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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 October-2 November 2012 and 6 to 9 November 2012
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
BADGE-MXDA (#42) possesses multiple secondary amines. The dissociation constant are estimated in the range of 8 to 9.5 which indicates that BADGE-MXDA (#42) has cationic properties. The testing regime followed OECD (2000) guidance with respect to testing such substances and the test solutions were amended with either 0 or 10 mg/L humic acid. Humic acid is commonly used when testing cationic substances because the dissolved organic carbon binds with cationic chemicals, neutralizes the charges, reduces toxicity and makes cationic chemicals less bioavailable.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Because BADGE MXDA (#42) is a multi-component substance of limited solubility, WAFs were created and analyzed via non-purgable organic carbon (NPOC) as a measure of dissolved organic carbon, and all values reported are nominal. This method is outlined in the OECD guidance (No. 23) on Aquatic Testing of Difficult Substances and Mixtures. NPOC was measured in test solutions at test initiation and termination. The lowest level quantifiable (LLQ) for this method is approximately 1 mg NPOC/L. Because the LLQ is low relative to the loading rates of BADGE-MXDA (#42), samples were taken from the control and two highest loading rates in the test with humic acid. Samples were collected from the bulk test solutions at test initation. At test termination, samples were collected from pooled replicate test vessels containing algae. Blank replicates were analyzed separately at termination.
Vehicle:
no
Details on test solutions:
Algal medium (AAP) was used as the diluent. A primary stock was prepared at the highest dose level via direct addition of the test material on a glass cover slip directly into the appropriate volume of AAP. After the primary stock solution was created, the clear and colorless portions were carefully poured into separate glass vessels. For the definitive test with humic acid, the prepared WAF (30 mg/L loading rate) was adjusted from pH 8.7 to 7.2 using 0.200 mL of HCL 1N prior to dilution or the addition of humic acid. Portions of the WAFs were added to volumes of AAP to achieve the nominal loading rates of 0 (control), 0.012, 0.026, 0.056, 0.12, 0.27, and 0.60 mg/L for the test without humic acid and 0 (control), 0.58, 1.3, 2.8, 6.2, 14, and 30 mg/L for the test with humic acid. Humic acid was then added in the amended test to achieve 10 mg humic acid/L. For the definitive tests, all solutions without humic were clear and colorless. All loading rates and the control containing humic acid were clear and amber.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The algal inoculum for the test was prepared from a 4-day old stock culture for both the test without humic acid and the test with 10 mg humic acid/L. Enough of the inoculum was used to achieve an initial cell density of approximately 10,000 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Test temperature:
Test without humic acid: 22 – 23°C
Test with humic acid: 22 – 24°C
pH:
Test without humic acid: 6.8-7.4
Test with humic acid: 6.9-7.2
Nominal and measured concentrations:
Test without humic acid: 0, 0.012, 0.026, 0.056, 0.12, 0.27, and 0.60 mg BADGE-MXDA (#42) /L, prepared as dilutions of a 0.60 mg/L WAF
Test with humic acid: 0, 0.58, 1.3, 2.8, 6.2, 14, and 30 mg BADGE-MXDA (#42)/L, prepared as dilutions of a 30 mg/L WAF
Details on test conditions:
Test without humic acid: Temperatures during the exposure period ranged from 22 to 23 ºC. Light intensity ranged from 4520 to 5700 lux. The pH was 7.0 at test initiation and ranged from 7.2 to 7.4 and 6.8 to 6.9 in replicates both with and without algae at test termination, respectively.
Test with humic acid: Temperatures during the exposure period ranged from 22 to 24 ºC. Light intensity ranged from 4420 to 5500 lux. The pH ranged from 6.9 to 7.2 at test initiation and was 7.0 and 7.0 to 7.2 in replicates both with and without algae at test termination, respectively. Prior to dilution, the 30 mg/L WAF was adjusted from pH 8.7 to 7.2 using 1N HCl, as described in Preparation of Test Solutions.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: Test with humic acid
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: cell yield
Remarks on result:
other: Test with humic acid
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Test with humic acid
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Test with humic acid
Details on results:
Test without humic acid: 72 hour cell density
EL50 >0.60 mg/L
NOELR ≥ 0.60 mg/L
0-72-hour cell yield
EyL50 >0.60 mg/L
NOELR ≥ 0.60 mg/L
0-72 hour growth inhibition
ErL50 >0.60 mg/L
NOELR ≥ 0.27 mg/L

Test with humic acid: 72 hour cell density
EL50 >30 mg/L
NOELR ≥ 30 mg/L
0-72-hour cell yield
EyL50 >30 mg/L
NOELR ≥ 30 mg/L
0-72 hour growth inhibition
ErL50 >30 mg/L
NOELR ≥ 30 mg/L
Results with reference substance (positive control):
None
Reported statistics and error estimates:
Test without humic acid: At 72 hours, no loading rates had cell densities significantly different from the control. Thus, the NOELR was ≥0.60 mg/L.
For cell density at 72 hours, the calculated EL50 and 95% confidence intervals was >0.60 (0.60 - >0.60) mg BADGE-MXDA (#42)/L. At 0-72 hours, no loading rates had yields significantly different from the control. Thus, the NOELR was ≥0.60 mg/L. For cell yield at 0-72 hours, the calculated EyL50 (95% confidence intervals) was >0.60 (0.60 - >0.60) mg BADGE-MXDA (#42)/L. The mean specific growth rate for 0.60 mg BADGE-MXDA (#42)/L was significantly different from the control. Thus, the NOELR was 0.27 mg/L. Between 0 and 72 hours, the calculated ErL50 and 95% confidence intervals were all >0.60 mg BADGE-MXDA (#42).
Test with humic acid: At 72 hours, no loading rates had mean cell densities significantly different from the control. Therefore, the NOELR was ≥30 mg/L. For cell density at 72 hours, the calculated EL50 (95% confidence intervals) was >30 (not calculable) mg BADGE-MXDA (#42)/L. At 0-72 hours, no loading rates had mean yields significantly different from the control. Therefore, the NOELR was ≥30 mg/L. For cell yield at 0-72 hours, the calculated EyL50 (95% confidence intervals) was >30 (not calculable) mg BADGE-MXDA (#42)/L. At 0-72 hours, no loading rates had average specific growth rates significantly different from the control. Therefore, the NOELR was ≥30 mg/L. Between 0 and 72 hours, the calculated ErL50 (95% confidence intervals) was >30 (not calculable) mg BADGE-MXDA (#42)/L.



