Document ID: EPA-HQ-OPPT-2013-0399-0213
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2019-07-24T04:00Z

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
                                                   					WASHINGTON, D.C. 20460 

                             OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION
                                        															  May 11, 2018										
MEMORANDUM

SUBJECT: 	Review of the Chironomid Toxicity Test and Environmental Risk Assessment for S-15-0009

FROM: 	J. Gallagher
		Toxicologist
		Assessment Branch 1
		Risk Assessment Division (7403M)

TO:		J. Alwood
      Program Manager
      Chemical Control Division 
      New Chemicals Management Branch (7405M)

THRU:	T. Wright
		Biologist
		Assessment Branch 5
		Risk Assessment Division (7403M)

Conclusions
   A. The submitted 28-day Chironomid sediment toxicity test on S-15-0009 is acceptable.
   B. In conjunction with results from submitted acute and chronic aquatic studies, physical/chemical/fate properties and discussions with the sponsor (                 ), the acute and chronic concentrations of concern for S-15-0009 are no effects at saturation (low environmental hazard).
   C. Environmental risks were not identified for S-15-0009.

Background
S-15-0009 (		;                               was submitted by                     in 2015. This amide (S-15-0009) has a reported maximum production volume of                 kg/yr and will be used 
         The reported new use with this significant new use notice is for "releases to water (above 0.001 mg/L)."  In 2013,                 was dropped with a non 5(e) SNUR with a water trigger of 0.001 mg/L based on ecological hazard estimates.  Ecotoxicity hazard concerns were high based on the predicted chronic fish endpoint (ECOSAR v. 1.1). S-15-0009 is expected to be slightly soluble with a measured water solubility of 0.19 mg/L and a predicted log Kow of 6.33.  The estimated Henry's law constant is 1x10[-6] (slightly volatile from water) and sorption to soils/sediments is predicted to be strong.  The time to complete ultimate aerobic biodegradation for S-15-0009 is predicted to be weeks.                 submitted acceptable ecotoxicity test data (acute fish, chronic daphnia, and algae) to EPA in 2015, with the S-15-0009 submission, to fill some data endpoints (Attachment A).  

When S-15-0009 went to FOCUS (July 30, 2015), it was placed into standard review for CCD to inform the submitter that the significant new use of releases to surface water exceeding 0.001 mg/L would not be permitted. Chronic risks to the environment were high due to releases to water where the chronic COC of 0.001 mg/L was exceeded  
                days (SWC: 0.281 mg/L) during use operations. The required testing at FOCUS was the fish early-life stage toxicity test (FELS; OPPTS Test Guideline 850.1400).   

Fish Early-Life Stage Toxicity Test Discussion
On February 2, 2016,                    submitted a FELS toxicity test protocol for S-15-0009.  The submitted protocol was not chemical-specific or tailored to S-15-0009 making it difficult to provide a detailed review. EPA reviewed the protocol and submitted it to         (See Attachment B).  Following EPA's review, the submitter encountered extensive challenges exposing fish to S-15-0009 in flow through or semi-static conditions, per test guidelines, while developing the experimental design for the FELS toxicity test.              had several conversations with EPA (see separate attachment entitled, "EPA Discussions FELS test for SNUN substance.") to discuss their challenges.  Due to the multiple problems that the company was having keeping S-15-0009 in test solution (using many approaches) in conjunction with the physical/chemical/fate properties, EPA agreed to waive the chronic fish study (and assessed S-15-0009/                   as no effects at saturation in the aqueous environment) in place of a Chironomid sediment/water toxicity test with robust analytical methods. On March 29, 2018,            submitted a Chironomid toxicity test which is reviewed below.

