Reaction mass of N,N'-ethylenebis[N-methyl-2-[(1-oxo-9-octadecenyl)oxy]-N-[2-[(1-oxo-9-octadecenyl)oxy]propyl]propylammonium] dimethyl disulphate and [2-[bis(2-hydroxypropyl)amino]ethyl]bis(2-hydroxypropyl)(methyl)ammonium methyl sulphate, dioleate (ester)

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18.06.1992-17.11.1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OTS 797.1400 (Fish Acute Toxicity Test)

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DuPont / 383
- Expiration date of the lot/batch: not specified
- Purity test date: not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: low solubility
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not expected

Analytical monitoring:

not specified

Details on sampling:

- Concentrations:
- Sampling method: not specified
- Sample storage conditions before analysis: not specified

Vehicle:

not specified

Details on test solutions:

Eight test concentrations and a water control were used. Nominal concentrations were 0.44, 0.55, 0.69, 0.86, 1.08, 1.34, 1.68, and 3.02 mg/L.

A 2mg/L stock solution was prepared in dilution water, mixed for 45 minutes, and used to prepare 0.44, 0.55, 0.69, and 0.86 mg/L test solutions. The remaining test solutions (1.08, 1.34, 1.68, and 3.02 mg/L) were prepared by direct addition of the appropiate amount of test substance to dilution water, mixed for 45 minutes and used immidiately. Stability and solubility of the test itme in dilution water and under storage conditions were not determined.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Strain: not applicable
- Source: not specified
- Age at study initiation (mean and range, SD): not specified
- Length at study initiation (length definition, mean, range and SD): 2.0 to 4.0 cm (mean 2.9 cm)
- Weight at study initiation (mean and range, SD): 0.103 to 0.972 g (mean 0.393 g)
- Method of breeding: not specified
- Maintenance of the brood fish: not specified

ACCLIMATION
- Acclimation period: not specified
- Acclimation conditions (same as test or not): same
- Type and amount of food during acclimation: fish were not fed 48 hours prior to nor during the test
- Feeding frequency during acclimation: not applicable
- Health during acclimation (any mortality observed): no moratlity was observed during aclimation

FEEDING DURING TEST
- Food type: fish were not fed 48 hours prior to nor during the test
- Amount: not applicable
- Frequency: not applicablä


Fathead Minnows, Pimephales promelas, were reared in contionously flowing well water at Haskell Laboratory. Adult fish reside in 29L aquaria at approximately 22 C and were fed frozen brine shrimp (Artemia sp., Golden Gate Brand, Kordon Division, Novalek Inc., Hayward, CA) generally twice daily, once on saturday and once on Sunday. Eggs were deposited on polyvinyl chloride tiles coated with sand.Tiles with eggs were aerated and incubated at approximately 22 C in a water bath . Larvae were reared in 29L aquaria at approximately 22 C and were fed newly hatched Artemia sp. (Aquarium products, Glen Burnie, MD) generally twice daily on weekdays and daily on weekends. Juveniles were held either either in 272L circular, fiberglass tanks or in 39L glass aquaria (23cm depth; 50 (L) x 25 (W) x 30 cm (H)) at approximately 22 C and were fed newly hatched Artemia sp. and/or frozen brine shrimp generally twice daily on weekdays, once on Saturday and once on Sunday. Sickness, injury and abnormalities were not observed. Animals were identified by lables on aquaria and test vessels.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

not specified

Hardness:

80 mg/L as CaCO3

Test temperature:

ca 21 C

pH:

ca 7.7

Dissolved oxygen:

ca 8.7 mg/L

Salinity:

N/A

Conductivity:

180 umhos/cm

Nominal and measured concentrations:

0.44, 0.55, 0.69, 0.86, 1.08, 1.34, 1.68, 3.02

Details on test conditions:

TEST SYSTEM
- Test vessel: aquarium, tank
- Material, size, headspace, fill volume: glass 20L 39(L) x 21(W) x 25 cm (H), 20cm liquid depth
- Aeration: not specified
- Type of flow-through (e.g. peristaltic or proportional diluter): not specified
- Renewal rate of test solution (frequency/flow rate): not specified
- No. of organisms per vessel: 20 fish per concentrations
- No. of vessels per concentration (replicates): two
- No. of vessels per control (replicates): one
- No. of vessels per vehicle control (replicates): not specified
- Biomass loading rate: not specified

