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EC number: 203-699-2 | CAS number: 109-73-9
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- Ecotoxicological Summary
- Aquatic toxicity
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- Short-term toxicity to fish
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- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
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Long-term toxicity to aquatic invertebrates
Administrative data
Link to relevant study record(s)
- Endpoint:
- long-term toxicity to aquatic invertebrates
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 25 June - 2 July 1994
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: Method 1002.0 (EPA 1989)
- Deviations:
- yes
- Remarks:
- Hardness and alkalinity of the highest treatment level and the control solutions were monitored at test initiation only, instead of beginning of each renewal period. T monitoring restricted to new solutions, but T control warranted (water bath).
- Qualifier:
- according to guideline
- Guideline:
- other: Standard guide for conducting acute toxicity tests with fishes, macroinvertebrates and amphibians (ASTM - 1993)
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 211 (Daphnia magna Reproduction Test)
- Deviations:
- yes
- Remarks:
- other Daphnia species used, 7d instead of 21d reproduction
- GLP compliance:
- yes (incl. QA statement)
- Analytical monitoring:
- yes
- Details on sampling:
- A semi-static test design with renewal of test media after 24 hours was applied.
During the definitive exposure period, water samples were removed from the test solution of each treatment level and the control at 0, 24 and 48 hours (fresh solutions) and at test termination (day 7, i.e. aged solution) for analysis of substance concentration. Each treatment level was sampled in duplicate using a volumetric pipet.
Samples analyzed at the 0, 24 and 48 hour sampling intervals were removed from an additional replicate vessel (replicate k, without test organisms) while samples analyzed at test termination were removed from a composite of test solutions from all 10 test replicates.
Each exposure solution sample was collected from the approximate midpoint of the test vessel. - Vehicle:
- no
- Details on test solutions:
- Prior to test initiation, a 1.0 mg A.I./mL stock solution was prepared by dissolving 0.1006 g of test material (0.1004 g as active ingredient) with 100 mL of dilution water in a 100-mL volumetric flask. The stock solution was observed to be clear and colorless with no visible signs of undissolved test material (e.g., precipitate, surface film).
Exposure solutions were prepared individually by adding the appropriate volume of the 1.0 mg A.I./mL stock solution to 500 mL of dilution water in a glass beaker. The resultant solution was briefly stirred with a glass rod and then decanted into the eleven individual replicate test vessels. Test vessels were capped immediately to avoid any loss of test material due to volatilization. The remaining test solution was used for initial water quality analyses. One set of eleven control vials was also established which contained the same dilution water and was maintained under the same conditions as the treatment level solutions but contained no monobutylamine. Test solutions were renewed daily. Test organisms were carefully transferred into freshly prepared solutions. - Test organisms (species):
- Ceriodaphnia dubia
- Details on test organisms:
- Source: Springborn Laboratories culture facility
Culturing water: well water adjusted to a total hardness range of 80 to 100 mg/L (as CaCO3).
The culture area photoperiod: 16 hours of light and 8 hours of darkness;
Culturing temperature: temperature controlled waterbath maintained the culture solution temperature at 25 ± 1 °C.
Feeding: combination of a unicellular green algae (Selenastrum capricornutum) and YCT mixture (yeast, cereal leaves and trout chow), once daily; food sources were considered to be of acceptable quality since the total concentration of pesticides measured (trout food source) was less than 0.3 mg/kg, As, Cd, Pb and Hg 2.6, 0.6, 0.4 and < 0.02 ppm, respectively.
Twenty-four hours before test initiation, all immature daphnids were removed from the culture beakers. Offspring produced over the first 8-hours period were culled individually using a glass pipette and were used to initiate the short-term test. This procedure ensured that the difference in age of the test population did not exceed 8 hours.
Age: <= 24hours
Feeding during test: combination of a unicellular green algae (Selenastrum capricornutum) and YCT mixture (yeast, cereal leaves and trout chow) once daily with each media renewal. - Test type:
- semi-static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 7 d
- Hardness:
- Moderately hard reconstituted water (96 to 100 mg/L as CaCO3)
- Test temperature:
- 23 to 26 °C
- pH:
- 7.8 to 8
- Dissolved oxygen:
- 62 - 104 %
- Salinity:
- no data
- Nominal and measured concentrations:
- Nominal concentrations are based on acute daphnia test results.