Chironomid Toxicity Test Review
Scymaris, Ltd. conducted a 28-day sediment-water toxicity test with the midge (Chironomus riparius) and S-15-0009 (purity: 99.62% Amide).  This study followed OECD 218 (2004).  Four replicates containing 20 larvae were exposed to a blank control, solvent control (tetrahydrofuran; THF), or the test substance at a nominal concentration of 62.5, 125, 250, 500, or 1000 mg/kg (dry weight).  Corresponding time-weighted mean measured concentrations of the test substance were 39.5, 74.0, 172, 363, and 788 mg/kg (dry weight), as determined via LC-MS analysis (robust analytical method).  The LOQs for sediment analysis were 0.04 mg/kg for the blank control, 0.04-0.06 mg/kg for the solvent control, 4.10-5.24 mg/kg for the 62.5 mg/kg treatment, 3.79-5.31 for the 125 mg/kg treatment, 4.02-4.81 mg/kg for the 250 mg/kg treatment, 3.81-7.48 mg/kg for the 500 mg/kg treatment, and 36.63-42.06 mg/kg for the 1000 mg/kg treatment.  The composition of the test sediment by dry weight was 5% sphagnum peat moss, 75% quartz sand, and 20% kaolinite clay.  A proportion (50%) of the dry sand component of the sediment for each treatment was added to a glass crystallizing dish where it received 10 mL of test item dissolved in tetrahydrofuran (THF) at the appropriate nominal concentration.  The stock solutions were pipetted onto the surface of the sand, and the solvent was allowed to evaporate overnight.  The remaining quantity of sand was then mixed with kaolinite clay, conditioned peat, and reconstituted water in individual bottles.  The amount of reconstituted water added to the sediment was that which liquefied it sufficiently to obtain good mixing on a tumble mill, using known and equal additions to all bottles.  The contents of the bottles were mixed on a tumble mill for approximately 15.5 hours before being dispensed to the test vessels.  For each treatment, approximately 133.5 g (wet weight) spiked sediment was weighed into each test vessel and reconstituted water was added to a total volume of 400 mL.  After a settlement period, vessels were provided with gentle aeration and then allowed to stabilize and equilibrate for 7 days prior to the exposure phase.  The test vessels were glass beakers fitted with lids consisting of inverted, plastic beakers with meshed air vents acting as traps to retain emerged adults.  Over the course of the study in the overlying water, temperatures ranged from 19.3-19.7ºC, pH ranged from 7.2-8.0, and dissolved oxygen ranged from 8.5-9.2 mg/L.  The water hardness of the overlying water ranged from 18-26 mg CaCO3/L.  Facility background data for the overlying water indicated a hardness of 89 mg CaCO3/L and TOC < 0.7 mg/L.  No significant effects were observed on development rates, total number emerged, or sex ratio of adults.  All test replicates showed no visible non-emerged larvae with the exception of one replicate at the 250 mg/kg treatment level, which showed no emergence; the 19 non-emergent larvae in this replicate were excluded from all result determinations.  The mean time to first emergence ranged from 16-18 days in the test groups and was 16 days in the control group.  Based on time-weighted mean measured test concentrations, the 28-day NOEC and LOEC were 788 mg/kg (dry weight) and > 788 mg/kg (dry weight), respectively. This is an acceptable test.
28-day NOEC = 788 mg/kg (dry weight)
28-day LOEC > 788 mg/kg (dry weight)

Environmental Hazard and Risk: Summary
Since 2015,                        submitted four acceptable toxicity tests (acute fish, algae, chronic aquatic invertebrate, and a chronic sediment toxicity test) to EPA on S-15-0009.  Based on the results from these tests, in conjunction with physical/chemical/fate properties and discussions between                         and EPA, the acute and chronic concentrations of concern for S-15-0009 are no effects at saturation (low environmental hazard). From the July 29, 2015 exposure report, the predicted surface water concentrations of S-15-0009 are 0.486 mg/L (Use: max acute eco) and 0.281 mg/L (Use: probabilistic dilution model).  As a result, environmental risks were not identified for S-15-0009.

                                 Attachment A
                                       
Ecotoxicity Test Data Results for S-15-0009: 

Fish Ecotoxicity Test:
Harlan Laboratories, Ltd. conducted a 96-hr acute toxicity test with rainbow trout (Oncorhynchus mykiss) and S-15-0009 (purity: 99.3%) under semi-static conditions with daily renewal.  The water solubility of the test substance was reported to be 0.0056 mg/L under test conditions.  This study followed OECD test guideline No. 203 (1992) and Method C.1 of Commission Regulation (EC) No. 440/2008.  Following a range-finding test, single replicates of seven O. mykiss were exposed to a dilution water control (dechlorinated laboratory tap water) or the test substance at a single nominal concentration of 100% v/v saturated solution.  Measured concentrations of test substance were below the limit of quantitation of the analytical method (HPLC-MS; LOQ = 0.010 mg/L).  To prepare the saturated solution (100 mg/L), a nominal amount of test item (2250 mg) was dispersed in 22.5 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours.  After 24 hours, the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 um Sartorious Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter).  The test vessels were covered to reduce evaporation.  The test item preparations were observed to be clear colorless solutions throughout the test.  Over the course of testing, temperature ranged from 13-15°C, pH ranged from 7.4-8.1, and dissolved oxygen ranged from 9.0-10.7 mg/L.  The dilution water hardness was 140 mg CaCO3/L.  A loading rate of 0.58 g fish/L was reported.  No mortality was observed in the control and test replicates at the saturation limit.  Based on the nominal concentration, the 96-hour LC50 was greater than 100% v/v saturated solution and determined to be no effects at saturation (NES).  This was an acceptable test.
96-hour LC50 = NES

