D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C8-16 (even numbered) and C18 unsaturated acyl] derivs.

Long-term toxicity to fish

Administrative data

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From April 3, 1991 to May 8, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study report, followed guideline, GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

Reliability, and validity evaluated against current OECD guideline, 26 July 2013

Deviations:

no

GLP compliance:

yes

Remarks:

EPA GLP

Test material

Test material information

Constituent 1

Reference substance name:

287735-50-6

Cas Number:

287735-50-6

IUPAC Name:

287735-50-6

Constituent 2

Reference substance name:

C12/14 Glucamide

IUPAC Name:

C12/14 Glucamide

Test material form:

other: Solid Gel

Details on test material:

-Name of test material : C12/14 Glucamide
-TSIN: P2721.01
-Substance type: Pure active substance
- Physical state: Opaque white solid gel
-Storage condition of test material: Room temperature
- Solubility: Moderate at room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples from 0, 0.63, 1.3, 2.5, 5.0, and 10 mg A.I/L test concentration were collected during in-life phase. The control and the high, middle, and low test concentrations were sampled and analyzed for test material concentration twice prior to the start of the definitive exposure.
- Sampling frequency: During the in-life phase of the definitive study, water samples were removed from both replicate test solutions of each treatment level and the controls on test days 0, 5, 7, 12, 14, 19, 26, 33 and 35 for analysis of test material concentration.
- Sampling method: At each sampling interval water samples were removed from both replicate test solutions of each treatment level and the controls, three Quality Control (QC) samples were also prepared at each sampling interval and remained with the set of exposure solution samples throughout the analytical process. The results of these analyses were used to judge the precision and quality control maintained during the analysis of exposure solution samples. All samples were analyzed by HPLC.
- Sample storage conditions before analysis: Not reported

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solutions of 9.85 mg A.l./mL were prepared every other day by diluting 22.04 g of test material (9.8519 g as active ingredient) with distilled water to a total volume of 1000 mL. The solution was heated (approximately 30C) to completely solubilize the test material.
- Application of test solution: 2.0 mL of the stock solution (9.85 mg A.I./mL) was delivered by a Harvard Peristaltic pump during each diluter cycle Into the diluter's chemical mixing chamber containing 1.97 L of dilution water. The solution contained in the mixing chamber constituted the higher t test concentration (10 mg A.I./L, nominal) and was subsequently diluted (50%) to provide the range of nominal exposure concentrations.
-Control: Dilution water was used as control
- Evidence of un-dissolved material: No, however the clear stock solution turned cloudy white within a few hours.

Test organisms

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead Minnow
- Strain: Not reported
- Source: Fathead minnow eggs were obtained from brood stock maintained at Springborn Laboratories, Inc.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilized eggs: On the day prior to test initiation, spawning tiles were placed in the fathead minnow brood culture unit. The eggs were collected from the tiles and impartially distributed to the egg incubation cups.
- Subsequent handling of eggs: After collection, eggs were distributed in manner as follows twelve unlabeled, unassigned petri dishes were set in a shallow water bath maintained at 25 ±1 °C. A small amount of water from the control aquaria was placed in each dish. The collected eggs were then counted into each dish sequentially, five at a time, until each dish contained 60 eggs. Subsequently, a second count was conducted of the eggs to ensure accuracy, twelve labeled incubation cups were placed in control water at 25 ±1°C. Each group of 60 eggs in the petri dishes was impartially transferred to one of the twelve incubation cups.
- Feeding: During the definitive test, subsequent to the completion of hatch (Day 5), the fry were fed live brine shrimp nauplii (Artemis salina) three times daily on weekdays and two times daily on weekends.

