Prometryn

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Oct 1987 to 9 Nov 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 72-4 (Fish Early Life-Stage and Aquatic Invertebrate Life-Cycle Studies)

Deviations:

yes

Remarks:

See protocol deviations in "Principle of method if other than guideline"

Principles of method if other than guideline:

The test was conducted according to "Protocol for Conducting a Flow-Through Life Cycle Toxicity Test with Daphnia magna" (Protocol# 081087/DM.LC) from the test facility.

Protocol deviations
1. The protocol states that each test vessel will receive 0.5 mL of yeast and 2.0 mL of algal suspension at each feeding period. During this study the algal suspension was fed at a rate of 3.0 mL per feeding interval.
2. The protocol states that specific conductance is determined at test initiation and weekly thereafter during the exposure period. During this study, specific conductance was determined at test initiation and on days 7 and 14. Measurement of this water quality parameter was inadvertently
omitted on day 21.
These deviations were not expected to affect the results of this study.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Water samples were removed from two replicate vessels of the high, middle, and low test concentrations twice prior to initiating the definitive chronic exposure. Results of these pretest analyses were used to judge whether sufficient quantities of the test substance were being delivered to the test vessels and appropriate test concentrations were being maintained to initiate the chronic exposure. During the in-life phase of the definitive test, water samples were removed and analyzed for the test substance weekly from two replicate vessels of each test concentration and the controls.

Vehicle:

yes

Remarks:

acetonitrile

Details on test solutions:

Approximately 0.1 g of the test substance was weighed on a balance in a 100 mL volumetric flask and solubilized in acetonitrile. The stock solution (ca. 1 µg/µL) was stored refrigerated (4 - 10°C) in a 100 mL amber serum vial with a teflon-lined lid. This stock was then used, with appropriate dilution for this study.
A 200 mL Mount and Brungs (1967) proportional diluter, calibrated to provide 50% dilutions between adjacent concentrations, delivered the dilution water and the test substance to the test vessels during the chronic toxicity test. The diluter was constructed entirely of glass and silicone tubing, stoppers and sealant. The diluter was equipped with a metering pump which delivered 390 mL of a test substance stock solution (2.0 mg/L) directly into the diluter mixing chamber during each diluter cycle. This 390 mL solution (2.0 mg/L) served as the highest treatment level from which calibrated volumes were diluted diluter to provide the 50% nominal concentration gradient (0.13 to 2.0 mg/L of the test substance). Five-centimeter (cm) lengths of 1 mm (inside diameter) glass capillary tubing were inserted through silicone stoppers in the mixing/splitting chambers of the diluter and into the test solution delivery tubes. This tubing served to restrict the flow of the test solutions, minimizing potentially stressful turbulence in the test vessels and providing equal distribution of the test solutions to the replicate vessels.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: Obtained from populations cultured at the test facility
- Stage at study initiation: < 24 hour old

FEED DURING TEST
- Food type: A diet consisting of a suspension of Fleischmann's yeast (5 mg/mL), a suspension of green algae (Ankistrodesmus falcatus; 4.0E+07 cells/mL), and a suspension of Selco (0.6 mg/mL, a commercial mixture of proteins and fatty acids).
- Method: The food suspensions were added to each replicate vessel at a rate of 0.5 mL of yeast suspension, 3.0 mL of algal suspension, and 1.0 mL of Selco suspension
- Frequency: Three times daily on weekdays and twice daily on weekends.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

180 mg/L as CaCO3

Test temperature:

20 ± 1°C

pH:

7.9 - 8.3

Dissolved oxygen:

8.4 - 8.7 mg O2/ L

Nominal and measured concentrations:

Nominal concentration: Control, 0.13, 0.25, 0.50, 1.0 and 2.0 mg/L.
Measured concentration: < 0.049, 0.10, 0.27, 0.46, 1.0 and 2.0 mg/L, respectively. See Table 1 in "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: 1.8 L glass battery jars
- Type of flow-through: Proportional diluter
- Test solution deliver rate: Approximately 6 aquarium volumes per 24-hour period in order to provide a 90% test solution replacement rate of approximately 9 hours
- No. of organisms per vessel: 20
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- Light intensity and source: 38 to 70 footcandles, a combination of cool white and wide spectrum Gro-lux fluorescent light
- Photoperiod: 16 hours/8 hours (light/dark)

TEST MEDIUM
- Source/preparation of dilution water: Culture and test dilution water were prepared by fortifying well water according to the formula for hard water (ASTM, 1980) and filtering it through an Amberlite XAD-7 resin column to remove any potential organic contaminants.
- Total hardness: 160 - 180 mg/L as CaCO3
- Alkalinity: 110 - 130 mg/L
- pH: 7.9 - 8.3
- Temperature: 20 ± 1°C
- Dissolved oxygen: > 60% of saturation
- Conductivity: 400 - 600 micromhos per centimeter (µmhos/cm)

WATER PARAMETERS
The test solution temperature was measured daily in one replicate vessel of each treatment level and control solution throughout the definitive 21-day exposure. The dissolved oxygen concentration of the test solutions was measured every weekday in one replicate vessel of each treatment level and control solution. In addition, on one day of each week the pH, temperature and dissolved oxygen concentration were measured in each replicate exposure vessel of all treatment levels and the control. Total hardness, alkalinity and specific conductivity of the test solutions were monitored weekly in one aquarium from each treatment and control solution.