Nominal concentrations: 0.43 - 0.85 - 1.7 - 3.3 - 6.5 mg/L
Measured concentrations: 0.41 - 0.82 - 1.7 - 3.0 - 6.1 mg/L - Details on test conditions:
- TEST SYSTEM
- Material: 20 ml glass vial each containig 20 mL of test solution and sealed with a Teflon lined screw top cap and minimal headspace to avoid volatilization losses
- Renewal rate of test solution: 24 hours renewal period
OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light and 8 hours of darkness
- Light intensity: 50 to 100 footcandles at the surface of the culture solutions
WATER PARAMETERS
- Source/preparation of dilution water: well water, same source as the water used in the daphnid cultures
- Pesticides: no
- Toxic metals: no
- Alkalinity: 62 to 66 mg/L
- pH: 7.8 to 8.0
- Ca/Mg ratio: 2
- Conductivity: 340 to 370 µmhos/cm
- Total organic carbon: 0.89 mg/L for the month of June 1994
The test vessels used to house the exposed organisms were placed in stratified random order in a temperature controlled water bath designed to maintain exposure solution temperatures at 25 ± 1 °C.
Selection of nominal monobutylamine concentrations for the short term chronic toxicity test with Ceriodaphnia dubia was based on toxicity information developed during a 48-hour acute toxicity test. The nominal concentrations selected for the definitive test were 0.43, 0.85, 1.7, 3.3 and 6.5 mg A.I./L.
Eleven replicate test vessels (replicates A - K) were established for each treatment level and the control. The replicate K vessel was used to provide solution for analytical sampling to confirm the concentration of monobutylamine. This vessel (K) did not contain a test organism.
The test was initiated when daphnids, < 24 hours old, were impartially distributed to each concentration and the controls (each of 10 replicate vessels (A - J), one daphnid per vessel) and was terminated 7 days later. Food suspensions were introduced into each treatment and control vessel at each renewal period (daily) at a rate of 100 µL suspension of YCT and 100 µL of algae at a concentration of 3.0 to 3.5 x 10^7 cells/mL.
MONITORING:
The number of mortalities and number of offspring produced in each replicate test vessel was recorded at each solution renewal (24-hour intervals) and at test termination (day 7). Biological observations and observations of the physical characteristics of each replicate test solution were also made and recorded on day 0, at each renewal period and test termination. The pH and dissolved oxygen concentration were measured in each treatment and the control at the beginning (new solutions) and end (aged solutions) of each renewal period. Specific conductance and temperature were measured at each renewal period in the freshly prepared test solutions. Total alkalinity and hardness were measured in the freshly prepared solutions of the highest treatment level and control at test initiation. Water quality measurements in the freshly
prepared solutions were obtained from replicate vessel K. Water quality measurements in the aged solutions were obtained from composited solutions from replicate vessels A - J of each concentration and control. Continuous temperature monitoring was performed on the surrounding water in the waterbath throughout the exposure period. - Reference substance (positive control):
- not specified
- Duration:
- 7 d
- Dose descriptor:
- LOEC
- Effect conc.:
- 2.22 mg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- test mat.
- Basis for effect:
- reproduction
- Remarks:
- and mortality
- Remarks on result:
- other: corresponding to nominal 6.5 mg/L
- Key result
- Duration:
- 7 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 1.09 mg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- test mat.
- Basis for effect:
- reproduction
- Remarks:
- and mortality
- Remarks on result:
- other: Corresponding to nominal 3.3 mg/L
- Details on results:
- Results on technical conditions: pH and concentrations
The water quality parameters measured, with the exception of pH, were unaffected by the concentrations of monobutylamine tested and remained within acceptable ranges for the survival of Ceriodaphnia dubia.
The pH of the exposure solutions generally increased with increasing test material concentration, however, this increase was minimal and the pH in all solutions remained within an acceptable range for the survival and the reproduction of Ceriodaphnia dubia.
Analysis of the newly prepared (0, 24 and 48 hours) exposure solutions for monobutylamine resulted in measured concentrations which were consistent between replicate vessels, sampling intervals and in agreement with the nominal fortified levels.
Measured concentrations established for the newly prepared solutions averaged 96% of nominal with minimal variability between measurements.
Measurement of exposure solutions on day-7 (aged solutions), resulted in concentrations significantly lower than measured concentrations determined in newly prepared solutions.