Daphnid Ecotoxicity Test:
Harlan Laboratories, Ltd. conducted a 21-day reproduction test with Daphnia magna and S-15-0009 (purity: 99.3%) under semi-static conditions with renewal 3 times per week.  The water solubility of the test substance was reported to be 0.0056 mg/L under test conditions.  This study followed OECD test guideline No. 211 (2012), referenced as Method C.20 of Commission Regulation (EC) No. 440/2008 and OPPTS 850.1300.  Following a range-finding test, ten replicates of single daphnids were exposed to the dilution water control (Elendt M7 medium) or the test substance at a single nominal concentration of 100% v/v saturated solution.  The time-weighted mean measured test concentration was 0.0038 mg/L, based on HPLC-MS analysis (LOQ = 0.0037 mg/L).  To prepare the saturated solution (100 mg/L), a nominal amount of test item (1100 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours.  After 24 hours, the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 um Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter).  Test vessels were covered with plastic lids to reduce evaporation.  At the start and throughout the test, all control and test solutions were observed to be clear, colorless solutions.  Over the course of testing, temperature ranged from 20-21°C, pH ranged from 7.2-8.2, and dissolved oxygen ranged from 8.3-9.5 mg/L.  The dilution water hardness ranged from 226-344 mg CaCO3/L.  A loading rate of 10 daphnids/L was calculated.  No mortality was observed in the control group, and 10% mortality was observed in the test group.  No significant impairment of reproduction was observed in the test group.  Based on the time-weighted mean measured test concentration, the 21-day EC50 value for immobilization and reproduction was greater than 0.0038 mg/L, and therefore greater than saturation (NES).  The NOEC and LOEC values were 0.0038 and greater than 0.0038 mg/L, respectively. This was an acceptable test.
21-day EC50 (immobilization and reproduction) = NES
21-day NOEC = 0.0038 mg/L
21-day LOEC >= 0.0038 mg/L
Aquatic invertebrate ChV = NES

Algal Ecotoxicity Test:
Harlan Laboratories, Ltd. conducted a 72-hour growth inhibition test with green algae (Pseudokirchneriella subcapitata) and S-15-0009 (purity: 99.30%) under static conditions.  The water solubility of the test substance was reported to be 0.0056 mg/L under test conditions.  This study followed OECD test guideline No. 201 (2006), referenced as Method C.3 of Commission Regulation (EC) No 761/2009.  Following a range-finding test, three replicates of P. subcapitata (5 x 10[3] cells/mL) were exposed to the test substance at a nominal concentration of 6.25, 12.5, 25, 50, or 100% v/v saturated solution.  HPLC-MS analysis of the 10 and 100% v/v saturated solution test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantitation of (LOQ = 0.010 mg/L).  An additional six replicates of P. subcapitata were exposed to a culture medium control.  The algae were illuminated with a light intensity of approximately 7000 lux with constant shaking.  To prepare the test solutions (100 mg/L), a nominal amount of test item (1100 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours.  After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 um Sartorius Sartopore filter (first approximately 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution.  A series of dilutions was made from this saturated solution to give stock solutions of 50, 25, 12.5 and 6.25% v/v saturated solution.  The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.  Test flasks were plugged with polyurethane foam bungs.  At the start of the test, all control and test cultures were observed to be clear colorless solutions.  After the 72-hour test period, all control and test cultures were observed to be green dispersions.  Over the course of testing, temperature was maintained at 24+-1°C and pH ranged from 7.4-8.1.  The mean cell density of control cultures increased by a factor of 91 within 72 hours.  No abnormalities were detected in control and test cultures at microscopic inspection.  Based on nominal concentrations, the 72-hour EC50 was greater than 100% v/v saturated solution for growth rate and yield and determined to be no effects at saturation (NES).  The NOEC and LOEC were 100% v/v saturated solution and greater than 100% v/v saturated solution or NES, respectively. This was an acceptable test.
72-hour EC50 (growth rate and yield) = NES
72-hour NOEC (growth rate and yield) = 100% v/v saturated solution
72-hour LOEC (growth rate and yield) >= 100% v/v saturated solution
Algal ChV = NES