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

35 d

Remarks on exposure duration:

(30 d post-hatch)

Post exposure observation period:

None

Test conditions

Hardness:

26-56 mg/L as CaCO3

Test temperature:

25-26 °C

pH:

6.8-7.5

Dissolved oxygen:

5.8-8.9 mg/L (Percent saturation ranged from 69% to 106%)

Nominal and measured concentrations:

Nominal test concentrations: 0, 0.63,1.3, 2.5, 5.0 and 10 mg A.l./L
Mean measured concentrations: 0, 0.69, 1.5, 2.5, 4.8, and 10 mg A.I/L (96 to 115.4% of nominal)
For details refer to ‘Table 1’ under ‘Any other information on materials and methods incl. Tables’ section.

Details on test conditions:

TEST SYSTEM
- Emybro cups: Embryo Incubation cups were glass jars (5 cm O.D., 8 cm high) with 40-mesh Nitex® screen bottoms. A rocker arm apparatus was used to gently oscillate the incubation cups in the test solutions.
- Test vessel: Aquarium (arranged in a thermostatically heated water bath at 25± 1 °C )
- Type: Open
- Material, size, headspace, fill volume: Each test aquarium measured 39 x 20 x 25 cm with a 19.5 cm high side drain that maintained a constant exposure solution volume of 15 L.
- Aeration: Test solutions were not aerated
- Type of flow-through: Intermittent-flow proportional diluter.
- Renewal rate of test solution: Test solutions were delivered to the vessels at an approximate rate of 6.5 aquarium volumes per 24-hour period, with a 90% replacement time approximately nine hours.
- No. of fertilized eggs/embryo per vessel: 60 eggs/ embryo incubation cup
- No. of larvae per vessel: 40 larvae/each exposure aquaria
- No. of vessels per concentration : 2
- No. of vessels per control: 2
- No. of vessels per vehicle control: 2
- Biomass loading rate: 1 egg/0.25 mL; 1 larvae/0.375 mL

-Exposure of embryos: The test exposure of fathead minnow embryos and larvae initiated when the egg incubation cups, each containing sixty embryos were distributed to each of the twelve test aquaria. Dead embryos were counted daily until hatching was complete. Hatching was deemed complete on exposure Day 5 when no more than 5 unhatched viable embryos remained in any control or treatment level egg incubation cup. The percent survival of organism at hatch was calculated.

-Exposure of larvae: The 30-day post-hatch larval exposure was initiated when 40 live larvae were impartially selected from the surviving larvae in each incubation cup on test Day 5 and placed into their respective exposure aquaria. Dead larvae were removed when observed and behavior and appearance of larvae were observed and recorded dally. Larval survival was estimated at least twice weekly. At 30 days post-hatch exposure % of survival of organisms at hatch was determined. For calculation, number of live larvae and embryos per incubation cup were compared to the number of embryos per cup on test Day 0 after hatching was completed. The surviving larvae were anesthetized with MS-222 (tricaine methanesulfonate) and measured for mean total length, and mean wet weight.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution and control water was well water which was pumped into an epoxy-coated concrete reservoir where it was supplemented on demand with Town of Wareham untreated well water and aerated. During the study, weekly characterization of the well water established following characteristics:
Total hardness as calcium carbonate (CaCO3): 29-34 mg/L
Alkalinity as calcium carbonate (CaCO3): 21- 34 mg/L
pH: 6.8- 7.1
Temperature: 20 ± 1°C
Conductivity of 125-130 µmhos/cm
Total organic carbon: 0.63 mg/L for the month of April 1991
-Metals and pesticides: Routine analyses were conducted on representative samples of the dilution water source for the presence of pesticides and PCB's. None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed.
- Alkalinity (during the 35 day exposure): 22-28 mg/L as CaCO3
- Specific Conductivity (during the 35 day exposure): 150 - 190 µmhos/cm
- Intervals of water quality measurement: Dissolved oxygen concentration, pH and temperature were measured in each aquarium daily. The temperature was continuously monitored in one replicate of the dilution water control. Continuous monitoring of the control solution temperature was performed. Total hardness and total alkalinity as CaCO3 and specific conductance were measured on Day 0 and weekly thereafter in alternating replicates of the high and low concentrations and the dilution water control.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light and 8 h dark (Durotest Vitalite fluorescent lights centrally located above the test aquaria)
- Light intensity: 50-80 footcandles

