Phenol, isopropylated, phosphate (3:1)

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 21, 2014 to May 20, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & US EPA test guidelines in compliance with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

see below

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

An exception was noted on the stock solutions prepared for use on Days 18 to 21 of the test, when it was prepared at 3001 μg/mL rather than 3000 μg/mL. This primary stock solution resulted in the test concentration of 7.69 μg/L rather than 7.68 μg/L as stated in the protocol. Since the results of the study are based on the mean measured test concentrations, this deviation from the study protocol had no adverse impact on the results or the interpretation of the results of the study.

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Analytical monitoring:

yes

Details on sampling:

Samples were collected from each treatment and control group one day prior to the start of the test after conditioning the diluter for two days. Water samples also were collected from alternating replicate test chambers in each treatment and control group at the beginning of the test, at approximately weekly intervals during the test and at the end of the test to measure concentrations of the test substance. All samples were collected from mid-depth, placed in glass vials, and processed immediately for analysis.

Vehicle:

yes

Details on test solutions:

Individual stock solutions were prepared for each of the five concentrations tested, and were prepared four times during the study. A primary stock solution was prepared by mixing a calculated amount of test substance into HPLC-grade dimethylformamide (DMF) at a nominal concentration of 3000 μg/mL. Four secondary stock solutions were prepared in DMF at nominal concentrations of 76.8, 192, 480 and 1200 μg/mL by proportional dilution of the primary stock. The exception was noted on the stock solutions prepared for use on Days 18 to 21 of the test, when it was prepared at 3001 μg/mL rather than 3000 μg/mL. This primary stock solution resulted in the test concentration of 7.69 μg/L rather than 7.68 μg/L as stated in the protocol. Since the results of the study are based on the mean measured test concentrations, this deviation from the study protocol had no adverse impact on the results or the interpretation of the results of the study. The primary and secondary stock solutions were mixed by inversion, and appeared clear and colorless. Stock solutions were stored refrigerated in a glass graduate cylinder or glass amber bottles, and aliquots of each stock were placed in the syringe pump at least every two days during the study.
The five test substance stock solutions were injected into the diluter mixing chambers at a rate of 15.5 μL/minute where they were mixed with dilution water delivered at a rate of 155 mL/minute to achieve the desired test concentrations. The negative control received dilution water only. The solvent control was prepared by delivering HPLC-grade DMF to the mixing chamber for the solvent control. The concentration of DMF in the solvent control and all Reofos 35 treatment groups was 0.1 mL/L.

Test organisms (species):

Daphnia magna

Details on test organisms:

The cladoceran, Daphnia magna, was selected as the test species for this study. Daphnids are representative of an important group of aquatic invertebrates and were selected for use in the test based upon past history of use in the laboratory. Daphnid neonates used in the test were less than 24 hours old and were obtained from cultures maintained by Wildlife International, Easton, Maryland.
Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test. During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 19.9 to 20.8°C, measured with a hand-held, liquid-in-glass thermometer. The pH of the water ranged from 8.2 to 8.5, measured with a Thermo Orion Benchtop 4 Star Plus pH meter. Dissolved oxygen concentrations were ≥7.0 mg/L (≥78% of saturation), measured with a Thermo Orion Benchtop 3 Star Plus dissolved oxygen meter.
During culture and testing, daphnids were fed a mixture of yeast, cereal grass media, and trout chow (YCT), supplemented with a vitamin stock solution and a suspension of the freshwater green alga, Pseudokirchneriella subcapitata. Daphnids were fed three times per day through Day 6 of the test and then were fed four times per day from Day 7 until the last day of the test. At each feeding, each test chamber was fed 0.75 mL of YCT, 1.5 mL of algae. In addition, each test chamber also received 0.50 mL of vitamin solution once daily. This amount of feed is equal to approximately 0.54 to 0.71 mg C/daphnid/day, based on a non-GLP study. While this amount of feed exceeds the OECD guideline recommended amount of 0.1 to 0.2 mg C/daphnid/day, an excess amount was fed in order to maintain sufficient feed in the flow-through system to support acceptable reproduction rates.
The four adult daphnids used to supply neonates for the test were held for at least 14 days prior to collection of the juveniles for testing, and had each produced at least one previous brood. Adult daphnids in the culture had produced an average of at least three young per adult per day over the 7-day period prior to the test. The adults showed no signs of disease or stress and no ephippia were produced during the holding period. To initiate the test, the juvenile daphnids were collected from the cultures and indiscriminately transferred one or two at a time to transfer chambers until each chamber contained 5 daphnids. Each group of neonates then was impartially assigned to a control or treatment group and the neonates were transferred to the test compartments to initiate the test. All transfers were made below the water surface using wide-bore pipettes.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