The acute fish (96-hour LC50 = NES), chronic daphnid (ChV [immobilization and reproductions] = NES) and algal (72-hour EC50 (growth rate and yield) = NES and ChV = NES) toxicity tests conducted with S-15-0009 were considered acceptable for the purposes of concentration of concern determination within OPPTS.  It is worth noting that ecotoxicity test data (on an analog) were submitted with           in 2013.  The analog test data was not acceptable because the test chemical used in the toxicity tests was not an appropriate analog based on differences (2 log units) in the octanol/water partitioning coefficients (Kow).   For comparative purposes, the fish (96-hr LC50), acute daphnia (48-hr EC50), and algal (96-hr EC50) toxicity values, based on SARs for S-15-00009, are NES, NES, and 0.12 mg/L or NES, respectively.  In addition, based on the same SAR equations (Amides), the fish, daphnia, and algal chronic values for S-15-0009 are 0.001 mg/L, 0.01 mg/L, and 0.03 mg/L or NES, respectively.  The acute concentration of concern (CoC) is no effects at saturation, based on test data and SAR predictions.  The chronic concentration of concern for S-15-0009 is calculated by dividing the QSAR-derived fish chronic value (0.001 mg/L) by an uncertainty factor of 10 yielding 0.1 ug/L or 0.1 ppb.  The lowest default chronic CoC for S-15-0009 is 1 ppb and will be used for chronic CoC determination.

Acute COC = NES
Chronic COC = 1 ppb

                                 Attachment B
                                       
                                       
                                       
                                  UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
                                                   					WASHINGTON, D.C. 20460 

                             OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION
                                   															  February 23, 2016										
MEMORANDUM

SUBJECT: 	Fish Early-Life Stage Toxicity Test Protocol Review for S-15-0009

FROM: 	J. Gallagher
		Toxicologist
		Assessment Branch 1
		Risk Assessment Division (7403M)

TO:		J. Alwood
      Program Manager
      Chemical Control Division 
      New Chemicals Management Branch (7405M)

THRU:	K. Eisenreich
		Toxicologist
		Associate Branch Chief, AB4
		Risk Assessment Division (7403M)

Background
S-15-0009 (         ;                   was submitted by               in 2015 and went to FOCUS on July 30, 2015.  This amide (S-15-0009) has a reported maximum production volume of                             and will be used as a

The reported new use with this significant new use notice is for "releases to water (above 0.001 mg/L)."  S-15-0009 is expected to be slightly soluble with a measured water solubility of 0.19 mg/L and a predicted log Kow of 6.33.  The estimated Henry's law constant is 1x10[-6] (slightly volatile from water) and sorption to soils/sediments is predicted to be strong.  The time to complete ultimate aerobic biodegradation for S-15-0009 is predicted to be weeks.  When S-15-0009 went to FOCUS, it was placed into standard review for CCD to inform the submitter that the significant new use of releases to surface water exceeding 0.001 mg/L would not be permitted.  In 2013,             was dropped with a non 5(e) SNUR with a water trigger of 0.001 mg/L based on ecological hazard estimates.  Ecotoxicity hazard concerns were high based on the predicted chronic fish endpoint (ECOSAR v. 1.1).               submitted acceptable ecotoxicity test data (acute fish, chronic daphnia, and algae) to EPA in 2015 to fill some data endpoints.  Chronic risks to the environment were high due to releases to water where the chronic COC of 0.001 mg/L was exceeded 200/200 days (SWC: 0.280 mg/L) during use operations. The required testing at FOCUS was the fish early-life stage toxicity test (OPPTS Test Guideline 850.1400).

Fish Early-Life Stage Toxicity Test Protocol Review
On February 2, 2016,                 submitted a fish early-life stage toxicity test protocol for S-15-0009.  The submitted protocol is not chemical-specific or tailored to S-15-0009.  As a result, it is difficult to provide a detailed review.  Overall, the protocol follows OPPTS Test Guideline 850.1400 and OECD Test Guideline 210.  In addition, EPA recommends following OECD Test Guideline 23.  EPA strongly recommends analytical quantification of S-15-0009 and reporting of the toxicity endpoint values (EC50, NOEC, LOEC, etc.) as mean measured test concentrations.  In addition, the protocol references water accommodating fraction (WAF) testing if the test material is not very water soluble.  EPA does not recommend WAF testing for S-15-0009, but does recommend that the submitter/testing laboratory clearly describe if there are water solubility challenges with S-15-0009.  Earlier toxicity test submissions had water solubility challenges with S-15-0009 even though the "measured" water solubility (provided by                 ) was 0.19 mg/L (slightly soluble).  Furthermore, conducting the definitive test with flow-through conditions is recommended.  Finally, EPA does not recommend filtering of stock /test solutions.  If particulates are found in the media, EPA recommends centrifugation of the stock /test solution.  EPA looks forward to reviewing the aquatic toxicity test results for S-15-0009 in the near future.