EFFECT PARAMETERS MEASURED:
Adult survival and measurements of offspring production were made on days 1, 2 and 4, and every Monday, Wednesday and Friday from day 7 through 21. The offspring were removed, counted and discarded. Test vessels were brushed to remove algal growth and the solution filtered through a fine mesh net a minimum of twice each week.

RANGE FINDING STUDY
- Acute static test: During this preliminary test, daphnids were exposed for 120 hours to nominal concentrations of the test substance ranging from 20 to 1.0 mg/L.
- Results of acute static test: After 120 hours of exposure, 93% mortality was observed among organisms exposed to 20 mg/L of the test substance. Mortality of < 13% and no adverse effects (behavior or physical) were observed among daphnids in the remaining treatment levels (10, 5.0 and 1.0 mg/L).
- Initial chronic test: Based on the results of the acute static test, the following nominal concentrations were selected for the initial chronic exposure: 20, 10, 5.0, 2.5 and 1.3 mg/L.
- Results of initial chronic test: Results of these weekly analyses established that the measured concentrations averaged 90% of the nominal levels and defined the mean measured exposure concentrations as 19, 9.3, 4.1, 2.7 and 0.87 mg/L of the test substance.
Based on the observation of reproductive effects at all concentrations of the test substance tested, including the lowest treatment level (1.3 mg/L, nominal), the initial chronic exposure was terminated after 14 days. All daphnids in exposure solutions containing 20 mg/L of the test substance died within the initial 4 days of the exposure. By test day 14, daphnid survival was also significantly reduced (80% survival) at the 10 mg/L treatment level when compared to the survival of the control daphnids (99%). EC50 values (95% confidence interval) for test days 7 and 14 in this initial exposure were calculated by moving average angle analysis to be 11.8 (10.9 - 12.9) mg/L and 10.3 (9.5 - 11.2) mg/L, respectively. Evidence for a marked effect on the reproductive performance was produced by organisms at the measured concentrations of 9.3, 4.1 and 2.7 mg/L. The relationship was clearly concentration dependent and nearly linear based on the number of offspring produced by day 14.In addition to the observed reproductive effects, visual observation of the exposed organisms on day 14 established that the surviving daphnids in all of the tested concentrations were smaller than the control daphnids. Based on these results, it was apparent that the concentration of the test substance estimated to cause reproductive and growth effects was more than a factor of 5 less than the geometric mean MATC based on a survival effect (approximately 7.1 mg/L, nominal), and the life cycle test was restarted at lower test concentrations. For the definitive 21 day life cycle exposure a nominal concentration range of 2.0 to 0.13 mg/L of the test substance was selected and approved by the study sponsor.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth

Details on results:

Overview of results are provided in the "Any other information on results incl. tables"
SURVIVAL
At test termination survival of daphnids at the highest mean measured concentration, 2.0 mg/L of the test substance, was 91% and was not significantly different from the survival of the control organisms (93%). Survival of daphnids at the remaining lower test concentrations (1.0 to 0.10 mg/L) was between 90 and 95% and also statistically comparable to the survival of the control daphnids. Survival of daphnids throughout the 21 day exposure period was not an indicator of adverse effects of the test substance exposure at the concentrations tested.

REPRODUCTION
Control daphnids had begun to release offspring by test day 9. By test termination, control daphnids had produced an average of 42 ± 8.7 offspring. Time to first offspring released and total number of offspring produced by control organisms exceeded the minimum standard performance criteria (e.g. 40 offspring per female) established by the EPA (1985). During the 21 days of exposure daphnids in the highest test concentration (2.0 mg/L) released an average of 36 offspring per female which was statistically comparable to the number of offspring released by the control daphnids. During the same time period daphnids exposed to the remaining treatment levels (1.0 to 0.10 mg/L) released cumulative numbers of offspring (35 - 42 per female) which were statistically comparable to the reproductive performance of the control organisms. Similar to daphnid survival, the reproductive performance by organisms during this life cycle study was not an indicator of the toxicity of the test substance at the concentration range tested.

GROWTH
The body length of control daphnids measured at test termination established that the length of the surviving control organisms averaged 4.2 (S.D. 0.26) mm. The mean body length of daphnids in the highest test concentration (2.0 mg/L) averaged 3.5 (S.D. = 0.27) mm, which represented a 17% reduction in growth. Daphnids at the lower test concentrations had mean body lengths ranging from 3.7 to 3.9 mm. The data on body length failed to pass Bartlett's test for homogeneity of variance (p < 0.01), and therefore the nonparametric Kruskal-Wallis test was used to determine significant differences among treatment groups. The results of this test indicated that the body lengths of daphnids exposed to 2.0 mg/L the test substance were significantly less than body lengths of controls. Body lengths of daphnids exposed to lower test concentrations were not significantly different from controls.
Data generated during the initial exposure established that EC50 values (95% confidence interval) for test days 7 and 14 for the test substance and D. magna were 11.8 (10.9-12.9) mg/L and 10.3 (9.5-11.2) mg/L, respectively. Based on the reduced growth (measured as body length) observed at the highest test concentration during the definitive chronic study, the estimated maximum acceptable toxicant concentration (MATC after 21 days exposure was > 1.0 mg/L and < 2.0 mg/L of the test substance (geometric mean MATC = 1.4 mg/L).

