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Long-term toxicity to fish

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Endpoint:
fish life cycle toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 1974 to October 1975
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study follows acceptable scientific methods and principles. Assessed all phases of the fish life-cycle, included spawning and egg hatchability as well as growth. Histopathological analyses and strong analytical monitoring of test substance concentrations were performed.
Qualifier:
according to
Guideline:
other: U.S. Environmental Protection Agency. July 1973. Biological Field and Laboratory Methods for Measuring the Quality of Surface Waters and Effluents. Edited by C. I. Weber
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 850.1500 (Fish Life Cycle Toxicity)
Deviations:
no
Principles of method if other than guideline:
The study was conducted in two phases. The initial phase consisted of static acute range-finding tests, and 96 hour static acute bioassays under varying water quality conditions (pH, hardness, age of test solution); both used 60-day old fry. The second phase consisted of two tests: a 15 day flow-through exposure of 12-day old fry, and a 10-month (302 day) test beginning with fry (F0), grown to sexual maturity, and ending with their (F1) offspring.
GLP compliance:
no
Remarks:
This study pre-dates GLPs.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: In the 15-day and 302-day tests, all concentrations were measured. The Phase 1 range-finding and acute toxicity tests were conducted at nominal test concentrations, without analytical monitoring.
- Sampling method: During the initial 15-day flow-through exposure, 100 mL samples were taken from each test tank and analyzed weekly. During the 10-month chronic exposure, 100 mL samples from each tank were analyzed for NPS 74.004 concentrations each week. This procedure was followed for the first 120 days of exposure; thereafter, the concentration of NPS 74.004 was measured in one tank at each concentration every week for each set of duplicate tanks. Weekly larval tank concentration analysis was begun one month after the beginning of the chronic study. Initially, all larval tanks were analyzed to establish concentration patterns; with the advent of spawning, the NPS 74,004 measurements were reduced to one tank at each concentration weekly for each set of duplicate tanks.
- Sample storage conditions before analysis: N/A
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:

1. Phase 1 range-finding and acute toxicity tests were conducted with NPS 74.004 (25.43% active). Stock solutions were prepared by weighing out the substance and bringing the stock solution to final volume with distilled water. All test concentrations were made by adding the appropriate amount of stock solution by volume to the diluent water. All test concentrations were based upon active ingredient concentrations..

2. Phase 2 tests. A Mount and Brungs type proportional diluter produced five concentrations of NPS 74.004 plus a control. Each concentration was split into 2 sets of duplicate flows in the mixing cell. Individual delivery tubes went to each of two duplicate spawning tanks and into each of two duplicate larvae tanks. The 15-day flow-through exposure test was also conducted using NPS 74.004 (25.43% active, 6/27/74). A stock solution was made by bringing 51.9 g NPS 74.004 (25.43 % active, 6/27/74) up to a volume of 2 liters using distilled water. The stock solution was injected by a peristaltic pump into the proportional diluter. The stock solution was renewed every 48 hours to ensure fresh stock and was kept in constant agitation during the injection period by a magnetic stir bar within the 2 liter stock solution reservoir. Stock solution concentration was calculated to provide theoretical NPS 74.004 concentrations of 2.0 ppm, 1.0 ppm, 0.50 ppm, 0.25 ppm, 0.13 ppm and 0.0 ppm from the diluter.

The chronic (full life cycle) test was conducted in the same manner as the 15-day flow-through exposure. Because of the length of time required to conduct this test (10 months) it was necessary to use more than one lot of test chemical for testing. The lots used in the chronic test were: NPS 74.004 (25.43% active, 6/27/74), NPS 74.004 (25.83% active, 1/16/75) and NPS 74.004 (25.8% active, 4/23/75). In the chronic test stock solution was made by bringing 51.9 g NPS 74.004 up to a volume of 2 liters using distilled water. Stock solution concentration for the chronic test was calculated to provide theoretical NPS 74.004 concentrations of 1.0 ppm, 0.50 ppm, 0.25 ppm, 0.13 ppm, 0.06 ppm and 0.0 ppm.
- Eluate: N/A
- Differential loading: N/A
- Controls: N/A
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc): N/A
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnow
- Strain: N/A
- Source: Cultured in the Aquatic Environmental Sciences Laboratory (AES) from an original stock supplied by the National Water Quality Laboratory, Duluth, Minnesota.
- Pre-exposure reproductive performance: N/A
- Age: Fish used in the 15 day flow through test were 11-12 days of age, and approximately 7 mm in length. The chronic test (302 day test) started with 18 hour old eggs collected from four different spawns in the AES culture stock.