- EFFECT PARAMETERS MEASURED:
The effects on embryo survival at hatch and on survival and growth (wet weight and total length) of larvae after 30 days of post hatch exposure were measured. Dead larvae were removed when observed and behavior and appearance of larvae were observed and recorded daily.
RANGE-FINDING STUDY
-Range finding study: Yes,
- Test concentrations: 0.31,0.63, 1.3, 2.5, 5.0, and 10 mg A.I./L
- Results used to determine the conditions for the definitive study: Following 13 days of exposure, 0% survival was recorded among fathead minnows exposed to the 10 mg A.I./L treatment level, while 100% survival was observed In the remaining concentrations tested {5.0-0.31 mg A.I./L). Sub-lethal effects (e.g., lethargy) were observed at the 5.0 mg A.I./L treatment level.
Based on the results of range finding study, nominal concentrations of 10, 5.0, 2.5, 1.3 and 0.63 mg A.I./L were selected for the definitive exposure.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

4.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

- Survival of embryo at completion of hatch: At the completion of the hatching period (Day 5) in the highest test concentration of test material (10 mg A.I./L) was 0% and was significantly reduced when compared to the survival of the control organisms (81 %). The survival of organisms exposed to the remaining treatment levels from 4.8-0.69 mg A.I./L ranged from 78 to 89% and was statistically comparable to the survival of the control organisms.
- Mortality/survival of larvae: As no fathead minnow survived the initial five days of exposure to 10 mg A.I./L, data for this treatment level were unavailable for statistical analysis. Following 30 days of post-hatch exposure, survival of fathead minnow larvae in concentrations of test material 4.8-0.69 mg A.I./L ranged from 93 to 95% and was comparable to survival of the control larvae (98%).
- Total length of surviving larvae: At the termination of test, mean total length observed at test concentrations from 4.8-0.69 mg A.I./L ranged from 32-33 mm, and was statistically similar to the performance of control larvae 33 mm.
-Total length of surviving larvae: At the termination of test, mean wet weight observed at test concentrations from 4.8-0.69 mg A.I./L ranged from 0.33-0.36 g, and was statistically similar to the performance of control larvae 0.36 g.
- Weekly measurement of temperature, dissolved oxygen, pH, specific conductance, total hardness and alkalinity in the exposure solutions established that these water quality parameters remained within acceptable limits for the survival and growth of fathead minnow.
- Statistical comparison of the growth data (total length and wet weight) determined at test termination demonstrated that exposure to concentrations ≤4.8 mg A.I./L did not adversely affect larval growth.

For details refer to Table 2 under 'Any other information including results.

Reported statistics and error estimates:

At the termination of the study, data obtained on organism survival at hatch, larval survival and larval growth (wet weight and total length at test termination) were statistically analyzed to establish significant differences between the treatment level and control organisms. Analyses were performed using the mean organism response in each replicate aquarium rather than Individual response values. All statistical conclusions were made at the 95% level of certainty except In the case of the Chi-Square Goodness of Fit Test and the Bartlett's Test, in which the 99% level of certainty was applied. The following procedures were used:

1) Significant differences in the percentage survival were determined after transformation (e.g. arsine square-root percentage) of the data,
2) As a check on the assumption of homogeneity of variance, implicit in parametric statistics, data for each endpoint were analyzed using Bartlett's Test (Horning and Weber, 1985).
3) For each endpoint, the performance at each treatment level of P2721.01 was compared with the performance of the control using the Williams' Test (Williams, 1971, 1972) or the Dunnett's Test (Dunnett, 1955, 1964). The Williams' Test and the Dunnetts' Test are parametric procedures. The Williams' Test is preferred for chronic toxicity tests and is more powerful than the Dunnett's Test (Rand and Petrocelli, 1985). However the Williams' Test, by design, assumes a concentration response due to increasing concentration of toxicant. If this assumption is violated, then the Dunnett's Test may be more appropriate. For this study all statistical comparisons were made using the Dunnett's Test.
4) Organism survival at hatch and larval survival data were analyzed before larval growth (length and weight); treatment levels that caused significant survival effects were excluded from further statistical analysis.