Reported statistics and error estimates:

See statistical analysis in "Any other information on materials and methods incl. tables"

Table 2. Weekly mean percentage survival of daphnids (Daphnia magna) during the 21-day chronic exposure to the test substance

Mean measured concentration (mg/L)	Mean percentage survival (S.D.)
	Day 7	Day 14	Day 21
Control	96 (8)	96 (8)	93 (9)
0.10	96 (5)	95 (7)	90 (7)
0.27	99 (3)	95 (4)	95 (4)
0.46	95 (4)	93 (6)	93 (6)
1.0	93 (6)	93 (6)	91 (6)
2.0	98 (3)	95 (7)	91 (9)

 

Table 3. Cumulative number of offspring produced per female daphnids (Daphnia magna) during the 21-day chronic exposure to the test substance

Mean measured concentration (mg/L)	Mean (standard deviation) cumulative number of offspring/female
	Day 7	Day 9	Day 11	Day 14	Day 16	Day 18	Day 21
Control	0	2 (0.6)	3 (0.5)	17 (1.3)	30 (7.0)	35 (8.9)	42 (8.7)
0.10	0	4 (1.9)	6 (3.0)	15 (2.4)	25 (5.9)	31 (8.3)	36 (7.8)
0.27	0	3 (1.3)	4 (1.3)	14 (3.1)	25 (7.0)	28 (7.5)	35 (8.2)
0.46	0	1 (1.5)	2 (2.2)	16 (2.2)	29 (2.9)	35 (5.1)	42 (5.3)
1.0	0	1 (0.8)	3 (2.2)	16 (3.9)	26 (3.9)	32 (6.9)	41 (8.2)
2.0	0	2 (0.5)	5 (1.3)	17 (2.1)	28 (3.3)	31 (3.4)	36 (4.3)

Table 4. Body length of daphnids (Daphnia magna) measured after 21 days of exposure to the test substance

Mean measured concentration (mg/L)		Body length (mm)	% of the mean control organism length
Control	A	4.3	---
	B	4.0
	C	4.3
	D	4.0
	Mean	4.2 (0.25)
0.10	A	3.8	93
	B	3.9
	C	4.2
	D	3.9
	Mean	3.9 (0.25)
0.27	A	3.6	88
	B	3.8
	C	3.7
	D	3.8
	Mean	3.7 (0.22)
0.46	A	3.8	93
	B	3.9
	C	3.8
	D	3.8
	Mean	3.9 (0.18)
1.0	A	3.7	90
	B	3.8
	C	3.8
	D	3.8
	Mean	3.8 (0.21)
2.0	A	3.5	83
	B	3.5
	C	3.6
	D	3.6
	Mean	3.5 (0.27)^a

a Values presented in parentheses equal standard deviation

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the 21-day NOEC was determined to be 1.0 mg/L.

Executive summary:

The chronic effect of continuous exposure to the test substance on aquatic invertebrates was evaluated in a single generation (21 day) flow-through life-cycle toxicity test with the freshwater invertebrate, Daphnia magna. The test was performed according to EPA OPP No. 72 – 4 guideline and in compliance with GLP. The following test conditions were determined: pH 7.9 – 8.3, 20 ± 1 °C and 8.4 – 8. 7 mg/L dissolved oxygen concentration. During the definitive 21-day chronic study, results of weekly analyses for the test substance established that the measured concentrations in the exposure solutions followed the expected concentration gradient and were consistent between replicate treatments and sampling intervals. Mean measured concentrations of the test substance in the test aquaria averaged 95% of the nominal levels (0.13, 0.25, 0.50, 1.0 and 2.0 mg/L) and were 0.10, 0.27, 0.46, 1.0 and 2.0 mg/L.  

At the termination of the 21-day definitive test, reduced organism growth, as determined by the measurement of individual body lengths, was the only indicator of toxicity of the test substance to Daphnia magna in the concentration range tested. On day 21, measurement of the surviving daphnids’ body length established that the control organisms had an average body length of 4.2 (S.D. 0.26) mm. The mean body length of daphnids exposed to the highest mean measured test concentration, 2.0 mg/L, averaged 3.5 (S.D. 0.27) mm which was significantly less than the length of the control daphnids and represented a 17% reduction in growth. No adverse effect was observed in the remaining lower treatment levels. Measurements at test termination established that daphnids in the remaining lower test concentrations had mean body lengths ranging from 3.7 to 3.9 mm and were not statistically different to the length of the control organisms. Survival and reproduction of daphnids throughout the 21-day exposure period were not affected in any treatment group. Mean survival in the 0.10 to 2.0 mg/L treatments was between 90 and 95% at test termination, compared to 93%in the control group. Similarly, no adverse effects on daphnid reproduction was observed in any of the tested concentrations during the 21-day exposure period. The NOEC was determined to be 1.0 mg/L  

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Nov 2012 to 20 Dec 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1350 (Mysid Chronic Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Prior to initiation of the test, analytical chemistry confirmed that the diluter test system was functioning properly and was adequately equilibrated. Water samples were collected from alternating replicate test chambers in each treatment and control group at the beginning of the test, approximately weekly during the test and at test termination to measure concentrations of the test substance. An additional set of samples were collected on Day 9 to confirm concentrations in the 1.0 mg a.i./L treatment group. The samples were collected from middepth, placed in glass scintillation vials, and processed immediately for analysis or stored refrigerated until they could be analyzed. An additional set of samples were collected on Day 28, test termination, and stored refrigerated as back-up samples for possible future analysis, if needed.