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Haskell Laboratory well
- Total organic carbon: <0.5
- Particulate matter: see tables
- Metals: see tables
- Pesticides: ND
- Chlorine: 6.2 mg/L
- Alkalinity: 78 mg/L as CaCO3
- Ca/mg ratio: 24.5 mg/L Ca and 3.8 mg/L Mg
- Culture medium different from test medium: -
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Adjustment of pH: not specified
- Photoperiod: 16 hour light 8 hour dark with 25 minutes transition
- Light intensity: 269.0 to 333.5 lux; 2.2 lux (transition)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

not specified

Key result

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

ca. 1.39 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

ca. 1.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

ca. 1.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

ca. 1.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: not observed
- Observations on body length and weight: not specified
- Other biological observations: not observed
- Mortality of control: not specified
- Other adverse effects control: not specified
- Abnormal responses: not specified
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not specified
- Effect concentrations exceeding solubility of substance in test medium: not specified

Sublethal observations / clinical signs:

TABLE 1
Chemical Characteristics of Haskell Laboratory Well Water
Parameter	Concentration	Parameter	Concentration
BOD, mg/L	<2	Iron, mg/L	0.025
COD, mg/L	<10	Fluoride, mg/L	<0.1
DOC, mg/L	2.8	Lead, mg/L	<0.075
TOC, mg/L	<0.5	Magnesium, mg/L	3.8
Total Kjeldahl N, mg/L	<0.2	MBAS/LAS, mg/L	<0.05
Ammonia N, mg/L	<0.05	Mercury, mg/L	<0.00020
Turbidity, NTU	1.6	Nickel, mg/L	<0.020
Phenolics, mg/L	<0.050	Nitrite, mg/L	<0.1
Color, Co/Pt	<5	Nitrate, mg/L	2.1
Solids		Phosphate, mg/L	<0.1
Total suspended, mg/L	<4	Potassium, mg/L	1.8
Aluminium, mg/L	<0.100	Selenium, mg/L	<0.005
Antimony, mg/L	<0.060	Silver, mg/L	<0.010
Arsenic, mg/L	<0.010	Sodium, mg/L	8
Beryllium, mg/L	<0.001	Sulfate, mg/L	4.8
Cadmium, mg/L	<0.002	Sulfide, mg/L	<0.05
Calcium, mg/L	24.5	Thallium, mg/L	<0.01
Chloride, mg/L	6.2	Zinc, mg/L	<0.020
Chromium, mg/L	<0.010	Volatile priority pollutants	ND
Cobalt, mg/L	<0.020	Acid extractable priority pollutants	ND
Copper, mg/L	<0.010	Base/neutral priority pollutants	ND
Cyanide, mg/L	<0.025	Pesticides/PCBs	ND
		Organophosphate pesticides, ug/L	<0.50

TABLE 2
Water Chemistry of the Dilution Water at Test Start
Total Alkalinity (mg/L as CaCO3)	EDTA Hardness (mg/L as CaCO3)	Conductivity (umhos/cm)
78	80	180