No post exposure observation period specified

Hardness:

Hardness (mg/L as CaCO3) 140 ± 7 (132 - 148)

Test temperature:

Water temperatures were within the 20 ± 1°C range established for the test.

pH:

Measurements of pH ranged from 8.0 to 8.1 during the test.

Dissolved oxygen:

Dissolved oxygen concentrations remained ≥ 80% of saturation (7.2 mg/L).

Salinity:

Not required - freshwater study

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal concentrations selected for use in the study were 7.68, 19.2, 48.0, 120 and 300 μg/L.
The measured concentrations of samples collected to verify the diluter system prior to the test ranged from 86.3 to 96.7% of nominal concentrations.

Details on test conditions:

Dilution Water
The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International site. The well water was passed through a sand filter to remove particles greater than approximately 25 μm, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45 μm to remove fine particles and was passed through an ultraviolet (UV) sterilizer.
The well water is characterized as moderately-hard water.

Test Apparatus
The toxicity test was conducted using an exposure system consisting of a continuous-flow diluter used to deliver each concentration of the test substance, a solvent control and a negative control (dilution water) to test chambers. Syringe pumps (Harvard Apparatus, South Natick, Massachusetts) were used to deliver test substance stock solutions or solvent to impartially assigned mixing chambers where the stocks or solvent were mixed with dilution water prior to delivery to the test chambers. The flow of dilution water into each mixing chamber was controlled using rotameters and was adjusted to provide approximately five volume additions of test water in each test chamber per day. After mixing, the flow from each mixing chamber was split to deliver test water to two replicate test chambers.
The syringe pumps used to deliver stock solutions or solvent to the mixing chambers were calibrated prior to the test. The rotameters used to control the flow of dilution water to the mixing chambers were calibrated prior to the test and calibrated/verified approximately weekly during the test. The proportion of the test water that was split into each replicate test chamber was checked prior to the test and approximately weekly during the test to ensure that flow rates varied by no more than ± 10% of the mean flow rate for the two replicates. Delivery of test solutions to the test chambers was initiated three days prior to the introduction of the test organisms to the test water in order to achieve equilibrium of the test substance. The general operation of the exposure system was checked visually at least once on the first and last days of the test and at least two times per day during the test.
The test chambers were placed in a temperature-controlled water bath to maintain the target water temperature throughout the test period. Test chambers were 25-L Teflon®-lined stainless steel aquaria filled with approximately 22 L of test water. The daphnids were held in two test compartments suspended in each of two test chambers. Test compartments were 300 mL glass beakers, approximately 6.5 cm in diameter and 12 cm in height. Nylon mesh screens covered two holes on opposite sides of each test compartment to permit test solution to flow in and out of the compartment. The depth of the test water in a representative compartment was approximately 8 cm, while the depth of water in a representative test chamber was approximately 29 cm. All test chambers were labeled with the project number, test concentration and replicate designation.