Vehicle:

yes

Remarks:

Dimethylformamide (DMF)

Details on test solutions:

Individual stock solutions were prepared for each of the concentrations tested, and were prepared two times during the test. All test solutions were adjusted to 100% active ingredient during preparation, based on the test substance purity (97.8%).
A primary stock solution was prepared by mixing a calculated amount of test substance into HPLC-grade dimethylformamide (DMF) at a nominal concentration of 50 mg a.i./mL. Four secondary stock solutions were prepared in DMF at nominal concentrations of 3.15, 6.5, 12.5 and 25 mg a.i./mL by proportional dilution of the primary stock. The stock solutions were mixed by inversion and all solutions appeared clear and colorless. Stock solutions were stored refrigerated in glass amber bottles, and aliquots of each stock were placed in the syringe pump every two to five days during the study.
Prior to pairing of the mysids, the five test substance stock solutions were injected into the diluter mixing chambers at a rate of 2.50 µL/minute where they were mixed with dilution water delivered at a rate of 125 mL/minute to achieve the desired test concentrations. Following pairing, the stock solutions were injected into the diluter mixing chambers at a rate of 5.00 µL/minute where they were mixed with dilution water delivered at a rate of 250 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 the test substance treatment groups was 0.02 mL/L.

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Details on test organisms:

TEST ORGANISM
- Common name: Saltwater mysid
- Age at study initiation: < 24 hours old

FEEDING
- Food type during cultivation and test: Live brine shrimp nauplii (Artemia sp.); The brine shrimp periodically were enriched with a nutrient enrichment
- Feeding frequency during cultivation: Daily
- Feeding frequency during test: 4 times/day
- The mysids in the cultures and test were fed the enriched brine shrimp for one of the daily feedings during the test. Mysid food was also periodically supplemented with Skeletonema costatum, a saltwater alga. Excess food and waste were siphoned out daily during observations.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
Adult mysids in the cultures were held in the laboratory for at least 14 days before juveniles were collected for testing. The culture was maintained in a flow-through saltwater system using water from the same source as used during the test. During the 2-week period immediately preceding the test, water parameters in the cultures were:
- Temperature: 25.1 - 28.6ºC
- pH: 8.0 - 8.2
- Dissolved oxygen: ≥ 7.1 mg O2/L (≥97% of saturation)
- Salinity: 19 - 20‰

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

28 d

Test temperature:

25 ± 2°C

pH:

7.9 - 8.1

Dissolved oxygen:

- 6.4 mg O2/L
- ≥ 87% of saturation

Salinity:

19 - 21‰

Nominal and measured concentrations:

Nominal concentrations: 0.063, 0.13, 0.25, 0.50 and 1.0 mg a.i./L
Measured concentrations: 0.055, 0.11, 0.22, 0.45 and 0.84 mg a.i./L, respectively. See Table 1 in "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
Prior to pairing
- Test compartment: 2 L glass container (12 cm in dameter and 19 cm in height with 2 nylon mesh covered holes on opposite sides)
- No. of organisms per compartment: 15 mysids/container
- Test vessel: 9 L glass aquaria (one test compartment was placed in each test vessel)
- Test solution volume: Approximately 2.5 L
- Water depth: 8.3 cm in test chamber and 7.6 cm in text compartment
After pairing
- Reproductive compartment: Approximately 10 cm diameter glass petri dishes (with sides of nylon mesh screen)
- No. of organisms per compartment: 1 pair/compartment
- Test vessel: 19 L glass aquaria (up to 5 reproductive compartments in each test vessel)
- Test solution volume: Approximately 14.5 L
- Water depth: 18.1 cm in test vessel and 17.8 cm in reproductive compartment

OTHER INFORMATION ON TEST APPARATUS
- Aeration: Yes (before test initiation)
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Flow rate: Each juvenile test chamber with at least 18 volume additions of test water per day and adult test chambers with at least 6 volume additions of test water per day.
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- No. of vessels per vehicle control: 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for culturing and testing was natural seawater collected at Indian River Inlet, Delaware. The freshly-collected seawater was passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800-L storage tank. The filtered saltwater then was diluted to a salinity of approximately 20‰ with freshwater from a well on the test facility site and was aerated with spray nozzles. Prior to use, the 20‰ water was filtered to 0.44 µm to remove fine particles and was passed through an ultraviolet (UV) sterilizer.

INTERVALS OF WATER QUALITY MEASUREMENT
- Temperature: Temperature was measured in each test chamber at the beginning and end of the test, and approximately weekly during the test. Temperature also was monitored continuously in one negative control test chamber (meters were calibrated prior to test initiation and verified or calibrated approximately weekly during the test with a hand-held liquid-in-glass thermometer).
- Dissolved oxygen: Prior to pairing, dissolved oxygen was measured in one replicate test chamber of each treatment and control group at the beginning of the test and approximately weekly during the test period, with measurements rotating among the replicates in each group at each measurement interval. After mysids attained sexual maturity and were paired on Day 14, dissolved oxygen was measured daily until the end of the test in one replicate test chamber of each treatment and control group, with measurements rotating among the replicates in each group at each measurement interval.
- pH: Measurements of pH were made in one replicate test chamber of each treatment and control group at the beginning and end of the test, and approximately weekly during the test, with measurements rotating among the replicates in each group at each measurement interval.
- Salinity: Salinity was measured daily in one replicate of the negative control, with measurements rotating among the replicates in the group at each measurement interval.