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): N/A
- Method of collection of fertilised eggs: Eggs used to begin the chronic test were removed from the spawning substrates and transferred immediately, without any chemical treatment, into glass and nylon egg cups suspended in the chronic test tanks.
- Subsequent handling of eggs: N/A


POST-HATCH FEEDING
- Start date: N/A
- Type/source of feed: Brine shrimp cultured in the laboratory.
- Amount given: N/A
- Frequency of feeding: Fish in all tanks were fed ad libitum twice daily
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
302 d
Remarks on exposure duration:
A 15 day flow-through assay was also conducted. Both were preceded by a range-finding test, and acute static tests under varying water quality conditions.
Post exposure observation period:
N/A
Hardness:
197-203 mg/L as CaCO3 (15-day assay)
221-222 mg/L as CaCO3 (chronic full life cycle assay)
Test temperature:
The mean temperature during the 15-day exposure and the 302 day chronic test was 24.3 degrees C with a range of 22.8 to 26.1 degrees C.
pH:
15-day test: pH ranged from 7.84 to 8.08. In the 302-day chronic test, pH ranged from 7.47 to 8.40.
Dissolved oxygen:
The mean dissolved oxygen level for both the 15-day exposure and chronic tests was 7.22 mg/L (range 5.0 - 8.6) representing a mean percent saturation of 85.0%. The lowest DO (5.0) was 59% of saturation.
Salinity:
N/A
Nominal and measured concentrations:
Nominal concentrations in the 302-day chronic test were: 1.0 ppm, 0.50 ppm, 0.25 ppm, 0.13 ppm, 0.06 ppm and 0.00 ppm (active ingredient basis).
Mean measured concentrations (averages of at least 40 samples) were: 0.88 ppm, 0.42 ppm, 0.21 ppm, 0.11 ppm, 0.065 ppm, and 0.015 mg/L (active ingredient).
For more details on the analytical results, see Table in "any other information on materials and methods" below, and attached docment.
Nominal concentrations in the 15-day test were: 2.0, 1.0, 0.50, 0.25, 0.13, and 0 mg/L (active ingredient basis).
Measured concentrations in the 15-day test were: 1.89, 0.98, 0.50, 0.28, 0.17, and 0.05 mg/L (active).
For more details, see Table in "any other information on materials and methods" below, and attached document.
Details on test conditions:
TEST SYSTEM

Both the 15 and 302-day tests:
- Emybro cups (if used, type/material, size, fill volume): glass and nylon mesh egg cups. Two egg cups were suspended in each of the 12 spawning tanks (5 concentrations and one control, in duplicate). Cups were kept in constant motion during egg incubation by a rocker arm assembly.
- Test vessel: Both the 15-day flow-through exposure and the chronic test were conducted in identical physical systems. A Mount and Brungs type proportional diluter produced five concentrations of NPS 74.004, plus a control. Each concentration was split into 2 sets of duplicate flows in the mixing cell. Individual delivery tubes went to each of two duplicate spawning tanks and into each of two duplicate larvae tanks.
- Type (delete if not applicable): Open
- Material, size, headspace, fill volume: Each of the duplicate spawning tanks were of glass and slate construction and measured 2' x 1' x 1'. They were filled to a volume of 42.53 liters and had an approximate adjusted depth of 9 inches. Each of the duplicate all glass larvae tanks was further divided into two identical chambers each measuring 1 ' x .5' x .5'. They were filled to a volume of 6.52 liters and had an approximate adjusted depth of 5 inches.
- Aeration: All well water entering the laboratory was aerated at a central unit. Supplementary aeration was used in both the 15-day and 302-day chronic tests by providing the distributing reservoir with air stones. However, the individual test tanks themselves were not directly aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter): Proportional diluter
- Renewal rate of test solution (frequency/flow rate): The flow from the diluter was adjusted to yield a flow rate of approximately 11 .7 volumes/24 hours (498 L per 24 hours) for the spawning tanks, and 12.72 volumes/24 hours (82 L per 24 hours) for the larval chambers.
- No. of fertilized eggs/embryos per vessel: 40 in each of the 24 egg cups for the chronic test
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): N/A
- Biomass loading rate: N/A