Environmental Conditions
Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity was measured at the water surface of one representative test chamber at the beginning of the test using a SPER Scientific Model 840006 light meter.
The target test temperature during the test was 20 ± 1°C. Temperature was measured in each test chamber at the beginning of the test, approximately weekly during the test, and at the end of the test using a liquid-in-glass thermometer. Temperature also was monitored continuously in one negative control test chamber using a validated environmental monitoring system (Amegaview Central Monitoring System), which were calibrated prior to exposure initiation and verified or calibrated approximately weekly during the test with a hand-held liquid-in-glass thermometer.
Dissolved oxygen was measured in one replicate test chamber of each treatment and control group at the beginning of the test, approximately three times per week during the test, and at the end of the test. Measurements typically rotated between the two replicates in each treatment or control group at each measurement interval. Measurements of pH were made in one replicate test chamber of each treatment and control group at the beginning of the test, approximately weekly during the test, and at the end of the test. Measurements typically rotated between the two replicates in each treatment or control group at each measurement interval. Dissolved oxygen was measured using a Thermo Orion 850Aplus dissolved oxygen meter, and measurements of pH were made using a Thermo Orion 525Aplus pH meter.
Hardness, alkalinity and specific conductance were measured in alternating replicates of the negative control (dilution water) and the highest test concentration at the beginning of the test, approximately weekly during the test and at the end of the test. Hardness and alkalinity were measured by titration based on procedures in Standard Methods for the Examination of Water and Wastewater. Specific conductance was measured using an Acorn Series Model CON6 Conductivity-Temperature meter. Total organic carbon (TOC) in the dilution water at the beginning and end of the test was measured using a Shimadzu Model TOC-VCSH total organic carbon analyzer, based on procedures in Standard Methods for the Examination of Water and Wastewater.

Biological Observations and Measurements
Observations of each first-generation daphnid were made daily during the test. At these times, the numbers of immobile daphnids were recorded along with any clinical signs of toxicity (e.g., inability to maintain position in the water column, uncoordinated swimming or cessation of feeding). Immobility was defined as a lack of movement, except for minor spontaneous random movement of the appendages. The presence of eggs in the brood pouch, aborted eggs, males or ephippia also were recorded daily. With the onset of reproduction, neonates produced by the first-generation daphnids were counted and then discarded every Monday, Wednesday and Friday during the test. The body length and the dry weight of each surviving first-generation daphnid were measured at the end of the test.

Statistical Analyses
Test endpoints analyzed statistically for first-generation daphnids were survival, reproduction, and growth (length and dry weight). Reproduction was based on the number of live young produced per reproductive day and the number of live young produced per parent animal alive at the beginning of the test. Reproductive days were defined as the number of days that the adult daphnid was alive from the day the first brood was released from any adult daphnid in the test until test termination. If an adult daphnid died, the number of reproductive days, for that adult, ended on the last day it was alive.
Negative control and solvent control data for each parameter were compared using an appropriate statistical test. No significant differences between the control groups were found for any parameter tested (α = 0.05). Therefore, the control data were pooled for comparison with the treatment groups.
Survival data was considered to be discrete-variable data, while reproduction and growth data were considered continuous-variable data. Discrete-variable data were analyzed using Chi-square and Fisher’s Exact test to identify treatment groups that showed a statistically significant difference (α = 0.05) from the control. All continuous-variable data were evaluated for normality using Shapiro-Wilk’s and for homogeneity of variance using Levene’s test (α = 0.01). When the data passed the assumptions of normality and homogeneity, those treatments that were significantly different from the control means were identified using Dunnett’s test (α = 0.05). All statistical tests were performed using a personal computer with TOXSTAT or SAS software.
The results of the statistical analyses were used to aid in the determination of the NOEC, LOEC and MATC. However, scientific judgement was used to determine if statistical differences were biologically meaningful, and if the data followed a concentration-dependent response. The NOEC was defined as the highest test concentration that produced no significant treatment-related effects on survival, reproduction or growth. The LOEC was defined as the lowest test concentration that produced a significant treatment-related effect on survival, reproduction or growth. The MATC was calculated as the geometric mean of the NOEC and LOEC. EC50 values were determined based on reproduction and on the immobility observed in the first-generation daphnids at the end of the test.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