OTHER TEST CONDITIONS
- Photoperiod: 14 hours/10 hours (light/dark); A 120-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting.
- Light intensity: 195 lux (at the surface of the water of one representative test chamber)
- Light source: Fluorescent light bulbs that emit wavelengths similar to natural sunlight
- Light intensity was measured at the water surface of one representative test chamber at test initiation

EFFECT PARAMETERS MEASURED:
Observations of the survival and behavior of each first-generation mysid were made daily throughout the test. The criteria for death included lack of movement, absence of respiratory movements, and lack of reaction to gentle prodding. At pairing on Day 14, the sex and maturity of each mysid was determined by microscopic examination, and when possible, five male/female pairs were assigned to reproductive compartments in each replicate test
chamber, with one pair per compartment. Any immature mysids or extra females were discarded at this time. Any sexually mature males remaining after pairing were maintained in a separate compartment within the respective replicate test chamber. If a male in a reproductive compartment died, it was replaced with a male from the pool of males maintained in the same replicate, if available.
Following pairing, reproductive females were observed daily for presence of eggs in the brood pouch. Second-generation mysids produced in each compartment were counted, recorded and removed daily. Second-generation mysids were also observed for abnormal development and abnormal behavior. The test was terminated on Day 28, which was at least seven days past the median time of first brood release for the negative and solvent controls (Day 20). At test termination, the sex of each surviving first-generation mysid was confirmed and the total length of each mysid was measured using calipers. The mysids then were placed in a drying oven, set at approximately 60°C, to obtain dry weight data. The mysids were dried for approximately 41 to 45 hours.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

0.22 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

Overview of the results are provided in "Any other information on results incl. tables"
JUVENILE SURVIVAL TO PARING (DAYS 0 - 14)
In general, surviving mysids in the negative control, solvent control, 0.055 and 0.11 mg a.i./L treatment groups appeared normal during the period from test initiation to pairing on Day 14. There were few observations of organisms in the 0.055 and 0.11 mg a.i./L treatment groups that displayed lethargy. These observations were infrequent, not concentration-responsive and comparable to the controls, therefore, they were not considered to be treatment-related. Mysids in the 0.22, 0.45 and 0.84 mg a.i./L treatment groups exhibited treatment-related signs of toxicity including lethargy, erratic swimming behavior and loss of equilibrium.
After 14 days of exposure, survival of juvenile mysids in the negative and solvent control groups was 100 and 95.0%, respectively. Since there were no statistically significant differences in survival between the negative and solvent control groups (p > 0.05), the control data were pooled for comparisons to the treatment groups. Survival in the pooled control and in the 0.055, 0.11, 0.22, 0.45 and 0.84 mg a.i./L treatment groups was 97.5, 80.0, 98.3, 75.0, 90.0 and 85.0%, respectively. Fisher’s Exact test indicated there was a statistically significant decrease in survival in the 0.055, 0.22, 0.45 and 0.84 mg a.i./L treatment groups when compared to the pooled control (p ≤ 0.05). While statistically significant, the decrease in survival in the 0.055, 0.22, 0.45 and 0.84 mg a.i./L treatment groups was not considered to be treatment related, since the decrease was slight and was not dose-responsive over the range of concentrations tested. Consequently, the NOEC for juvenile survival was 0.84 mg a.i./L, the highest concentration tested.

ADULT SURVIVAL TO PAIRING (DAY 15 - 28)
In general, surviving mysids in the negative control, solvent control, 0.055, 0.11 and the 0.22 mg a.i./L treatment groups appeared normal during this period. There were a few observations of organisms that exhibited lethargy, however, these observations were infrequent and were not considered to be treatment related. Mysids in the 0.45 and 0.84 mg a.i./L treatment groups exhibited treatment-related signs of toxicity including lethargy, erratic swimming and appearing smaller than those in the controls.
At test termination, survival of adult mysids in the negative and solvent control groups was 96.0 and 83.3%, respectively. Since there was a statistically significant difference in survival between the negative and solvent control groups (p < 0.05), the data for the negative control group were used for comparison to the treatment groups. Survival in the 0.055, 0.11, 0.22, 0.45 and 0.84 mg a.i./L treatment groups was 91.2, 75.0, 88.9, 91.7 and 62.9%, respectively. Fisher’s Exact test indicated there was a statistically significant decrease in survival in the 0.11 and 0.84 mg a.i./L treatment group when compared to the negative control (p ≤ 0.05). While statistically significant, the decrease in survival in the 0.11 mg a.i./L treatment group was not considered to be treatment related, since the decrease was slight and not dose-responsive. Consequently, the NOEC for adult survival (Days 15 – 28) was 0.45 mg a.i./L and the LOEC was 0.84 mg a.i./L.