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water for all phases of the investigation was provided by a 50-ft well located on the Tarrytown, New York laboratory site. Water from the well was pumped into the laboratory through black iron and PVC pipe. The water was then sterilized at the laboratory by use of ultraviolet light and proportioned into lines of ambient (11- 13 degrees C) and heated water for distribution to testing stations. Heated water was softened by exchange of sodium for calcium. Mean hardness and conductivity of the well water were 222 mg/L as CaCO3, and 807 umhos/cm, respectively.
- Total organic carbon: N/A
- Particulate matter: N/A
- Metals: N/A
- Pesticides: N/A
- Chlorine: N/A
- Alkalinity: N/A
- Ca/mg ratio: N/A
- Conductivity: 807.08 umhos/cm
- Salinity: N/A
- Culture medium different from test medium: N/A
- Intervals of water quality measurement: N/A


OTHER TEST CONDITIONS
- Adjustment of pH and water hardess: No.
- Photoperiod: The photoperiod for both the 15-day exposure and the chronic test was 16 hours light, 8 hours dark.
- Light intensity: Illumination for all tests was provided by overhead banks of 40 watt Duro-Test Optima lamps. The lamps at a vertical distance of approximately 7 feet from the surface of the test chambers provided roughly 80-85 foot candles of illumination.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : N/A

15-day test: At the termination of the 15-day exposure, mean survival and mean lengths were recorded for all fish in each test concentration.

302-day chronic test: After 30-days and 60-days exposure, all fry in both the spawning and larval tanks were counted and photographed. After 120 days, the number of fish in the spawning tanks were reduced to 15 fish per tank in preparation for spawning. All extra fish were measured (weight, length) and preserved in Bouin's solution for histological examination. The histological exam was conducted on five, 120-day old fish from the controls and from the 0.5 and 1.0 mg/L concentrations.

Within 24 hours after the 120-day thinning, five spawning substrates were placed in each of the duplicate spawning tanks. (Substrates were etches safety glass measuring 4 X 3 X 1.5 inches (L X W X H). Percent hatchability was recorded for each hatch after a 6-day egg incubation period. All fish in the spawning tanks were sacrificed at 302 days exposure, and lengths, weights, sex and condition were recorded.

VEHICLE CONTROL PERFORMED: N/A


RANGE-FINDING STUDY
- Test concentrations: N/A
- Results used to determine the conditions for the definitive study: Results of the initial, static acute range-finding test, 4 fish/concentration, indicated that the 96 hour LC50 was in the range of 3-4 mg/L. Results of the range-finding tests to determine the toxicity of aged solutions of NPS 74.004 indicated that non-aged and aged (48 hrs) test solutions had similar toxicities. These tests were conducted in partially softened well water with a pH of 7.8.


POST-HATCH DETAILS
- Begin of post-hatch period: N/A
- No. of hatched eggs (alevins)/treatment released to the test chamber: See table below in results.
- Release of alevins from incubation cups to test chamber on day no.: N/A


