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Ecotoxicological information

Long-term toxicity to fish

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Link to relevant study record(s)

Reference
Endpoint:
fish early-life stage toxicity
Type of information:
read-across based on grouping of substances (category approach)
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
Justification for type of information:
Information on the category justification can be found in the Quaternary ammonium salts (QAS) category and section 13.2 of IUCLID.
Qualifier:
according to guideline
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
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
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
ca. 273.2 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
Remarks:
(post hatch)
Effect conc.:
ca. 94 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95% confidence interval: 64-126 µg a.i./L
Key result
Duration:
28 d
Dose descriptor:
NOEC
Remarks:
(post hatch)
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
Remarks:
(post hatch)
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.
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

For further details on results, please refer to the attachment under 'attached background material'.

Validity criteria fulfilled:
not specified
Conclusions:
Based on the results of the source study, the 7-d NOEC for hatchability was 273.2 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 of the source substance, C12-16 ADBAC (30% active in water, radiochemical purity: 96.5% (TLC) / 95.5% (HPLC)) to Fathead minnow (Pimephales promelas), according to U.S. EPA FIFRA 72-4(a), in compliance with GLP. Eighty fish eggs per concentration were exposed to the radiolabeled source substance for 34 d (6 d pre-hatching and 28-d post-hatching) at nominal concentrations of 0, 27, 74, 135, 180, 270 and 490 µg/L. Analytical determination was performed, and the sample concentrations were verified by liquid scintillation counting. The mean measured concentrations were determined to be 0, 32.2, 75.9, 134.2, 186.8, 273.2 and 488.7 µg a.i./L of the radiolabelled source substance (with 95.5-96.5% radiolabelled purity). After 7 d, 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 28 d (post-hatch period) under daily static renewal test conditions. Observations of symptoms and mortality were conducted daily. Under the conditions of the study, the NOEC for hatchability was 273.2 µg/L and the 34-d NOEC and LC50 for survival were determined to be 32.2 and 94 µg a.i./L, respectively. Further, no reduction in growth was observed at any of the tested concentration, therefore the 28-d NOEC for growth was considered to be greater than 32.2 µg a.i./L (lowest concentration tested without a significant decrease in survival) (McIntyre and Pate, 1992). Based on the results of the source study, similar effect levels can be considered for the target substance.

Description of key information

Based on the results of the study with a structurally similar substance, the 28-d NOEC value of 32.2 µg a.i./L for long-term toxicity to fish has been considered further for hazard/risk assessment.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
NOEC
Effect concentration:
32.2 µg/L

Additional information

A study was conducted to determine the long-term toxicity of the source substance, C12-16 ADBAC (30% active in water, radiochemical purity: 96.5% (TLC) / 95.5% (HPLC)) to Fathead minnow (Pimephales promelas), according to U.S. EPA FIFRA 72-4(a), in compliance with GLP. Eighty fish eggs per concentration were exposed to the radiolabeled source substance for 34 d (6 d pre-hatching and 28-d post-hatching) at nominal concentrations of 0, 27, 74, 135, 180, 270 and 490 µg/L. Analytical determination was performed, and the sample concentrations were verified by liquid scintillation counting. The mean measured concentrations were determined to be 0, 32.2, 75.9, 134.2, 186.8, 273.2 and 488.7 µg a.i./L of the radiolabelled source substance (with 95.5-96.5% radiolabelled purity). After 7 d, 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 28 d (post-hatch period) under daily static renewal test conditions. Observations of symptoms and mortality were conducted daily. Under the conditions of the study, the NOEC for hatchability was 273.2 µg/L and the 34-d NOEC and LC50 for survival were determined to be 32.2 and 94 µg a.i./L, respectively. Further, no reduction in growth was observed at any of the tested concentration, therefore the 28-d NOEC for growth was considered to be greater than 32.2 µg a.i./L (lowest concentration tested without a significant decrease in survival) (McIntyre and Pate, 1992).


Based on the above study, same effect levels were concluded in the biocide assessment report on C12-16 ADBAC by RMS Italy (ECHA biocides assessment report, 2015). While in the biocides assessment report on TMAC C, a similar chronic NOEC value (i.e., 0.0322 mg a.i./L) for fish was concluded based on a read across study to DDAC. Applying a MW correction factor of 0.75, the chronic NOEC value for Coco TMAC was extrapolated to 0.024 mg a.i./L (ECHA biocides assessment report, 2016). In line with the C12 -16 ADBAC biocides assessment report and given that the read across to C12-16 ADBAC can be justified for the target substance based on a category approach, the 28-d NOEC value of 32.2 µg a.i./L has been considered further for hazard/risk assessment.