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Long-term toxicity to fish

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Reference
Endpoint:
long-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 14, 1991 to May 28, 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: U.S. EPA FIFRA 72-4(a)
Deviations:
yes
Remarks:
the slight deviations from the protocol (such as hardness level, renewal of test solutions, temperature) did not have any impact on the test results
GLP compliance:
yes
Remarks:
US EPA GLP
Specific details on test material used for the study:
- Active substance (30% a.s.), alkyl(C12-C16)dimethylbenzylammonium chloride (ADBAC)
- Physical state: Light beige solid
- Analytical purity: 30% w/w a.s. in aqueous solution;
- Lot/batch No.: 05-6K, (non-labelled material)
- Radiochemical purity (if radiolabelling): 98.4% [96.5% (by TLC) and 95.5% (by HPLC)]
- Specific activity (if radiolabelling): 25mCi/mmol
- Locations of the label (if radiolabelling): Labelled on the benzene ring
- Stability under test conditions: The non-radiolabelled a.s., ADBAC, is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five years under standard laboratory conditions.
- Storage condition of test material: Under refrigeration
Analytical monitoring:
yes
Details on sampling:
See below under 'Details on test solutions'
Vehicle:
yes
Details on test solutions:
- The test solutions used in this study were prepared from a single primary stock solution. The primary stock was prepared by mixing appropriate amounts of 14C-a.s. test substance and non-radiolabelled a.s. test substance in de-ionized water to yield a nominal concentration of 3.93 mg/mL 14C-a.s. test substance/a.s. test substance. The concentration of the radiolabelled a.s. test substance in the stock solution was verified by liquid scintillation counting (LSC).
- Test solutions were prepared by adding measure aliquots of the primary stock solution to dilution water. All test solutions were re-newed daily and prepared immediately before use. The 24h old test solutions were siphoned remained in the vessel. To complete the renewal process, newly prepared test solution or control water was gently poured along the side of the appropriate vessel.
- Triplicate one mL samples were collected and counted for all LSC analyses.
Test organisms (species):
Pimephales promelas
Details on test organisms:
Test organism:
- Common name: Fathead minnow
- Source: The U. S. EPA-Environmental Monitoring and Support Laboratory (Newtown, OH)
- Age at study initiation: <24 h

Method for preparation and collection of fertilized eggs:
- Method of collection of fertilised eggs: Eggs were obtained from in-house culture
- Subsequent handling of eggs: Fertilized eggs ≤24 h old were exposed in prepared test containers. They were examined daily to monitor hatching. For 5-7 d after test initiation, fry were observed for development, hatching and survival on a daily basis.

Post-hatch feeding:
- Type/source of feed: Newly hatched brine shrimp (Artemia salina) nauplii was used as food source
- Frequency of feeding: At least twice per day (second feeding prior to test water renewal)
Test type:
other: Daily static-renewal without aeration
Water media type:
freshwater
Total exposure duration:
34 d
Remarks on exposure duration:
(6d pre-hatch + 28d post-hatch)
Hardness:
75 mg/L CaCO3
Test temperature:
24.2 to 26.3°C
pH:
6.7 to 7.6
Dissolved oxygen:
5.0 to 9.2 mg/L (61 to 111% air saturation) at 25°C
Nominal and measured concentrations:
Nominal concentrations: 0, 27, 74, 135, 180, 270 and 490 µg/L
Measured concentrations: 0, 32.2, 75.9, 134.2, 186.8, 273.2 and 488.7 µg a.i./L
Details on test conditions:
Test sytem:
- Test vessel: 1 L glass beakers containing 0.5 L of test solution with an approx. depth of 65 cm.
- Type: Closed - to minimise evaporation and keep out foreign matter
- Aeration: No aeration
- Renewal rate of test solution (frequency/flow rate): Daily
- No. of fertilized eggs/embryos per vessel: 20 eggs per replicate. After 7 d of exposure, surviving fry from two replicates were thinned to 10 animals per replicate for each test concentration and control.
- No. of vessels per concentration (replicates): 4 replicates per group

Test medium / water parameters:
- Source/preparation of dilution water: Dilution water consisted of water from two deep wells. Water was treated to remove iron and organic impurities, passed through reverse-osmosis purifiers, and treated to obtain a hardness of approximately 75 mg/L as CaCO3.
- Alkalinity: 80-84 mg/L
- Conductivity: 206-211 umhos

Other test conditions:
- Photoperiod: 16 h light : 8 h dark
- Light intensity: Fluorescent light of 76.4 to 80.5 fc intensity

Effect parameters measured (with observation intervals if applicable) : Toxicity, mortality, effects on hatchability and growth

Vehicle control: Yes

Range-fingind study:
- Test concentrations: 100, 300, 600 and 900 µg/L
- Duration of exposure: 10d
- Number of eggs/vessel: 30 eggs for each test concentration, transferred using a wide-bore pipette
- Results used to determine the conditions for the definitive study: Yes; fry mortality was ≤10% in the control and 100 ug/L; 80% in 300 µg/L and 100% in the 600 and 900 µg/L nominal test concentrations during the 10-d exposure.