TABLE 3
Physical and Chemical Characteristics of H-19,521 Test Solutions
Nominal concentrations (mg/L)	Time
	0 hours		24hours		48 hours		72 hours		96 hours
	A	B	A	B	A	B	A	B	A	B
Dissolved Oxygen (mg/L)
H2O Control	8.7	8.7	8	8.1	7.1	7.2	6.9	7	7.5	7
0.44	8.7	8.7	8	8	7.2	7.1	7	7	7.2	7.1
0.55	8.7	8.7	8.1	8.3	7.4	7.5	7.1	7	7	7
0.69	8.7	8.7	8.4	8.3	7.4	7.5	7.2	7.1	6.9	7.2
0.86	8.7	8.7	8.4	8.4	7.5	7.5	7.3	7.3	7	7.1
1.08	8.7	8.7	8.4	8.2	7.8	7.9	7.6	7.5	7.2	7.4
1.34	8.7	8.7	8.3	8.3	7.6	7.4	7.4	7.2	7.4	7.1
1.68	8.7	8.7	8.6	8.6	b	b	b	b	b	b
3.02	8.7	8.7	8.6	8.6	b	b	b	b	b	b
pH
H2O Control	7.8	7.8	8.1	8.1	7.5	7.5	7.5	7.5	7.7	7.7
0.44	7.7	7.7	7.5	7.5	7.6	7.6	7.5	7.5	7.7	7.7
0.55	7.7	7.7	7.6	7.6	7.7	7.7	7.5	7.5	7.7	7.7
0.69	7.7	7.7	7.8	7.8	7.6	7.6	7.5	7.5	7.7	7.7
0.86	7.7	7.7	7.9	7.9	7.7	7.7	7.6	7.6	7.7	7.7
1.08	7.7	7.7	7.6	7.6	7.5	7.5	7.6	7.6	7.7	7.7
1.34	7.7	7.7	7.6	7.6	7.6	7.6	7.6	7.6	7.7	7.7
1.68	7.7	7.7	7.6	7.6	b	b	b	b	b	b
3.02	7.7	7.7	7.5	7.5	b	b	b	b	b	b
Temperature (C)
H2O Control	21.5	21.5	21.1	21.1	21.2	21.2	21.2	21.2	21.2	21.4
0.44	21.3	21.3	21.1	21.2	21.2	21.3	21.1	21.3	21.5	21.5
0.55	21.4	21.2	21	21.2	21.3	21.3	21.2	21.1	21.5	21.7
0.69	21.3	21.1	21.2	21.1	21.2	21.2	21.1	21.1	21.2	21.2
0.86	21.4	21.5	21.1	21.1	21.2	21.2	21.2	21.2	21.3	21.4
1.08	21.3	21.1	21.1	21.2	21.1	21.1	21.2	21.2	21.4	21.3
1.34	21.1	21.2	21.1	21.1	21.2	21.1	21.1	21.2	21.3	21.2
1.68	21.3	21.3	21.3	21.2	b	b	b	b	b	b
3.02	21.1	21.1	21.1	21.2	b	b	b	b	b	b

TABLE 4
Observed Mortality of Fathead Minnows, Pimephales promelas, Exposed to H-19,521 for 96 hours in a Static, Unaerated Test
Nominal Concentrations (mg/L)	Cumulative Mortality in Percent
	24Hours		48 Hours		72 Hours		96 Hours
	A	B	A	B	A	B	A	B
H2O Control	0	0	0	0	0	0	0	0
0.44	0	0	0	0	0	0	0	0
0.55	0	0	0	0	0	0	0	0
0.69	0	0	0	0	0	0	0	0
0.86	0	0	0	0	0	0	0	0
1.08	0	0	10	20	10	30	10	30
1.34	60	0	90	30	90	30	90	30
1.68	100	100	100	100		100	100	100
3.02	100	100	100	100	100	100	100	100

TABLE 5
Observed Effects of H-19,521 on Fathead Minnows, Pimephales promelas, Exposed for 96 Hours in a Static, Unaerated Test
Nominal Concentrations (mg/L)	Number of Fish With Effect
	24 Hours		48 Hours		72 Hours		96 Hours
	A	B	A	B	A	B	A	B
H2O Control	0/10	0/10	0/10	0/10	0/10	0/10	0/10	0/10
0.44	0/10	0/10	0/10	0/10	0/10	0/10	0/10	0/10
0.55	0/10	0/10	0/10	0/10	0/10	0/10	0/10	0/10
0.69	0/10	0/10	0/10	0/10	0/10	0/10	0/10	0/10
0.86	0/10	0/10	0/10	0/10	0/10	0/10	0/10	0/10
1.08	0/10	0/10	0/9	1^cd/8	0/9	0/7	0/9	0/7
1.34	0/4	0/10	0/1	0/7	0/1	0/7	0/1	0/7
1.68	b	b	b	b	b	b	b	b
3.02	b	b	b	b	b	b	b	b

TABLE 6
H-19,521 Concentrations Causing Mortality to 50% (LC50) of Fathead Minnows, Pimephales promelas, at Specific Time Intervals
Time	LC50 (mg/L)	95% Fiducial Limits (mg/L)		Slope	Y-intercept
24 Hours	1.39	1.3	1.47	N/A	N/A
48 Hours	1.25	1.18	1.34	15.8	-1.55
72 Hours	1.25	1.18	1.34	15.8	-1.55
96 Hours	1.25	1.18	1.34	15.8	-1.55

Validity criteria fulfilled:

yes

Conclusions:

The test substance (based on nominal concentrations) was moderately toxic to fathead minnows, Pimephales promelas, in a 96-hour, static, unaerated test.

Executive summary:

The goal of the study was to assess the acute toxicity of the test substance to fathead minnows, Pimephales promelas.