41.5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: reproduction & growth

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

106 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: reproduction & growth

Duration:

21 d

Dose descriptor:

other: MATC

Effect conc.:

66.3 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: reproduction & growth

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

198 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: 95% confidence interval of 106 to 268 μg/L

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

150 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

per reproductive day

Remarks on result:

other: 95% confidence interval of 125 to 167 μg/L

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

151 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

number of live young produced per adult at the start of the test

Remarks on result:

other: 95% confidence interval of 117 to 170 μg/L.

Details on results:

Measurement of Test Concentrations
Nominal concentrations selected for use in the study were 7.68, 19.2, 48.0, 120 and 300 μg/L. During the course of the test, the appearance of the test solutions at these nominal concentrations was observed in both the diluter mixing chambers, where test substance stocks and dilution water were combined prior to delivery to the test chambers, and in the test chambers. Two days prior to the beginning of the exposure, a very slight slick was noted on the surface of the solution in the mixing chamber of the 300 μg/L nominal concentration. Therefore, an impellor was placed in the mixing chamber to aid in the mixing of the test stock solution and the dilution water in the mixing chamber prior to delivery to the test chambers. Following the addition of the impellor, the slick was no longer observed in the in the mixing chamber of the 300 μg/L nominal concentration, and the test solutions in the mixing chambers and test chambers appeared clear and colorless during the test, with no evidence of precipitation observed in any control or treatment solution.
The measured concentrations of samples collected to verify the diluter system prior to the test ranged from 86.3 to 96.7% of nominal concentrations. Samples of the test solutions collected during the test had measured concentrations that ranged from 82.1 to 95.2% of nominal concentrations. When the measured concentrations of test solution samples collected on Days 0, 7, 14 and 21 of the test were averaged for each treatment group, the mean measured test concentrations were 6.77, 17.0, 41.5, 106 and 268 μg/L, which represented 88, 89, 86, 88 and 89% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations.

Water Chemistry Measurements
Water temperatures were within the 20 ± 1°C range established for the test. Dissolved oxygen concentrations remained ≥ 80% of saturation (7.2 mg/L). Measurements of pH ranged from 8.0 to 8.1 during the test. Measurements of specific conductance, hardness and alkalinity were comparable between the control and treatment group and did not appear to be influenced by Reofos 35 concentration. TOC in the dilution water at test initiation and termination was <1 mg C/L. Light intensity at test initiation was 1351 lux at the surface of the water of one representative test chamber.

Survival and Clinical Observations
After 21 days of exposure, survival in the negative control groups was 90. There was no significant difference in survival between the negative and solvent control groups. Therefore, the control data were pooled for comparison with the treatment groups. Survival in the pooled control, 6.77, 17.0, 41.5, 106 and 268 μg/L treatment groups at test termination was 93, 95, 100, 90, 95 and 25%, respectively. Fisher’s Exact test indicated that the decrease in survival in the 268 μg/L treatment group was statistically significant in comparison to the pooled control (p ≤ 0.05). Consequently, the NOEC for survival was 106 μg/L and the LOEC was 268 μg/L. Based on the mortalities observed in the treatment groups, the 21-day EC50 value was 198 μg/L, with a 95% confidence interval of 106 to 268 μg/L.
Daphnids in the 6.77, 17.0, 41.5 and 106 μg/L treatment groups that survived to test termination generally appeared normal. In the 268 μg/L treatment group, all surviving first-generation daphnids were considered to be small in stature from Day 5 through Day 21 of the test. Lethargy and/or discoloration were also noted in the daphnids in this treatment group during the test.