Post-hatching details:
- Begin of post-hatch period: After 6d
Reference substance (positive control):
no
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
273.7 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
number hatched
Key result
Duration:
28 d
Dose descriptor:
LC50
Effect conc.:
94 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 64-126 ug/L
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
32.2 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
> 32.2 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
Hatchability:
- Hatchability in the mean measure test concentrations 32.2 through 273.2 µg/L ranged from 78 to 91 percent. None of these differences were significantly (P>0.05) different from the control (89% hatchability).
- Percent hatchability in the 488.7 µg/L was 68 percent which was significantly (P <0.05) lower than the control.
- The NOEC for hatchability, therefore was 273.2 µg/L.

Mortality:
- The 28-d LC50 value was calculated to be 94 ug/L with a 95% confidence interval of 64 to 126 µg/L.. See below Table 1 under ány other information on results incl. tables'
- 10% mortality occured in the dilution water control during the 28-d post-hatch exposure period.
- The survival after 28 d post-hatch in the mean measure test concentration 32.2 µg/L (74%) was not significantly lower (P>0.05) when compared to control survival (90%); however survival in all other test concentrations was significantly lower (P<0.05) than the control.

Growth:
- The average dry weight of the surviving fish generally increased as the concentration of the test substance increased (range, 0.972 mg/fish to 1.908 mg/fish), and in each case was higher than the control (0.800 mg/fish). The trend of increased growth also correlated with the decreased numbers of fish present in each test concentration suggesting food and space may have been the reason for the increased weights in the higher concentrations which were lethal to the less tolerant or weaker fish.
- Since no reduction in growth was observed at any of the test concentrations, th IC50 and LOEC values were not determined.
- The NOEC for gowth was determined to be >32.2 ug/L, the lowest concentration tested without significant decrease in survival.
Results with reference substance (positive control):
-
Reported statistics and error estimates:
Estimate of LC50 values were obtained using version 2 of the computer program TOXSTAT (Peltier and Weber). IC50calculated using monotonic regression analysis. The 28-d post-hatch survival data were analyzed using Fisher’s Exact Test. Analysis of variance (ANOVA) and Dunnett’s multiple comparison tests were used (SAS-Version 6.03) for comparing hatchability and weight data.

Table 1. LC50

LC50 (µg/L)

7-d post hatch

14-d post hatch

21 -d post hatch

28-d post hatch

 

198

104

98

94

95% confidence

limits (µg/L)

(164-231)

(72-138)

(68-130)

(64-126)

Table 2. NOECs LOECs values

LOEC (µg/L) hatchability

488.7

NOEC (µg/L) hatchability

273.2

NOEC(µg/L) survival

32.2

NOEC (µg/L) growth

>32.2

Validity criteria fulfilled:
not specified
Conclusions:
Under the study conditions, the 28 d NOEC for hatchability was 273.7 µg/L, the 28 d NOEC for survival was 32.2 µg a.i./L and the 28 d NOEC for growth was > 32.2 µg a.i./L
Executive summary:

A study was conducted to determine the long-term toxicity to fish of the test substance according to U.S. EPA FIFRA 72-4(a), in compliance with GLP. The experiment assessed toxicity, mortality, hatchability and growth of Fathead minnow (Pimephales promelas). Eggs (80 per concentration) were exposed to mean measured concentrations of 0, 32.2, 75.9, 134.2, 186.8, 273.2 and 488.7 µg a.i./L of radiolabelled test substance. Analytical determination was performed and the sample concentrations were verified by liquid scintillation counting. After 7 days, surviving fry from two replicates were thinned to 10 animals per replicate for each exposure group (total of 20 animals per concentration) and exposed to the same concentrations for a 28 day post-hatch static renewal toxicity test. Observations of symptoms and mortality were conducted daily. Under the study conditions, the 28 d NOEC for hatchability was 273.7 µg/L, the 28 d NOEC for survival was 32.2 µg a.i./L and the 28 d NOEC for growth was > 32.2 µg a.i./L (McIntyre, 1992).

Description of key information

The 28 d NOEC for Fathead minno wmortality due to the test substance was determined to be 0.032 mg a.i./L (measured).

Key value for chemical safety assessment

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

Additional information

A study was conducted to determine the long-term toxicity to fish of the test substance according to U.S. EPA FIFRA 72-4(a), in compliance with GLP. The experiment assessed toxicity, mortality, hatchability and growth of Fathead minnow (Pimephales promelas). Eggs (80 per concentration) were exposed to mean measured concentrations of 0, 32.2, 75.9, 134.2, 186.8, 273.2 and 488.7 µg a.i./L of radiolabelled test substance. Analytical determination was performed and the sample concentrations were verified by liquid scintillation counting. After 7 days, surviving fry from two replicates were thinned to 10 animals per replicate for each exposure group (total of 20 animals per concentration) and exposed to the same concentrations for a 28 day post-hatch static renewal toxicity test. Observations of symptoms and mortality were conducted daily. Under the study conditions, the 28 d NOEC for hatchability was 273.7 µg/L, the 28 d NOEC for survival was 32.2 µg a.i./L and the 28 d NOEC for growth was > 32.2 µg a.i./L (McIntyre, 1992).