REPRODUCTION
Number of reproductive days is the number of days that the female was alive from the day of the first brood release of any female in the test, therefore if a female dies, the number of reproductive days ends on the last day she was alive. The day of first brood release in this study was Day 16.
The mean percent of surviving females producing young in the negative and solvent control groups was 100 and 94.4, respectively. Since there were no statistically significant differences in the percent of females producing young between the negative and solvent control groups (p > 0.05), the control data were pooled for comparisons among the treatment groups. The mean percent of females producing young in the pooled controls and in the 0.055, 0.11, 0.22, 0.45 and 0.84 mg a.i./L treatment groups was 97.4, 93.8, 100, 86.7, 31.6 and 9.1 %, respectively. Fisher’s Exact test indicated there were statistically significant decreases in mean percent of females producing young in the 0.45 and 0.84 mg a.i./L treatment groups, in comparison to the pooled controls (p ≤ 0.05).
The mean number of young produced per surviving female in the negative control and solvent control groups was 5.5 and 6.5, respectively. Since there were no statistically significant differences in reproduction between the negative and solvent control groups (p > 0.05), the control data were pooled for comparisons among the treatment groups. The mean number of young produced per female in the pooled control group and the 0.055, 0.11, 0.22, 0.45 and 0.84 mg a.i./L treatment groups was 6.0, 7.3, 6.6, 2.7, 0.85 and 0.33, respectively. Wilcoxon’s rank sum test with a Bonferroni adjustment indicated there were statistically significant decreases in the mean number of young produced per female in the 0.22, 0.45 and 0.84 mg a.i./L treatment groups when compared to the pooled control (p ≤ 0.05).
Both of these endpoints were calculated based on the total number of surviving females present at test termination. Females that died during the test prior to test termination and the young that they produced were excluded from the calculation of the mean percent of females producing young and the mean number of young per female. Control reproductive performance in this study met the ASTM Standard E 1191-03a Guideline (2) acceptability criteria, which states that at least 75% of the first-generation females in the control(s) must produce young and that the average be at least three young produced per first-generation female.
The mean number of young produced per reproductive day in the negative and solvent control groups was 0.419 and 0.488, respectively. Since there were no statistically significant differences in reproduction between the negative and solvent control groups (p > 0.05), the control data were pooled for comparisons among the treatment groups. The mean number of young produced per reproductive day in the pooled control and in the 0.055, 0.11, 0.22, 0.45 and 0.84 mg a.i./L treatment groups was 0.454, 0.562, 0.512, 0.210, 0.060 and 0.021, respectively. Wilcoxon’s rank sum test with a Bonferroni adjustment indicated there were statistically significant decreases in reproduction in the 0.22, 0.45 and 0.84 mg a.i./L treatment groups in comparison to the pooled control (p ≤ 0.05). Consequently, the LOEC for reproduction was 0.22 mg a.i./L and the NOEC was 0.11 mg a.i./L.
Observations of second-generation mysids revealed no abnormal development or abnormal behavior, except for an occasional lethargic organism, in the negative control, solvent control, 0.055, 0.11, 0.22, 0.45 and 0.84 mg a.i./L treatment groups.

GROWTH OF MALES
The mean total length and dry weight of male mysids in the negative control group was 8.03 mm and 0.81 mg, respectively. In the solvent control group, the mean total length and dry weight of males was 7.92 mm and 0.79 mg, respectively. Since there were no statistically significant differences between the negative and solvent control groups for males for total length or dry weight (p > 0.05), the control data were pooled for comparisons among the treatment groups for dry weight and length.
The mean total length of male mysids in the pooled control and the 0.055, 0.11, 0.22, 0.45 and 0.84 mg a.i./L treatment groups was 7.97, 7.95, 8.20, 7.94, 7.83 and 7.43 mm, respectively. The mean dry weight of males in the pooled control and the 0.055, 0.11, 0.22, 0.45 and 0.84 mg a.i./L treatment groups was 0.80, 0.77, 0.89, 0.82, 0.76 and 0.69 mg, respectively. Dunnett’s test indicated there were statistically significant decreases in mean total length and mean dry weight for males in the 0.84 mg a.i./L treatment group, in comparison to the pooled control (p ≤ 0.05).

GROWTH OF FEMALE
The mean total length and dry weight of female mysids in the negative control group was 8.12 mm and 1.06 mg, respectively. In the solvent control group, the mean total length and dry weight of females was 7.97 mm and 0.94 mg, respectively. Since there were no statistically significant differences between the negative and solvent control groups for female total length (p > 0.05), the control data were pooled for comparisons among the treatment groups for total length. There was a statistically significant (p ≤ 0.05) difference between the negative and solvent control groups for female dry weight. Therefore, treatment groups were compared to the negative control group for female dry weight. The mean total length of female mysids in the pooled control and the 0.055, 0.11, 0.22, 0.45 and 0.84 mg a.i./L treatment groups was 8.05, 8.08, 8.36, 7.99, 7.96 and 7.24 mm, respectively. Dunnett’s test indicated there was a statistically significant decrease in mean total length for females in the 0.84 mg a.i./L treatment group , in comparison to the pooled control (p ≤ 0.05). The mean dry weight of females in the 0.055, 0.11, 0.22, 0.45 and 0.84 mg a.i./L treatment groups was 0.99, 1.06, 0.96, 0.99 and 0.66 mg, respectively. Dunnett’s test indicated there was a statistically significant decrease in mean dry weight for females in the 0.84 mg a.i./L treatment group , in comparison to the negative control (p ≤ 0.05).
Based on the effects on growth observed in the 0.84 mg a.i./L treatment group, the LOEC for growth (total length and dry weight) was 0.84 mg a.i./L and the NOEC was 0.45 mg a.i./L.

Reported statistics and error estimates:

See statistical analysis in "Any other information on materials and methods incl. tables"

Table 2. Summary of survival of saltwater mysids exposed to the test substance

Survival of Saltwater Mysids
Mean Measured Concentration (mg a.i./L)	Juvenile Survival to Pairing on Day 14			Adult Survival to Test Termination on Day 28
	Number Originally Exposed	Number Surviving	Percent Survival	Number Alive at Pairing^1	Number Surviving	Percent Survival
Negative Control	60	60	100	50	48	96.0
Solvent Control	60	57	95.0	48	40	83.3
Pooled Control	120	117	97.5	98	88	89.8
0.055	60	48	80.0*^2	34	31	91.2
0.11	60	59	98.3	48	36	75.0**^3
0.22	60	45	75.0*^2	36	32	88.9
0.45	60	54	90.0*^2	48	44	91.7
0.84	60	51	85.0*^2	35	22	62.9**