As a consequence, time weighted mean values were calculated for the LOEC and the NOEC according to the methodology given in OECD 211, Annex 6.
All validity criteria of US EPA method 1002.0 were fulfilled. - Results with reference substance (positive control):
- no data
- Reported statistics and error estimates:
- Data on survival were analyzed using Fisher's Exact test (Steel and Torrie, 1980). Survival data were analyzed prior to data for reproductive performance.
Test concentrations which adversely affected survival were excluded from subsequent analysis of the reproduction data.
Reproduction data from test concentrations where mortality was not significantly different from the controls was analyzed using the Dunnett's test (Dunnett, 1955, 1964). Test organisms that died during the test were included in the analysis of the reproduction data. Fisher's and Dunnett's tests identify the treatment levels that produce organism responses significantly different (p <=0.05) from those of the test controls.
The Chi-square test was used to compare the observed sample distribution with normal distribution. As a check on the assumption of homogeneity of variance implicit in parametric statistics, data for reproduction were analyzed using Bartlett's Test. - Validity criteria fulfilled:
- yes
- Conclusions:
- Long-term toxicity to aquatic invertebrates was determined according to US EPA method 1002.0 (1989) using Ceriodaphnia dubia:
NOEC (7d; reproduction & parental survival; TWM measured): 1.09 mg/L n-butylamine;
LOEC (7d; reproduction & parental survival; TWM measured): 2.22 mg/L n-butylamine. - Executive summary:
Inhibition of reproduction and mortality of Ceriodaphnia dubia by n-butylamine was assessed according to US EPA method 1002.0 ("DAPHNID, CERIODAPHNIA DUBIA, SURVIVAL AND REPRODUCTION TEST", 1989) as well as U.S. standard guide for conducting acute toxicity tests with fishes, macroinvertebrates and amphibians (ASTM - 1993), applying a semistatic test design (24 hours renewal period) with analytical verification of test item concentrations during the test. The valid and reliable study was performed compliant with GLP and fulfilled all validity criteria of the guideline. 5 concentration levels were tested (nominal concentrations: 0.43 - 0.85 - 1.7 - 3.3 - 6.5 mg/L) and measured concentrations determined from freshly prepared solutions were between 89.4% and 96.5% of nominal values. A rapid decline in n-butylamine concentration was observed during 24 hours. To account for this, time weighted mean measured conentrations were calculated for the NOEC (nominal 3.3 mg/L) and the LOEC (nominal 6.5 mg/L) according to the methodology given in OECD 211 (October 2012), Annex 6.
The pH of the exposure solutions generally increased with increasing test material concentration, however, this increase was minimal and the pH in all solutions remained within an acceptable range for the survival and the reproduction of Ceriodaphnia dubia.
Results:
Mortality levels observed (nominal concentrations): 0 % in control, 30% at 0.43 mg/L, 10% at 0.85 mg/L, 10% at 1.7 mg/L, 30% at 3.3 mg/L, 50% at 6.5 mg/L.
Mean number of offspring per female (SD), based on nominal concentrations: 35(6.1) in control, 28(14) at 0.43 mg/L, 36(12) at 0.85 mg/L, 35(11) at 1.7 mg/L, 24(16) at 3.3 mg/L, 11(12) at 6.5 mg/L.
Based on these data, the following results were obtained based on time weighted mean measured concentrations:
NOEC (7d; reproduction & parental survival; TWM measured): 1.09 mg/L n-butylamine;
LOEC (7d; reproduction & parental survival; TWM measured): 2.22 mg/L n-butylamine.
Reference
Details on results
Analytical results:
Analysis of the newly prepared (0, 24 and 48 hours) exposure solutions for monobutylamine resulted in measured concentrations which were consistent between replicate vessels, sampling intervals
and in agreement with the nominal fortified levels. Measurement of exposure solutions on day 7, which represented exposure conditions maintained in aged (24-hour old) solutions, resulted in measured monobutylamine concentrations which were significantly lower than measured concentrations determined in the newly prepared solutions: all levels were < LOQ, except for the highest concentration of nominally 6.5 mg/L for which ca. 7.7% of the mean measured concentration determined for the freshly prepared solutions was found after 24 hours (for detailed results see table below). This was unexpected based on the preliminary testing on stability over 24 hours performed beforehand. In an effort to define conditions which resulted in the decreased exposure concentrations during the 24 hours between solution preparation and renewal, additional investigations were conducted comparing stability of two amines (monobutylamine and monoethylamine) in standard dilution water when prepared in clean vessels versus vessels which were conditioned in the presence of monoethylamine to simulate the biological activity in the vessels during the chronic toxicity test. Using clean vessels, results of the preliminary testing on n-butylamine stability over 24 hours could be confirmed. Results with the preconditioned vessels led to the conclusion that preconditioning analogous to standard conditions required to support the survival and reproduction of Ceriodaphnia dubia provided biological activity capable of biodegradation of the test material, which was more rapidly if conditioning had been performed with the specific amine compared to the non-specific one.