All data and the final report will be maintened either at Haskell Laboartory for Toxicology and Industrial Medicine (Newark, Delaware) or be archived at the DuPont Records Management Center (Wilmington, Delaware).

A 2mg/L stock solution was prepared in dilution water, mixed for 45 minutes and used to prepare 0.44, 0.55, 0.69, and 0.86 mg/L test solutions. The remaining test solutions (1.08, 1.34, 1.68, and 3.02 mg/L) were prepared by direct addition of the appropriate amount of test substance to dilution water, mixed for 45 minutes and used immidiately. Stability and solubility of the test substance in dilution water and under storage conditions were not determined.

Water originated from the Haskell Laboratory well.

Fathead Minnows, Pimephales promelas, were reared in continously flowing well water at Haskell Laboratory. Adult fish resided in 29L aquaria at approximately 22 C and were fed frozen brine shrimp (Artemia sp., Golden Gate Brand, Novalek Inc.) generally twice daily, once on Saturday and once on Sunday. Eggs were deposited on polyvinyl chloride tiles coated with sand. Tiles with eggs were aerated and incubated at approximately 22 C in a water bath. Larvae were reared in 29L aquaria at approximetely 22 C and were fed newly hatched Artemia sp. generally twice daily on weekdays and daily on weekends. Juveniles were held either in 272L circular fiberglass tanks or in 39L glass aquaria at approximately 22 C and were fed newly hatched Artemia sp. and/or frozen brine shrimp generally twice daily on weekdays once on Saturday and once on Sunday. Sickness, injury and abnormalities were not observed. Animals were identified by labels on aquaria and test vessels.

Eight test concentrations and a water control were used. Nominal concentrations were 0.44, 0.55, 0.69, 0.86, 1.08, 1.34, 1.68 and 3.02 mg/L. Control fish ranged from 2.0 to 4.0 cm standard lenght (mean 2.9 cm) and 0.103 to 0.972 g wet weight (mean 0.393 g)

Glass aquaria (20L; 39(L) x 21(W) x 25(H) cm) containing 16 L of test solution (approximately 20 -cm liquid depth) were employed. Ten fish were added to each replicate using random numbers (two replicate per concentration, total 20 fish per concentration). Control loading was 0.246 g/L at test conclusion. Test solutions ranged from 21.0 to 21.7 C (mean 21.2 C). Fish were not fed 48 hours prior to nor during the test. A photoperiod of 16 hours light (269.0 to 333.5 LUX) versus 8 hours darkness was employed with 25 minutes of transitional light (approximately 2.2 LUX) preceding and following the 16 hour light interval. Mortality and behavioral effects were noted daily.

Dissolved oxygen (YSI Model 54A, Yellow Springs Instruments), pH (Corning Model 610A, Corning Scientifc Instruments) and temperature (American Society of Testing Materials registered mercury thermometer) were mesaured in the water control and all test concentrations before fish were added at the beginning of the test and daily thereafter. Total alkalinity (Hach kit, Hach Chemical Company), EDTA hardness ( Hach kit, Hach Chemical Company) and conductivity (YSI Model 33, Yellow Springs Instruments) of the dilution water were measured at the beginning of the test. A continously-recording thermometer in a control vessel was used to check for temperature variation during the 96 -hour test.

Concentrations of the test substance in the test solutions were not measured.

The 24 -hour LC50 was calculated by the moving average angle method. The 48, 72 and 96 hour LC50´s were calculated by the SAS Probit method.

A 96 -hour static rangefinding test was conducted using a control and 1, 100, 500, 1000 mg/L, (five fish per nominal concentration). Mortalities were 0, 0, 100, 100 and 100% respectively. An earlier definitive study was conducted but was repeated due to mortality at all concentrations.

Nominal concentrationsin the definitive test were 0.44, 0.55, 0.69, 0.86, 1.08, 1.34, 1.68 and 3.02 mg/L. The test substance appeared to be in solution at all concentrations except 3.02 mg/L. All chemicals and physical parameters were within expected ranges.

Behavioral observations appear has been made. The 96 -hour LC50 based on nominal concentrations was 1.25 mg/L with a 95% fiducal interval of 1.18 to 1.34 mg/L.

The test substance (based on nominal concentrations) was moderately toxic to fathead minnows, Pimephales promelas, in a 96 -hour static, anaerated test.

Description of key information

The goal of the study was to assess the acute toxicity of the test substance to fathead minnows, Pimephales promelas.