The NOELR for growth rate was at least 100 times greater in the test with humic acid, indicating a mitigating effect of humic acid on BADGE-MXDA (#42).

Validity criteria fulfilled:
yes
Conclusions:
In line with the recommendation by relevant guidelines (OECD, 2000 and USEPA, 2010), the effects observed in the series amended with humic acid are perceived more predictive of the potential the effects exerted by the test material in the environment.
Without Humic Acid
o 72 hour cell density
EL50 >0.60 mg/L
NOELR ≥ 0.60 mg/L
o 0-72-hour cell yield
EyL50 >0.60 mg/L
NOELR ≥ 0.60 mg/L


o 0-72 hour growth inhibition
ErL50 >0.60 mg/L
NOELR = 0.27 mg/L
• With 10 mg Humic Acid/L
o 72 hour cell density
EL50 >30 mg/L
NOELR ≥ 30 mg/L
o 0-72-hour cell yield
EyL50 >30 mg/L
NOELR ≥ 30 mg/L
o 0-72 hour growth inhibition
ErL50 >30 mg/L
NOELR ≥ 30 mg/L
Executive summary:

The purpose of this study was to assess the effects of BADGE-MXDA (#42) to the freshwater green alga, Pseudokirchneriella subcapitata, over a 72-hour static exposure period. These data were used to determine EL50 (the loading rate causing 50% inhibition) values for 72-hour cell density, 0-72-hour cell yield, and average specific growth rate from 0 to 72 hours. When possible, NOELR (no-observable-effect loading rate, the highest loading rate not significantly different from the controls) values were determined. BADGE-MXDA (#42) has low aqueous solubility and therefore test solutions were prepared from a water accommodated fraction (WAF). BADGE-MXDA (#42) possesses cationic properties and was, therefore, tested in line with the OECD (2000) recommendation for testing substances with cationic properties for the mitigating effect of humic acid. The results of the test are as follows: Without Humic Acid: 72 hour cell density EL50 >0.60 mg/L NOELR ≥ 0.60 mg/L; 0-72-hour cell yield EyL50 >0.60 mg/L; NOELR ≥ 0.60 mg/L; 0 -72 hour growth inhibition ErL50 >0.60 mg/L NOELR = 0.27 mg/L. With 10 mg Humic Acid/L: 72 hour cell density EL50 >30 mg/L; NOELR ≥ 30 mg/L; 0 -72 -hour cell yield EyL50 >30 mg/L NOELR ≥ 30 mg/L; 0 -72 hour growth inhibition ErL50 >30 mg/L; NOELR ≥ 30 mg/L. The NOELR for growth rate was at least 100 times greater in the test with humic acid, indicating a mitigating effect of humic acid on BADGE-MXDA (#42).

Description of key information

One freashwater algal study (Pseudokirchneriella subcapitata)

Key value for chemical safety assessment

EC50 for freshwater algae:
30 mg/L
EC10 or NOEC for freshwater algae:
30 mg/L

Additional information

The purpose of this study was to assess the effects of BADGE-MXDA (#42) to the freshwater green alga, Pseudokirchneriella subcapitata, over a 72-hour static exposure period. These data were used to determine EL50 (the loading rate causing 50% inhibition) values for 72-hour cell density, 0-72-hour cell yield, and average specific growth rate from 0 to 72 hours. When possible, NOELR (no-observable-effect loading rate, the highest loading rate not significantly different from the controls) values were determined. BADGE-MXDA (#42) has low aqueous solubility and therefore test solutions were prepared from a water accommodated fraction (WAF). BADGE-MXDA (#42) possesses cationic properties and was, therefore, tested in line with the OECD (2000) recommendation for testing substances with cationic properties for the mitigating effect of humic acid. The results of the test are as follows: Without Humic Acid: 72 hour cell density EL50 >0.60 mg/L NOELR ≥ 0.60 mg/L; 0-72-hour cell yield EyL50 >0.60 mg/L; NOELR ≥ 0.60 mg/L; 0 -72 hour growth inhibition ErL50 >0.60 mg/L NOELR = 0.27 mg/L. With 10 mg Humic Acid/L: 72 hour cell density EL50 >30 mg/L; NOELR ≥ 30 mg/L; 0 -72 -hour cell yield EyL50 >30 mg/L NOELR ≥ 30 mg/L; 0 -72 hour growth inhibition ErL50 >30 mg/L; NOELR ≥ 30 mg/L. The NOELR for growth rate was at least 100 times greater in the test with humic acid, indicating a mitigating effect of humic acid on BADGE-MXDA (#42).