FERTILIZATION SUCCESS STUDY
- Number of eggs used: N/A
- Removal of eggs to check the embryonic development on day no.: N/A
Reference substance (positive control):
no
Duration:
15 d
Dose descriptor:
LOEC
Effect conc.:
0.98 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: survival and mean length
Remarks on result:
other: See Tables 2 and 10 for analytical data and 15-day survival and length data.
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
0.495 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: survival and mean length
Remarks on result:
other: See Tables 2 and 10 for analytical data and 15-day survival and length data.
Duration:
120 d
Dose descriptor:
LC10
Effect conc.:
0.46 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks:
Total fry survival for F0 and F1 generations in the 302-day chronic test.
Remarks on result:
other: 95% Confidence Limits 0.20-0.60mg/L. See Tables 12A and B, 13, 14, and 20 in the attached document.
Duration:
120 d
Dose descriptor:
LC50
Effect conc.:
0.87 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks:
Total fry survival for F0 and F1 generations in the 302-day chronic test.
Remarks on result:
other: 95% Confidence Limits 0.68-1.29mg/L. See Tables 12A and B, 13, 14, and 20 in the attached document.
Duration:
302 d
Dose descriptor:
NOEC
Effect conc.:
0.42 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Reduced fry survival; reduced hatchability of F1 eggs; and an occluding abnormality of the eye, at the highest treatment, 0.88 mg/L (LOEC).
Remarks on result:
other: See Tables 12A and B, 13, 14, and 20 in the attached document.
Duration:
302 d
Dose descriptor:
LOEC
Effect conc.:
0.88 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Reduced fry survival; reduced hatchability of F1 eggs; and an occluding abnormality of the eye, at the highest treatment, 0.88 mg/L (LOEC).
Remarks on result:
other: See Tables 12A and B, 13, 14, and 20 in the attached document.
Details on results:
- Mortality/survival at embryo, larval, juvenile, and adult stages:
1. 15-day exposure. At the termination of the 15-day flow-through exposure examination of the data revealed that mean survival was high (85%-95%) in all concentrations lower than 0.98 mg/L (mean measured active ingredient). Mean survival at 0.98 mg/L and 1.89 mg/L was notably low, 47.0% and 0.0% respectively.

2. 302-day chronic exposure. Mean survival data for fathead minnows in the chronic parental test tanks and the larval tank reveal that in both, survival seemed adversely affected at 30-day exposure to a mean measured concentration of 0.88 mg/L. Concentrations below 0.88 mg/L showed no observable effect on fry in either the parental or larval tanks at 30-day exposure. The 60-day exposure data for both series revealed no additional mortality at any of the concentrations tested. Similarly, 120 day exposure resulted in no change or negligible change in survival for all concentrations. Examination of the 302 day survival data collected at the termination of the parental tank chronic exposure showed slight reductions in survival in one test chamber only at concentrations of 0.24 mg/L (73% survival) and 0.42 mg/L (80%). 100% and 87% survival were observed in the other chambers. Both duplicates were reduced (73 and 80%) at the highest concentration, 0.88 mg/L, although survival was still relatively high, 77%.
- Days to hatch or time to release of young: N/A
- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: N/A
The 0.88 mg/L exposure exhibited a greater percent of spawns resulting in low hatchability than other exposure groups.
- Number of fish in swim-up stage at one or more time periods (e.g., day x1, x2): N/A
- Observations on body length and weight of young and/or exposed parents at one or more time periods:
An examination of length data for parental tank fry and the larval tank duplicate fry indicates the possibility of a toxicity related growth effect at 30 and 60 day exposure for fry in the parental tank series. During the 30 day exposure the "t" test indicates that fish in the 0.42 mg/L and 0.88 mg/L concentration tanks had lengths significantly smaller than the controls. The ANOVA and Duncan's tests further revealed that the lengths of fish in the 0.88 mg/L concentration were significantly smaller than lengths in all other concentrations. Similarly, "t" test analysis of the 60 day exposure lengths for the parental tank fish reveals that the lengths of fish in the 0.42 mg/L and 0.88 mg/L were significantly different from that of the controls.
- Number of healthy fish at end of test: N/A
- Type of and number with morphological abnormalities: N/A
- Type of and number with behavioural abnormalities:
Fish from the highest treatment exhibited erratic swimming behavior and did not consume as much food as fish in other treatments.
- Type and number of developmental/reproductive effects: N/A
- Detailed data on spawning, egg numbers, fertility, and fecundity: N/A
- Type and magnitude of hormonal changes: N/A
- Other biological observations:
Examination of the 30 day F1 fry survival analysis reveals that the mean number of survivors in the 0.88 mg/L 30 day fry exposures is significantly smaller than mean survival at all other concentrations plus controls. The 60 day F1 fry survival data analysis again indicates that only the 0.88 mg/L demonstrate survival significantly lower than the controls. Neither the 30 day nor 60 day F1 fry NPS 74.004 exposure growth analyses indicate a significant effect of the toxicant on growth at any of the concentration levels.
The histological examination performed on 120 day exposed fish from parental tank controls, 0.42 mg/L and 0.88 mg/L show no discernable evidence of tissue pathology due to toxicant exposure. At termination, after 302 days of NPS 74.004 exposure, a general morphological examination of all test fish revealed an occluding abnormality of the eyes in 10 of the 23 surviving fish in the 0.88 mg/L concentration.
- Effect concentrations exceeding solubility of substance in test medium: N/A
- Incidents in the course of the test which might have influenced the results: N/A
Results with reference substance (positive control):
N/A
Reported statistics and error estimates:
N/A