Reproduction
Reproduction data from the surviving daphnids in the 268 μg/L treatment group were excluded from statistical analyses due to the statistically significant effects on survival in that treatment group. The first day of brood production in the negative and solvent control replicates and in the 6.77, 17.0, 41.5 and 106 μg/L Reofos 35 treatment replicates was Day 7 or 8 of the test. The exception was noted in replicate C of the 106 μg/L where the first day of brood production was on Day 10 of the test. The first generation daphnids in the 268 μg/L Reofos 35 treatment replicates did not begin production until Day 15 of the test, indicating a delay in reproduction for this treatment group. With the exception of one immobile neonate in negative control replicate D, there were no immobile neonates observed in the controls or the 6.77, 17.0, 41.5 and 106 μg/L treatment groups.
Immobile neonates were observed in the 268 μg/L treatment group during the test. Aborted or shed eggs were also observed in replicates A, B and C of the 268 μg/L treatment group. No males or ephippia were produced in the controls or treatment groups during the test. The delay in brood production and the production of immobile neonates and aborted eggs in the 268 μg/L treatment group was considered a treatment related effect.
Adult daphnids in the negative and solvent control groups produced an average of 13.73 and 13.78 live young per reproductive day, respectively. There was no significant difference in reproduction between the negative and solvent control groups. Therefore, the control data were pooled for comparison with the treatment groups. Adult daphnids in the pooled control, 6.77, 17.0, 41.5, 106 and 268 μg/L treatment groups produced an average of 13.75, 15.48, 13.58, 14.30, 9.83 and 0.112 live young per reproductive day, respectively. Dunnett’s test indicated there was a statistically significant decrease in mean neonate production in the 106 μg/L treatment group in comparison to the pooled control (p ≤ 0.05). Consequently, the NOEC for reproduction was 41.5 μg/L and the LOEC was 106 μg/L. Based on the mean number live young per reproductive day, the 21-day EC50 value was 150 μg/L, with a 95% confidence interval of 125 to 167 μg/L.
Adult daphnids in the negative and solvent control groups produced an average of 193.7 and 205.8 live young per adult at the start of the test, respectively. There was no significant difference in reproduction between the negative and solvent control groups. Therefore, the control data were pooled for comparison with the treatment groups. Adult daphnids in the pooled control, 6.77, 17.0, 41.5, 106 and 268 μg/L treatment groups produced an average of 199.8, 221.0, 203.8, 201.1, 142.6 and 1.40 live young per reproductive day, respectively. Dunnett’s test indicated there was a statistically significant decrease in mean neonate production in the 106 μg/L treatment group in comparison to the pooled control (p ≤ 0.05). Consequently, the NOEC for reproduction per adult at the start of the test was 41.5 μg/L and the LOEC was 106 μg/L. Based on the mean number of live young produced per adult at the start of the test, the 21-day EC50 value was 151 μg/L, with a 95% confidence interval of 117 to 170 μg/L.

Growth
Daphnids in the negative control group averaged 4.65 mm in length and 1.38 mg in dry weight, while daphnids in the solvent control group averaged 4.73 mm in length and 1.26 mg in dry weight. There were no significant differences in the growth parameters between the negative and solvent control groups. Therefore, the control data were pooled for comparison with the treatment groups. Daphnids in the pooled control, 6.77, 17.0, 41.5, 106 and 268 μg/L treatment groups had mean lengths of 4.69, 4.73, 4.75, 4.73, 4.28 and 2.50 mm, respectively, and mean dry weights of 1.32, 1.36, 1.21, 1.26, 1.01 and 0.16 mg, respectively. Growth measurements from the surviving daphnids in the 268 μg/L treatment group were excluded from statistical analyses due to the statistically significant effects on survival in that treatment group. Dunnett’s test indicated there was a statistically significant decrease in mean length and mean dry weight in the 106 μg/L treatment group in comparison to the pooled control (p ≤ 0.05). Consequently, the NOEC for growth (length and dry weight) was 41.5 μg/L, and the LOEC was 106 μg/L.