Analytical results table:
Measured concentrations (mg A.I./L) referring to fresh media, with exception to day 7, referring to 24 hours old media |
Arithmetic mean (SD) |
Geometric mean |
||||||||
Time point |
0 h |
0 h |
24 h |
24 h |
48 h |
48 h |
7 d |
7 d |
fresh media |
fresh media |
Nominal Concentration (mg A.I./L) |
1 |
2 |
1 |
2 |
1 |
2 |
1 |
2 |
||
Control |
<0.11 |
<0.11 |
<0.12 |
<0.12 |
<0.13 |
<0.13 |
<0.11 |
<0.11 |
N.A. |
N.A. |
0.43 |
0.41 |
0.42 |
0.45 |
0.46 |
0.35 |
0.34 |
<0.11 |
<0.11 |
0.41 (0.051) |
0.40 |
0.85 |
0.85 |
0.87 |
0.87 |
0.9 |
0.7 |
0.74 |
<0.11 |
<0.11 |
0.82 (0.079) |
0.82 |
1.7 |
1.9 |
1.8 |
1.8 |
1.8 |
1.6 |
1.5 |
<0.11 |
<0.11 |
1.7 (0.13) |
1.73 |
3.3 |
3.3 |
3.3 |
3.1 |
3.1 |
2.7 |
2.7 |
<0.38 |
<0.38 |
3.0 (0.26) |
3.02 |
6.5 |
6.8 |
6.7 |
6.2 |
6.2 |
5.6 |
5.3 |
0.49 |
0.46 |
6.1 (0.57) |
6.11 |
Stock solution (1000) |
1100 |
1000 |
1000 |
NA |
1000 |
Quality control samples (QC) in mg/L A.I., value in parentheses represents the nominal fortified concentration:
QC #1 QC #2 QC #3 |
0.443 1.98 6.15 |
0.447 2.04 6.27 |
0.454 2.14 6.19 |
0.432 2.05 6.07 |
Biological results:
Throughout the 7-day study, no undissolved test material (e.g., precipitate, film on the solutions' surface) was observed in any of the exposure solutions. Cumulative mean percent survival of parental daphnids is presented in the table below. Survival among control organism was 100% and exceeded the minimal performance criteria (80%). Survival among organisms exposed to the highest treatment level (6.5 mg A.I./L nominal concentration) was 50%, which was significantly different as compared to the control. The percent survival in the remaining treatment levels after 7-days of exposure to monobutylamine ranged from 70 to 90% which was comparable to the survival of the control organisms (not significantly different). Sublethal effects (e.g., lethargy) were observed among one of the surviving daphnids exposed to the nominal treatment levels of 1.7, 3.3 and 6.5 mg A.I./L. No sublethal effects were observed among surviving daphnids exposed to the lower treatments or the control solutions.
Biological results table 1: Cumulative mean percent survival of parental daphnids (Ceriodaphnia dubia) during the 7-day static renewal exposure to monobutylamine.
Nominal |
|
Mean Cumulative Percent Survival |
Day 6 |
Day 7 |
|
Control |
100 |
100 |
0.43 |
80 |
70 |
0.85 |
90 |
90 |
1.7 |
90 |
90a |
3.3 |
70 |
70a |
6.5 |
50 |
50a* |
a) one of the mobile ceriodaphnids was observed to be lethargic.
*) significantly different from the control organisms.