N/A

Validity criteria fulfilled:
not specified
Remarks:
There are no validity criteria for a fish life cycle test (EPA). This study met the typical validity criteria for a chronic aquatic test (control mortality, measured test concentration, etc.).
Conclusions:
The full life cycle (302 day) toxicity test of C12/14 amine oxide to fathead minnows indicated a NOEC of 0.42 and a LOEC of 0.88 mg/L (mean measured active ingredient), based on reduced fry survival, reduced hatching of eggs, and occluded eyes in test fish. This was consistent with results of a 15-day test, in which the NOEC and LOEC were determined to be 0.495 and 0.98 mg/L (mean measured active ingredient), respectively.

Executive summary:

A full life-cycle toxicity test of C12/14 amine oxide was conducted with the fathead minnow, Pimephales promelas (302 day flow through test). The nominal test concentrations were 0, 0.06, 0.13, 0.25, 0.50, and 1.0 mg/L (active ingredient). Mean measured concentrations were 82 -117% of nominal. Endpoints included survival, growth, and hatchability.

In the full life cycle chronic test (302 days) of fathead minnows, the NOEC of C12/14 amine oxide was 0.42 mg/L (mean measured active ingredient), based on reduced fry survival, reduced egg hatch, and occluded eyes in test fish. This was consistent with results of a preliminary 15 -day test, in which the NOEC was 0.495 mg/L.

Endpoint:
fish life cycle toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Read across based on an study following the guideline EPA OPPTS 850.1500 (Fish Life Cycle Toxicity) and performed with C12/14 Amine Oxide. The read across justification document is attached in IUCLID Section 13.
Reason / purpose:
read-across source
Duration:
302 d
Dose descriptor:
NOEC
Effect conc.:
0.42 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other:
Remarks:
In the full life cycle chronic test (302 days) of fathead minnows, the NOEC of C12/14 amine oxide was 0.42 mg/L (mean measured active ingredient), based on reduced fry survival, reduced egg hatch, and occluded eyes in test fish. This was consistent with results of a preliminary 15 -day test, in which the NOEC was 0.495 mg/L.

Description of key information

No chronic fish toxicity studies have been performed using C12-C18 AAAO. However, data are available for the structural analogue C12-14 AO. Following the read-across strategy, it is considered appropriate to cover this endpoint by a key study performed with the structural analogue C12-C14 Amine Oxide (C12-C14 AO), yielding a 302d NOEC value of 0.42 mg a.i./L.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
0.42 mg/L

Additional information

No chronic fish toxicity studies have been performed using C12 -C18 AAAO. However, data are available for C12-14 AO, a similar substance to which C12 -C18 AAAO can be read across. In a full life-cycle toxicity test (similar to EPA OPPTS 850.1500) fathead minnows (Pimephales promelas) were exposed to the substance for 302 days under flow through conditions [Aquatic Environmental Services (1976)]. The nominal test concentrations were 0, 0.06, 0.13, 0.25, 0.50, and 1.0 mg AO/L . Mean measured concentrations were 82 -117% of nominal. Endpoints included survival, growth, and hatchability. The 302d NOEC was 0.42 mg a.i./L (mean measured concentration), based on reduced fry survival, reduced egg hatch, and occluded eyes in test fish. This was consistent with results of a preliminary 15 -day test, in which the NOEC was 0.495 mg AO/L.

The justification for read-across within this endpoint can be found in Annex 1 of the Chemical Safety Report: "Justification of the analogue approach".