Results with reference substance (positive control):

Reference substance not used in this study.

Measured Concentrations of Reofos 35 in Test Solution Samples

Nominal Test Concentration (μg/L)	Rep.	Sample Number (616A-117-)	Sampling Time (Days)	Measured Concentration (μg/L)^1,2	Percent of Nominal^2	Mean Measured Concentration (μg/L)	Mean Measured Percent of Nominal
Negative Control	A	1	0	<LOQ	--	<LOQ	--
	B	8	7	<LOQ	--
	A	15	14	<LOQ	--
	B	22	21	<LOQ	--
Solvent Control	A	2	0	<LOQ	--	<LOQ	--
	B	9	7	<LOQ	--
	A	16	14	<LOQ	--
	B	23	21	<LOQ	--
7.68	A	3	0	6.35	82.7	6.77 ± 0.328 CV = 4.84%	88
	B	10	7	7.15	93.1
	A	17	14	6.78	88.3
	B	24	21	6.81	88.7
19.2	A	4	0	17.4	90.6	17.0 ± 0.685 CV = 4.04%	89
	B	11	7	16.0	83.3
	A	18	14	17.5	91.1
	B	25	21	17.0	88.5
48.0	A	5	0	40.4	84.2	41.5 ± 2.86 CV = 6.89	86
	B	12	7	16.0	83.3
	A	18	14	17.5	91.1
	B	26	21	45.7	95.2
120	A	6	0	113	94.2	106 ± 5.29  CV = 4.99%	88
	B	13	7	103	85.8
	A	20	14	101	82.1
	B	26	21	107	89.2
300	A	7	0	265	88.3	268 ± 6.13  CV = 2.29%	89
	B	14	7	276	92.0
	A	21	14	270	90.0
	B	28	21	262	87.3

¹The limit of quantification (LOQ) was 2.50 μg/L, calculated as the product of the concentration of the lowest calibration standard (5.00 μg/L) and the dilution factor of the matrix blank samples (0.500).

²Results were generated using Excel 2010 in full precision mode. Manual calculations may differ slightly.

 

Means and Ranges of Water Quality Measurements Taken During the 210Day Exposure to Reofos 35

Mean Measured Concentration (μg/L)	Mean ± Std. Dev. And Range of Measured Parameters^1
	Temperature^2(°C)	Dissolved Oxygen^3(mg/L)	pH	Hardness^4(mg/L as CaCO3)	Alkalinity^4(mg/L as Ca CO3)	Conductivity^4(μS/cm)
Negative Control	19.9 ± 0.12 (19.8 – 20.1)	8.8 ± 0.16 (8.6 – 9.1)	8.1 ± 0.05 (8.0 – 8.1)	140 ± 7 (132 – 148)	179 ± 2 (177 – 180)	345 ± 34 (302 – 379)
Solvent Control	19.9 ± 0.16 (19.7 – 20.1)	8.3 ± 0.43 (7.3 – 8.7)	8.1 ± 0.06 (8.0 – 8.1)	-- --	-- --	-- --
6.77	19.9 ± 0.16 (19.7 – 20.1)	8.1 ± 0.41 (7.5 – 8.7)	8.1 ± 0.06 (8.0 – 8.1)	-- --	-- --	-- --
17.0	19.9 ± 0.19 (19.6 – 20.1)	8.2 ± 0.42 (7.4 – 8.7)	8.1 ± 0.06 (8.0 – 8.1)	-- --	-- --	-- --
41.5	19.8 ± 0.23 (19.5 – 20.1)	8.0 ± 0.40 (7.2 – 8.6)	8.1 ± 0.06 (8.0 – 8.1)	-- --	-- --	-- --
106	19.8 ± 0.21 (19.5 – 20.1)	8.0 ± 0.38 (7.3 – 8.6)	8.1 ± 0.06 (8.0 – 8.1)	-- --	-- --	-- --
268	19.8 ± 0.23 (19.5 – 20.1)	7.9 ± 0.43 (7.3 – 8.7)	8.1 ± 0.06 (8.0 – 8.1)	141 ± 7 (132 – 148)	180 ± 4  (177 – 184)	350 ± 33 (306 – 380)