Results on reproduction (see results table below):
Daphnids at all treatment levels released their first brood on day 3. All control organisms released more than three broods, exceeding the minimal performance criteria (60% of the surviving females must produce three broods). Reproduction among test organisms exposed to the highest treatment level (6.5 mg A.I./L nominal) averaged 11 offspring per female which was reduced compared to the reproduction of the control organisms (35 offspring/female). Since parental organism survival was adversely affected at this treatment level, the reproductive performance of organisms exposed to 6.5 mg A.I./L (nominal) monobutylamine was not statistically analysed. Reproduction among organisms exposed to the remaining treatment levels ranged from 24 to 36 offspring per female and was statistically comparable to the control data.
Results table 2: Mean cumulative number of offspring produced per female daphnid (Ceriodaphnia dubia) during the 7-day static renewal exposure to monobutylamine.
Nominal |
|
|
Mean Cumulative Number of Offspring/Female |
|
Mean Total |
||||
|
|
|
|
|
|
|
|||
(mg A.I./L) |
Day: |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
(SD)a) |
Control |
|
0 |
0 |
3 |
5 |
7 |
9 |
11 |
35 (6.1) |
0.43 |
|
0 |
0 |
5 |
6 |
3 |
6 |
10 |
28 (14) |
0.85 |
|
0 |
0 |
3 |
5 |
6 |
6 |
15 |
36 (12) |
1.7 |
|
0 |
0 |
4 |
6 |
4 |
7 |
13 |
35 (11) |
3.3 |
|
0 |
0 |
2 |
3 |
0 |
9 |
10 |
24 (16) |
6.5 |
|
0 |
0 |
0 |
2 |
0 |
4 |
6 |
11 (12) |
In conclusion, the NOEC (7 days) for parental mortality as well as reproduction was determined to be 3.3 mg/L n-butylamine (nominal concentration). The corresponding LOEC (7 days) for mortality and reproduction equals the highest concentration tested, i.e. 6.5 mg/L n-butylamine (nominal concentration).
Description of key information
Long-term toxicity to aquatic invertebrates was determined according to US EPA method 1002.0 (1989) using Ceriodaphnia dubia:
NOEC (7d; reproduction & parental survival; TWM measured): 1.09 mg/L n-butylamine;
LOEC (7d; reproduction & parental survival; TWM measured): 2.22 mg/L n-butylamine.
Key value for chemical safety assessment
Fresh water invertebrates
Fresh water invertebrates
- Effect concentration:
- 1.09 mg/L
Additional information
Inhibition of reproduction and mortality of Ceriodaphnia dubia by n-butylamine was assessed according to US EPA method 1002.0 ("DAPHNID, CERIODAPHNIA DUBIA, SURVIVAL AND REPRODUCTION TEST", 1989) as well as U.S. standard guide for conducting acute toxicity tests with fishes, macroinvertebrates and amphibians (ASTM - 1993), applying a semistatic test design (24 hours renewal period) with analytical verification of test item concentrations during the test. The valid and reliable study was performed compliant with GLP and fulfilled all validity criteria of the guideline. 5 concentration levels were tested (nominal concentrations: 0.43 - 0.85 - 1.7 - 3.3 - 6.5 mg/L) and measured concentrations determined from freshly prepared solutions were between 89.4% and 96.5% of nominal values. A rapid decline in n-butylamine concentration was observed during 24 hours. To account for this, time weighted mean measured conentrations were calculated for the NOEC (nominal 3.3 mg/L) and the LOEC (nominal 6.5 mg/L) according to the methodology given in OECD 211 (October 2012), Annex 6.
The pH of the exposure solutions generally increased with increasing test material concentration, however, this increase was minimal and the pH in all solutions remained within an acceptable range for the survival and the reproduction of Ceriodaphnia dubia.
Results:
Mortality levels observed (nominal concentrations): 0 % in control, 30% at 0.43 mg/L, 10% at 0.85 mg/L, 10% at 1.7 mg/L, 30% at 3.3 mg/L, 50% at 6.5 mg/L.
Mean number of offspring per female (SD), based on nominal concentrations: 35(6.1) in control, 28(14) at 0.43 mg/L, 36(12) at 0.85 mg/L, 35(11) at 1.7 mg/L, 24(16) at 3.3 mg/L, 11(12) at 6.5 mg/L.
Based on these data, the following results were obtained based on time weighted mean measured concentrations:
NOEC (7d; reproduction & parental survival; TWM measured): 1.09 mg/L n-butylamine;
LOEC (7d; reproduction & parental survival; TWM measured): 2.22 mg/L n-butylamine.
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