A computer program was used to perform the computations to determine NOEC and LOEC.

Any other information on results incl. tables

Table 1: Organism survival at hatch and of fathead minnow larvae(Pimephales promelas) following 35 days exposure (30 days post-hatch) to P2721.01 (Study # 35058)

Mean Measured Concentration (mg A.I./L)		Embryo Hatching Success (%)	Larval Survival at Termination (%)
10	Mean	0^a	0^b
4.8	Mean	89	93
2.5	Mean	80	94
1.5	Mean	88	95
0.69	Mean	78	94
Control	Mean	81	98

a = Significantly different (p ≤0.05) as compared to the control organisms

b = Data were excluded from statistical analysis since no fathead minnow survived the initial 5-day exposure for this treatment level

Table 2: Total length and wet weight of surviving larvae at test termination (30 days post-hatch) of the early life-stage exposure of fathead minnow(Pimephales promelas) to P2721.01 (Study # 35058)

Mean Measured Concentration (mg A.I./L)		Total Length(mm)	Wet Weight (g)
10	Mean	a	a
4.8	Mean	32(2.7)	0.33(0.081)
2.5	Mean	33(2.9)	0.35(0.096)
1.5	Mean	33(3.4)	0.36(0.10)
0.69	Mean	33(4.0)	0.35(0.12)
Control	Mean	33(2.8)	0.36(0.080)

a = Data were excluded from statistical analysis since no fathead minnow survived the initial 5-day exposure for this treatment level.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In a 35 d (30 d post-hatch) early-life stage toxicity test of C12/14 Glucamide to Pimephales promelas, the NOEC was established at 4.8 mg A.I/L, based on embryo mortality at hatch.
The result was based on mean measured concentration.

Executive summary:

A 35 d (30 d post-hatch) fish early-life stage toxicity test of C12/14 Glucamide to Pimephales promelas (fathead minnow) was determined under flow-through conditions following methods comparable to the OECD 210 guideline, Fish, Early-Life Stage Toxicity Test.

The nominal test concentrations were 0.63, 1.3, 2.5, 5.0 and 10 mg A.l./L. Mean measured concentrations were 0.69, 1.5, 2.5, 4.8, and 10 mg A.I/L (96 to 115.4% of nominal) .There were 2 replicates at each test concentration and control level. Results are based on measured concentration.

At the completion of the hatching period (Day 5), survival of fathead minnow was significantly reduced at 10 mg A.I./L and was 0. Survival in the remaining concentrations from 4.8-0.69 mg A.I./L ranged from 78 to 89% and was statistically comparable to the survival of control organisms. Since no fathead minnow survived the initial 5 days of exposure to the highest treatment level tested (10 mg A.I./L), data for this treatment level was not statistically analyzed.

After 30 d post-hatch period, mean larvae survival ranged from 93-95% at test concentrations 4.8-0.69 mg A.I./L and was comparable to 98% survival of the control larvae.

Statistical comparison of the growth data (total length and wet weight) determined at test termination (30 days post-hatch) demonstrated that exposure to concentrations of test substance 4.8 mg A.I./L did not adversely affect larval growth. The mean total length and wet weight of larvae ranged from 32-33 mm and 0.33-0.36 g, respectively, and was statistically comparable to the performance of the control larvae (33 mm and 0.36 g).

Based on the observed effects on organism survival at hatch, the NOEC of C12/14 Glucamide was established as 4.8 mg A.I./L

This fish early-life stage toxicity test is classified as acceptable, and satisfies the guideline requirements for the OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test).