** Statistically significant decrease in survival in comparison to the negative control group using Fisher’s Exact test (p ≤ 0.05).
1 The number alive at pairing may be less than the number surviving to Day 14 due to the fact that extra females that cannot be used to form pairs and any immature mysids are discarded at the time of pairing on Day 14.
*2 Statistically significant decrease in survival in comparison to the pooled control using Fisher’s Exact test (p ≤ 0.05). While the decrease in survival was statistically significant in comparison to the pooled control, it was not considered to be treatment-related since the difference was slight and not dose-responsive over the range of concentrations tested.
3 While the decrease in survival was statistically significant in comparison to the negative control, it was not considered to be treatment-related since the difference was slight and not dose-responsive. It is important to note that of the 12 mortalities that occurred in this level, 5 were due to mysids impinging themselves above the water-line and subsequently dying due to desiccation. This is a documented phenomenon in mysid behavior and since this high amount of death due to impingement was not observed in the other treatment groups, it does not appear to be a treatment related effect.

Table 3. Summary of reproduction of saltwater mysids exposed to the test substance

Nominal Concentration (mg a.i./L)	Mean Number of Young Produced Per Reproductive Day ± SD	Percent of Surviving Females Producing Young 1	Mean Number of Young Per Surviving Female ± SD^1
Negative Control	0.419 ± 0.029	100	5.5 ± 0.38
Solvent Control	0.488 ± 0.175	94.4	6.5 ± 2.21
Pooled Control	0.454 ± 0.121	97.4	6.0 ± 1.58
0.055	0.562 ± 0.332	93.8	7.3 ± 4.32
0.11	0.512 ± 0.084	100	6.6 ± 0.77
0.22	0.210 ± 0.093*	86.7	2.7 ± 1.21*
0.45	0.060 ± 0.061*	31.6**	0.85 ± 0.89*
0.84	0.021 ± 0.042*	9.1**	0.33 ± 0.65*

* Statistically significant decrease in number of young produced per reproductive day and number of young per surviving female in comparison to the pooled control using Wilcoxon’s rank sum test with a Bonferroni adjustment (p ≤ 0.05).
** Statistically significant decrease in percent of females producing young in comparison to the pooled control using Fisher’s Exact test (p ≤ 0.05).
1 Calculated based on the total number of surviving females present at test termination. Females that died prior to test termination and the young that they produced were excluded from the calculation of the mean percent of females producing young and the mean number of young per female.

Table 4. Summary of growth of saltwater mysids exposed to the test substance

Growth Parameters at Termination on Day 28
Mean Measured Concentration (mg a.i./L)	Mean Total Length ± SD (mm)		Mean Dry Weight ± SD (mg)
	Males	Females	Males	Females
Negative Control	8.03 ± 0.106	8.12 ± 0.158	0.81 ± 0.016	1.06 ± 0.043
Solvent Control	7.92 ± 0.301	7.97 ± 0.052	0.79 ± 0.066	0.94 ± 0.059
Pooled control	7.97 ± 0.216	8.05 ± 0.135	0.80 ± 0.045	--1
0.055	7.95 ± 0.278	8.08 ± 0.227	0.77 ± 0.124	0.99 ± 0.027
0.11	8.20 ± 0.190	8.36 ± 0.145	0.89 ± 0.024	1.06 ± 0.106
0.22	7.94 ± 0.152	7.99 ± 0.114	0.82 ± 0.026	0.96 ± 0.069
0.45	7.83 ± 0.112	7.96 ± 0.049	0.76 ± 0.021	0.99 ± 0.105
0.84	7.43 ± 0.245*	7.24 ± 0.298*	0.69 ± 0.114*	0.66 ± 0.120**

 * Statistically significant decrease in comparison to the pooled control using Dunnett’s test (p ≤ 0.05).
** Statistically significant decrease in comparison to the negative control using Dunnett’s test (p ≤ 0.05).
1 There was a statistically significant difference between the negative and solvent control groups in female mean dry weight measurements. The data was compared to both the solvent and negative control groups and results were reported to the comparisons made to the negative control.

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the 28-day NOEC (based on reproduction) was determined to be 0.11 mg a.i./L and the LOEC was determined to be 0.22 mg a.i./L.

Executive summary:

The chronic effect of continuous exposure to the test substance on marine aquatic invertebrates was evaluated under flow-through conditions with saltwater mysids (Americamysis bahia) over a period of 28 days. The test was performed according to OCSPP 850.1350 guideline and in compliance with GLP. The test conditions were as follows: salinity 19 - 21‰, pH 7.9 – 8.1, temperature 24.5 – 26.2 °C and dissolved oxygen concentration 6.4 – 7.4 mg/L (≥ 87% of saturation), 14 hours light (195 lux) and 10 hours darkness. The mean measured concentrations of the substance in the test medium were 0.055, 0.11, 0.22, 0.45 and 0.84 mg/L (measured by HPLC, nominal concentrations were 0.063, 0.13, 0.25, 0.50 and 1.0 mg/L, respectively). A negative control and a solvent control (0.02 mL/L DMF) group were included in the test as well. Mysids of age <24 hours were used at the start of the test and evaluated for survival, reproduction and growth over the study period.