All data and the final report will be maintened either at Haskell Laboartory for Toxicology and Industrial Medicine (Newark, Delaware) or be archived at the DuPont Records Management Center (Wilmington, Delaware).

A 2mg/L stock solution was prepared in dilution water, mixed for 45 minutes and used to prepare 0.44, 0.55, 0.69, and 0.86 mg/L test solutions. The remaining test solutions (1.08, 1.34, 1.68, and 3.02 mg/L) were prepared by direct addition of the appropriate amount of test substance to dilution water, mixed for 45 minutes and used immidiately. Stability and solubility of the test substance in dilution water and under storage conditions were not determined.

Water originated from the Haskell Laboratory well.

Fathead Minnows, Pimephales promelas, were reared in continously flowing well water at Haskell Laboratory. Adult fish resided in 29L aquaria at approximately 22 C and were fed frozen brine shrimp (Artemia sp., Golden Gate Brand, Novalek Inc.) generally twice daily, once on Saturday and once on Sunday. Eggs were deposited on polyvinyl chloride tiles coated with sand. Tiles with eggs were aerated and incubated at approximately 22 C in a water bath. Larvae were reared in 29L aquaria at approximetely 22 C and were fed newly hatched Artemia sp. generally twice daily on weekdays and daily on weekends. Juveniles were held either in 272L circular fiberglass tanks or in 39L glass aquaria at approximately 22 C and were fed newly hatched Artemia sp. and/or frozen brine shrimp generally twice daily on weekdays once on Saturday and once on Sunday. Sickness, injury and abnormalities were not observed. Animals were identified by labels on aquaria and test vessels.

Eight test concentrations and a water control were used. Nominal concentrations were 0.44, 0.55, 0.69, 0.86, 1.08, 1.34, 1.68 and 3.02 mg/L. Control fish ranged from 2.0 to 4.0 cm standard lenght (mean 2.9 cm) and 0.103 to 0.972 g wet weight (mean 0.393 g)

Glass aquaria (20L; 39(L) x 21(W) x 25(H) cm) containing 16 L of test solution (approximately 20 -cm liquid depth) were employed. Ten fish were added to each replicate using random numbers (two replicate per concentration, total 20 fish per concentration). Control loading was 0.246 g/L at test conclusion. Test solutions ranged from 21.0 to 21.7 C (mean 21.2 C). Fish were not fed 48 hours prior to nor during the test. A photoperiod of 16 hours light (269.0 to 333.5 LUX) versus 8 hours darkness was employed with 25 minutes of transitional light (approximately 2.2 LUX) preceding and following the 16 hour light interval. Mortality and behavioral effects were noted daily.

Dissolved oxygen (YSI Model 54A, Yellow Springs Instruments), pH (Corning Model 610A, Corning Scientifc Instruments) and temperature (American Society of Testing Materials registered mercury thermometer) were mesaured in the water control and all test concentrations before fish were added at the beginning of the test and daily thereafter. Total alkalinity (Hach kit, Hach Chemical Company), EDTA hardness ( Hach kit, Hach Chemical Company) and conductivity (YSI Model 33, Yellow Springs Instruments) of the dilution water were measured at the beginning of the test. A continously-recording thermometer in a control vessel was used to check for temperature variation during the 96 -hour test.

Concentrations of the test substance in the test solutions were not measured.

The 24 -hour LC50 was calculated by the moving average angle method. The 48, 72 and 96 hour LC50´s were calculated by the SAS Probit method.

A 96 -hour static rangefinding test was conducted using a control and 1, 100, 500, 1000 mg/L, (five fish per nominal concentration). Mortalities were 0, 0, 100, 100 and 100% respectively. An earlier definitive study was conducted but was repeated due to mortality at all concentrations.

Nominal concentrationsin the definitive test were 0.44, 0.55, 0.69, 0.86, 1.08, 1.34, 1.68 and 3.02 mg/L. The test substance appeared to be in solution at all concentrations except 3.02 mg/L. All chemicals and physical parameters were within expected ranges.

Behavioral observations appear has been made. The 96 -hour LC50 based on nominal concentrations was 1.25 mg/L with a 95% fiducal interval of 1.18 to 1.34 mg/L.

The test substance (based on nominal concentrations) was moderately toxic to fathead minnows, Pimephales promelas, in a 96 -hour static, anaerated test.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.25 mg/L