¹Total organic carbon measured in the dilution water at test initiation and termination was <1 mg C/L

²Temperature monitored continuously during the test ranged from approximately 19.41 to 20.14°C, measured to the nearest 0.01°C

³A dissolved oxygen concentration of 5.4 mg/L represents 60% saturation at 20°C in freshwater

⁴-- = no measurements scheduled

 

Summary of Survival, Reproduction and Growth ofDaphnia magnaExposed to Reofos 35 for 21 Days

Mean Measured Concentration^1(μg/L)	Percent Adult Survival	Mean No. Neonates Per Adult at Start ± Std. Dev.	Mean No. Neonates Per Reproductive Day ± Std. Dev.	Mean Length ± Std. Dev. (mm)	Mean Dry Weight ± Std. Dev. (mg)
Negative Control	90	193.7 ± 21.86	13.73 ± 1.821	4.65 ± 0.100	1.38 ± 0.145
Solvent Control	95	205.8 ± 14.59	13.78 ± 0.998	4.73 ± 0.0957	1.26 ± 0.109
Pooled Control	93	199.8 ± 18.29	13.75 ± 1.360	4.69 ± 0.0991	1.32 ± 0.135
6.77	95	221.0 ± 14.48	15.48 ± 0.818	4.73 ± 0.0957	1.36 ± 0.0850
17.0	100	203.8 ± 37.44	13.58 ± 2.497	4.75 ± 0.0577	1.21 ± 0.152
41.5	90	201.1 ± 23.74	14.30 ± 1.742	4.73 ± 0.0500	1.26 ± 0.0789
106	95	142.6 ± 34.70*	9.83 ± 1.884*	4.28 ± 0.150*	1.01 ± 0.0275*
268	25*	1.40 ± 2.406^1	0.112 ± 0.183^1	2.50 ± 0.656^1	0.16 ± 0.121^1

1 Reproduction and growth data from the 268 μg/L treatment group were excluded from the statistical analysis due to statistically significant effect on survival at test end.

* Indicates a significant difference (p≤0.05) from the pooled control using Fisher’s Exact test for survival endpoint and Dunnett’s one-tailed test for reproduction and growth endpoints

Validity criteria fulfilled:

yes

Conclusions:

The cladoceran, Daphnia magna, was exposed to Reofos 35 at mean measured concentrations of 6.77 to 268 μg/L under flow-through conditions for 21 days. There were statistically significant treatment-related effects on survival at concentrations ≥268 μg/L. Reproduction and growth, measured as length and dry weight, were the most sensitive biological endpoint measured in this study. Daphnids exposed to Reofos 35 at concentrations ≥106 μg/L had statistically significant reductions in reproduction, length and dry weight in comparison to the pooled control. Consequently, the NOEC, based on reproduction and growth, was 41.5 μg/L, the LOEC was 106 μg/L and the MATC was calculated to be 66.3 μg/L. The 21-day EC50 value for adult immobility was 198 μg/L, with a 95% confidence interval of 106 to 268 μg/L. The 21-day EC50 value for reproduction based on the number of live young produced per reproductive day was 150 μg/L, with a 95% confidence interval of 125 to 167 μg/L. The 21-day EC50 value for reproduction based on the number of live young produced per adult at the start of the test was 151 μg/L, with a 95% confidence interval of 117 to 170 μg/L.

Executive summary:

The objective of this study was to determine the effects of Reofos 35 on the survival, growth and reproduction of the cladoceran, Daphnia magna, during a 21-day exposure period under flow-through test conditions. The cladoceran, Daphnia magna, was exposed to Reofos 35 at mean measured concentrations. The study was performed in accordance with U.S. EPA OPPTS NUMBER 850.1300 and OECD GUIDELINE 211.

