate and yield were > 28.9 mg/L and 28.2 mg/L, respectively or NES. The 72-hour NOEC and LOEC were 7.56 mg/L and 28.9 mg/L, respectively. The 72-hour ChV was 14.8 mg/L. This study is acceptable although the highest mean measured concentration was slightly higher than the measured water solubility. 72-hour EC50 (growth rate) > 28.9 mg/L 72-hour EC50 (yield) = 28.2 mg/L 72-hour NOEC (growth rate and yield) = 7.56 mg/L 72-hour LOEC (growth rate and yield) = 28.9 mg/L 72-hour ChV (growth rate and yield) = 14.8 mg/L

The acute base set toxicity studies for the 96-hour fish, 48-hour Daphnia, and 72-hour algal studies are acceptable. For comparative purposes, p redictions based on SAR analysis for are 2.2, 0.35, and 0.16 for acute fish, daphnia, and green algae endpoints, respectively and 0.053, 0.038, and 0.066 mg/L for chronic fish, daphnia, and green algae endpoints, respectively. The acute CoC for is determined from the experimental value from the72-hour EC50 algal (yield) toxicity study of 28.2 mg/L (28,200 p pb) and divided by an assessment factor of 4 yielding an acute CoC of 7,050 p pb. There is significant toxicity variability between the chronic predicted value and the chronic experimental data as it relates to the algal endpoint. However, the most sensitive species in this case is based on the chronic daphnia endpoint. Therefore, the chronic CoC is derived by dividing the predicted chronic daphnia toxicity value of 0.0.38 mg/L by an uncertainty factor of 10, yielding 0.0038 mg/L or 4

p pb.

Acute CoC = 7,050 p pb
Chronic CoC = 4 p pb

Ecotox Study Reviewer: January 13, 2014

Factors Values Comments

Assessment Factor
10