Reproduction, measured as the mean number of young produced per reproductive day and the mean number of young per surviving female, was the most sensitive biological endpoint measured. The mean number of young produced per reproductive day in the negative and solvent control groups was 0.419 and 0.488, respectively. Since there were no statistically significant differences in reproduction between the two groups (p > 0.05), the control data were pooled (0.454) for comparison with the treatment groups. The mean number of young produced per reproductive day was 0.210, 0.060 and 0.021 in the 0.22, 0.45 and 0.84 mg/L treatment group, respectively and statistically, significantly reduced in comparison to the pooled control (p ≤ 0.05, Wilcoxon’s rank sum test with a Bonferroni adjustment). Consequently, the LOEC for reproduction was 0.22 mg/L and the NOEC was 0.11 mg/L.  

Other adverse effects were observed in the study, including lethargy, erratic swimming behaviour, loss of equilibrium, reduced survival of adult mysids, reduced number of females producing young, decreases in mean total length and mean dry weight for males and decrease in mean total length and mean dry weight for females. However, the observed LOEC and NOEC values for these effects were exceeding the effect levels for reproduction.

Description of key information

All available data was assessed and the studies representing the worst-case effects are included here as key. The results can be considered worst-case and are selected for the CSA.

Freshwater 21-d NOEC = 1.0 mg/L, Daphnia magna, growth, EPA OPP 72 - 4, Suprenant 1988

Saltwater 28-d NOEC = 0.11 mg/L, Americamysis bahia, reproduction, EPA OPPTS 850.1350, Claude et al. 2013

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

1 mg/L

Marine water invertebrates

Marine water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.11 mg/L

Additional information

Fresh water

The chronic effect of continuous exposure to the test substance on aquatic invertebrates was evaluated in a single generation (21 day) flow-through life-cycle toxicity test with the freshwater invertebrate, Daphnia magna. The test was performed according to EPA OPP No. 72 – 4 guideline and in compliance with GLP. The following test conditions were determined: pH 7.9 – 8.3, 20 ± 1 °C and 8.4 – 8. 7 mg/L dissolved oxygen concentration. During the definitive 21-day chronic study, results of weekly analyses for the test substance established that the measured concentrations in the exposure solutions followed the expected concentration gradient and were consistent between replicate treatments and sampling intervals. Mean measured concentrations of the test substance in the test aquaria averaged 95% of the nominal levels (0.13, 0.25, 0.50, 1.0 and 2.0 mg/L) and were 0.10, 0.27, 0.46, 1.0 and 2.0 mg/L.  

At the termination of the 21-day definitive test, reduced organism growth, as determined by the measurement of individual body lengths, was the only indicator of toxicity of the test substance to Daphnia magna in the concentration range tested. On day 21, measurement of the surviving daphnids’ body length established that the control organisms had an average body length of 4.2 (S.D. 0.26) mm. The mean body length of daphnids exposed to the highest mean measured test concentration, 2.0 mg/L, averaged 3.5 (S.D. 0.27) mm which was significantly less than the length of the control daphnids and represented a 17% reduction in growth. No adverse effect was observed in the remaining lower treatment levels. Measurements at test termination established that daphnids in the remaining lower test concentrations had mean body lengths ranging from 3.7 to 3.9 mm and were not statistically different to the length of the control organisms. Survival and reproduction of daphnids throughout the 21-day exposure period were not affected in any treatment group. Mean survival in the 0.10 to 2.0 mg/L treatments was between 90 and 95% at test termination, compared to 93%in the control group. Similarly, no adverse effects on daphnid reproduction was observed in any of the tested concentrations during the 21-day exposure period. The NOEC was determined to be 1.0 mg/L  

Marine water

The chronic effect of continuous exposure to the test substance on marine aquatic invertebrates was evaluated under flow-through conditions with saltwater mysids (Americamysis bahia) over a period of 28 days. The test was performed according to OCSPP 850.1350 guideline and in compliance with GLP. The test conditions were as follows: salinity 19 - 21‰, pH 7.9 – 8.1, temperature 24.5 – 26.2 °C and dissolved oxygen concentration 6.4 – 7.4 mg/L (≥ 87% of saturation), 14 hours light (195 lux) and 10 hours darkness. The mean measured concentrations of the substance in the test medium were 0.055, 0.11, 0.22, 0.45 and 0.84 mg/L (measured by HPLC, nominal concentrations were 0.063, 0.13, 0.25, 0.50 and 1.0 mg/L, respectively). A negative control and a solvent control (0.02 mL/L DMF) group were included in the test as well. Mysids of age <24 hours were used at the start of the test and evaluated for survival, reproduction and growth over the study period.

Reproduction, measured as the mean number of young produced per reproductive day and the mean number of young per surviving female, was the most sensitive biological endpoint measured. The mean number of young produced per reproductive day in the negative and solvent control groups was 0.419 and 0.488, respectively. Since there were no statistically significant differences in reproduction between the two groups (p > 0.05), the control data were pooled (0.454) for comparison with the treatment groups. The mean number of young produced per reproductive day was 0.210, 0.060 and 0.021 in the 0.22, 0.45 and 0.84 mg/L treatment group, respectively and statistically, significantly reduced in comparison to the pooled control (p ≤ 0.05, Wilcoxon’s rank sum test with a Bonferroni adjustment). Consequently, the LOEC for reproduction was 0.22 mg/L and the NOEC was 0.11 mg/L.  

Other adverse effects were observed in the study, including lethargy, erratic swimming behaviour, loss of equilibrium, reduced survival of adult mysids, reduced number of females producing young, decreases in mean total length and mean dry weight for males and decrease in mean total length and mean dry weight for females. However, the observed LOEC and NOEC values for these effects were exceeding the effect levels for reproduction.