 

The cladoceran, Daphnia magna, was exposed to Reofos 35 at mean measured concentrations of 6.77 to 268 μg/L under flow-through conditions for 21 days. There were statistically significant treatment-related effects on survival at concentrations ≥268 μg/L. Reproduction and growth, measured as length and dry weight, were the most sensitive biological endpoint measured in this study. Daphnids exposed to Reofos 35 at concentrations ≥106 μg/L had statistically significant reductions in reproduction, length and dry weight in comparison to the pooled control. Consequently, the NOEC, based on reproduction and growth, was 41.5 μg/L, the LOEC was 106 μg/L and the MATC was calculated to be 66.3 μg/L. The 21-day EC50 value for adult immobility was 198 μg/L, with a 95% confidence interval of 106 to 268 μg/L. The 21-day EC50 value for reproduction based on the number of live young produced per reproductive day was 150 μg/L, with a 95% confidence interval of 125 to 167 μg/L. The 21-day EC50 value for reproduction based on the number of live young produced per adult at the start of the test was 151 μg/L, with a 95% confidence interval of 117 to 170 μg/L.

Description of key information

The recent 21-d daphnia study is taken as the value for hazard assessment.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

41.5 µg/L

Additional information

The data set available for the phosphates as a group indicates that these do pose hazardous effects to Daphnia. However as detailed within the registration dossier, such effects are considered to be attributable to the content of the impurity, triphenyl phosphate, CAS 115-86-6, (EC No. 204-112-2). The content of this impurity is considered to adversely affect the toxicity of the substance within aquatic organisms. The registration dossier that two separate classifications applicable to the substance for this endpoint as follows:

• Phenol, isopropylated, phosphate (3:1) [Triphenyl phosphate >5%] - H410: Very toxic to aquatic life with long lasting effects.

• Phenol, isopropylated, phosphate (3:1) [Triphenyl phosphate < 5%] - H413: May cause long lasting harmful effects to aquatic life.

It is considered that given the age of this data, that the Triphenyl phosphate is considered to be present in these products at and above the 5% threshold proposed. As such, this data as presented is considered to represent the classification where the Triphenyl phosphate content is greater than 5%.

For this data where Triphenyl phosphate >5%, the 21– day NOEC from the recent Daphnia magna Reproduction Test (OECD 211) is taken as the value for hazard assessment. This gives a value of 41.5 µg/L.

The supporting data set available as follows is offered as an indication of the accuracy of the above study, and provides for the following values:

 

Daphnia
Substance	Endpoint	Result	M or N	Trade name	Basis for effect
Phenol, isopropylated, phosphate(3:1)	21 d NOEC	0.027 mg/L	nominal	Trade Name Kronitex 200	Survival
	21 d NOEC	0.006 mg/L	nominal	Trade Name Kronitex 200	reproduction
	21 d NOEC	0.028 mg/L	nominal	Trade Name Phosflex 31P	Survival
	21 d NOEC	0.028 mg/L	nominal	Trade Name Phosflex 31P	reproduction
Gammarus pseudolimnaeus
Phenol, isopropylated, phosphate(3:1)	90 d NOEC	0.011 mg/L	nominal	Trade Name Kronitex 200	Survival
	90 d NOEC	0.063 mg/L	nominal	Trade Name Kronitex 200	Growth
	90 d NOEC	0.019 mg/L	nominal	Trade Name Phosflex 31P	Survival
	90 d NOEC	>= 0.088 mg/L	nominal	Trade Name Phosflex 31P	Growth
Chironomus plumosus
Phenol, isopropylated, phosphate(3:1)	30 d NOEC	0.184 mg/L	nominal	Trade Name Kronitex 200	Emergence
	30 d NOEC	0.184 mg/L	nominal	Trade Name Phosflex 31P	Emergence